register interest

Dr Opher Gileadi

Research Area: Protein Science and Structural Biology
Technology Exchange: Crystallography
Scientific Themes: Protein Science & Structural Biology and Cancer Biology
Keywords: Structural genomics, DNA repair, RECQ Helicase and biotechnology
Web Links:
Human DNA Helicase RECQ1 in complex with DNA

Human DNA Helicase RECQ1 in complex with DNA

The search for understanding the mechanisms underlying human diseases and identifying targets for intervention has resulted in a deluge of data from genetic linkage analysis, which is likely to ramp up with current re-sequencing projects. Translating data on disease-linked genes and alleles into mechanistic insights and validated drug targets remains a huge challenge. In particular, many of the genes identified through genetic studies encode proteins with unknown function, or whose role in the disease is unclear.

Our approach is to initiate research on disease-linked genes and mutations by investigating the encoded proteins. The key is to develop and apply a systematic approach whereby both known and “orphan” proteins can be characterized to a satisfactory level. Based on our expertise in protein science, we express and purify relevant domains of the wild-type and mutated proteins, and use these to formulate and test hypotheses about the function of the proteins and the impact of the disease-linked mutations.

Purified proteins can be used to directly for enzymatic activity, binding to small molecules or binding to other proteins. Determination of protein structures by X-ray crystallography can give essential information on their function and evolutionary relationships.

Other Investigations include identification of molecular partners and post-translational modification by mass spectrometry, screening for small-molecule ligands, and generation of specific antibodies. The reagents and information can be used in partnership with scientists involved in the clinical investigation of the relevant diseases to further our understanding of the changes occurring in patients and to evaluate the therapeutic implications.

As part of the structural genomics effort, we continue to determine structures and characterize the biochemistry of medically-relevant human proteins. The group has focussed on DNA helicases and nucleases involved in DNA repair (pictured). DNA repair mechanisms are emerging as an potential new target for cancer therapy. Cancer cells are defective in DNA repair and are genetically unstable; it is thought that further inhibition of the remaining repair pathways will selectively kill cancer cells with little effect on other cells in the body. To explore this possibility, we have been screening and developing small-molecule inhibitors of the DNA helicases RECQ1 and BLM, which are currently evaluated for their impact on cultured cancer cells.

Name Department Institution Country
Prof Peter J McHugh (RDM) Weatherall Institute of Molecular Medicine Oxford University, Weatherall Institute of Molecular Medicine United Kingdom
Professor Paul Brennan Target Discovery Institute Oxford University, NDM Research Building United Kingdom
Dr Brian D Marsden Structural Genomics Consortium Oxford University, Old Road Campus Research Building United Kingdom
Professor Frank von Delft Structural Genomics Consortium Oxford University, Old Road Campus Research Building United Kingdom
Associate Professor David H Drewry SGC Eshelman School of Pharmacy, University of North Carolina United States
Bulbrook D, Brazier H, Mahajan P, Kliszczak M, Fedorov O, Marchese FP, Aubareda A, Chalk R, Picaud S, Strain-Damerell C et al. 2018. Tryptophan-Mediated Interactions between Tristetraprolin and the CNOT9 Subunit Are Required for CCR4-NOT Deadenylase Complex Recruitment. J Mol Biol, 430 (5), pp. 722-736. | Show Abstract | Read more

The zinc-finger protein tristetraprolin (TTP) binds to AU-rich elements present in the 3' untranslated regions of transcripts that mainly encode proteins of the inflammatory response. TTP-bound mRNAs are targeted for destruction via recruitment of the eight-subunit deadenylase complex "carbon catabolite repressor protein 4 (CCR4)-negative on TATA-less (NOT)," which catalyzes the removal of mRNA poly-(A) tails, the first obligatory step in mRNA decay. Here we show that a novel interaction between TTP and the CCR4-NOT subunit, CNOT9, is required for recruitment of the deadenylase complex. In addition to CNOT1, CNOT9 is now included in the identified CCR4-NOT subunits shown to interact with TTP. We find that both the N- and C-terminal domains of TTP are involved in an interaction with CNOT9. Through a combination of SPOT peptide array, site-directed mutagenesis, and bio-layer interferometry, we identified several conserved tryptophan residues in TTP that serve as major sites of interaction with two tryptophan-binding pockets of CNOT9, previously found to interact with another modulator GW182. We further demonstrate that these interactions are also required for recruitment of the CCR4-NOT complex and TTP-directed decay of an mRNA containing an AU-rich element in its 3'-untranslated region. Together the results reveal new molecular details for the TTP-CNOT interaction that shape an emerging mechanism whereby TTP targets inflammatory mRNAs for deadenylation and decay.

Bradley AR, Echalier A, Fairhead M, Strain-Damerell C, Brennan P, Bullock AN, Burgess-Brown NA, Carpenter EP, Gileadi O, Marsden BD et al. 2017. The SGC beyond structural genomics: redefining the role of 3D structures by coupling genomic stratification with fragment-based discovery. Essays Biochem, 61 (5), pp. 495-503. | Show Abstract | Read more

The ongoing explosion in genomics data has long since outpaced the capacity of conventional biochemical methodology to verify the large number of hypotheses that emerge from the analysis of such data. In contrast, it is still a gold-standard for early phenotypic validation towards small-molecule drug discovery to use probe molecules (or tool compounds), notwithstanding the difficulty and cost of generating them. Rational structure-based approaches to ligand discovery have long promised the efficiencies needed to close this divergence; in practice, however, this promise remains largely unfulfilled, for a host of well-rehearsed reasons and despite the huge technical advances spearheaded by the structural genomics initiatives of the noughties. Therefore the current, fourth funding phase of the Structural Genomics Consortium (SGC), building on its extensive experience in structural biology of novel targets and design of protein inhibitors, seeks to redefine what it means to do structural biology for drug discovery. We developed the concept of a Target Enabling Package (TEP) that provides, through reagents, assays and data, the missing link between genetic disease linkage and the development of usefully potent compounds. There are multiple prongs to the ambition: rigorously assessing targets' genetic disease linkages through crowdsourcing to a network of collaborating experts; establishing a systematic approach to generate the protocols and data that comprise each target's TEP; developing new, X-ray-based fragment technologies for generating high quality chemical matter quickly and cheaply; and exploiting a stringently open access model to build multidisciplinary partnerships throughout academia and industry. By learning how to scale these approaches, the SGC aims to make structures finally serve genomics, as originally intended, and demonstrate how 3D structures systematically allow new modes of druggability to be discovered for whole classes of targets.

Couñago RM, Allerston CK, Savitsky P, Azevedo H, Godoi PH, Wells CI, Mascarello A, de Souza Gama FH, Massirer KB, Zuercher WJ et al. 2017. Structural characterization of human Vaccinia-Related Kinases (VRK) bound to small-molecule inhibitors identifies different P-loop conformations. Sci Rep, 7 (1), pp. 7501. | Show Abstract | Read more

The human genome encodes two active Vaccinia-related protein kinases (VRK), VRK1 and VRK2. These proteins have been implicated in a number of cellular processes and linked to a variety of tumors. However, understanding the cellular role of VRKs and establishing their potential use as targets for therapeutic intervention has been limited by the lack of tool compounds that can specifically modulate the activity of these kinases in cells. Here we identified BI-D1870, a dihydropteridine inhibitor of RSK kinases, as a promising starting point for the development of chemical probes targeting the active VRKs. We solved co-crystal structures of both VRK1 and VRK2 bound to BI-D1870 and of VRK1 bound to two broad-spectrum inhibitors. These structures revealed that both VRKs can adopt a P-loop folded conformation, which is stabilized by different mechanisms on each protein. Based on these structures, we suggest modifications to the dihydropteridine scaffold that can be explored to produce potent and specific inhibitors towards VRK1 and VRK2.

Drewry DH, Wells CI, Andrews DM, Angell R, Al-Ali H, Axtman AD, Capuzzi SJ, Elkins JM, Ettmayer P, Frederiksen M et al. 2017. Progress towards a public chemogenomic set for protein kinases and a call for contributions. PLoS One, 12 (8), pp. e0181585. | Show Abstract | Read more

Protein kinases are highly tractable targets for drug discovery. However, the biological function and therapeutic potential of the majority of the 500+ human protein kinases remains unknown. We have developed physical and virtual collections of small molecule inhibitors, which we call chemogenomic sets, that are designed to inhibit the catalytic function of almost half the human protein kinases. In this manuscript we share our progress towards generation of a comprehensive kinase chemogenomic set (KCGS), release kinome profiling data of a large inhibitor set (Published Kinase Inhibitor Set 2 (PKIS2)), and outline a process through which the community can openly collaborate to create a KCGS that probes the full complement of human protein kinases.

Aquino B, Couñago RM, Verza N, Ferreira LM, Massirer KB, Gileadi O, Arruda P. 2017. Structural Characterization of Maize SIRK1 Kinase Domain Reveals an Unusual Architecture of the Activation Segment. Front Plant Sci, 8 pp. 852. | Show Abstract | Read more

Kinases are primary regulators of plant metabolism and excellent targets for plant breeding. However, most kinases, including the abundant receptor-like kinases (RLK), have no assigned role. SIRK1 is a leucine-rich repeat receptor-like kinase (LRR-RLK), the largest family of RLK. In Arabidopsis thaliana, SIRK1 (AtSIRK1) is phosphorylated after sucrose is resupplied to sucrose-starved seedlings and it modulates the sugar response by phosphorylating several substrates. In maize, the ZmSIRK1 expression is altered in response to drought stress. In neither Arabidopsis nor in maize has the function of SIRK1 been completely elucidated. As a first step toward the biochemical characterization of ZmSIRK1, we obtained its recombinant kinase domain, demonstrated that it binds AMP-PNP, a non-hydrolysable ATP-analog, and solved the structure of ZmSIRK1- AMP-PNP co-crystal. The ZmSIRK1 crystal structure revealed a unique conformation for the activation segment. In an attempt to find inhibitors for ZmSIRK1, we screened a focused small molecule library and identified six compounds that stabilized ZmSIRK1 against thermal melt. ITC analysis confirmed that three of these compounds bound to ZmSIRK1 with low micromolar affinity. Solving the 3D structure of ZmSIRK1-AMP-PNP co-crystal provided information on the molecular mechanism of ZmSIRK1 activity. Furthermore, the identification of small molecules that bind this kinase can serve as initial backbone for development of new potent and selective ZmSIRK1 antagonists.

Newman JA, Aitkenhead H, Savitsky P, Gileadi O. 2017. Insights into the RecQ helicase mechanism revealed by the structure of the helicase domain of human RECQL5. Nucleic Acids Res, 45 (7), pp. 4231-4243. | Show Abstract | Read more

RecQ helicases are important maintainers of genome integrity with distinct roles in almost every cellular process requiring access to DNA. RECQL5 is one of five human RecQ proteins and is particularly versatile in this regard, forming protein complexes with a diverse set of cellular partners in order to coordinate its helicase activity to various processes including replication, recombination and DNA repair. In this study, we have determined crystal structures of the core helicase domain of RECQL5 both with and without the nucleotide ADP in two distinctly different ('Open' and 'Closed') conformations. Small angle X-ray scattering studies show that the 'Open' form of the protein predominates in solution and we discuss implications of this with regards to the RECQL5 mechanism and conformational changes. We have measured the ATPase, helicase and DNA binding properties of various RECQL5 constructs and variants and discuss the role of these regions and residues in the various RECQL5 activities. Finally, we have performed a systematic comparison of the RECQL5 structures with other RecQ family structures and based on these comparisons we have constructed a model for the mechano-chemical cycle of the common catalytic core of these helicases.

Vaz B, Popovic M, Newman JA, Fielden J, Aitkenhead H, Halder S, Singh AN, Vendrell I, Fischer R, Torrecilla I et al. 2016. Metalloprotease SPRTN/DVC1 Orchestrates Replication-Coupled DNA-Protein Crosslink Repair. Mol Cell, 64 (4), pp. 704-719. | Show Abstract | Read more

The cytotoxicity of DNA-protein crosslinks (DPCs) is largely ascribed to their ability to block the progression of DNA replication. DPCs frequently occur in cells, either as a consequence of metabolism or exogenous agents, but the mechanism of DPC repair is not completely understood. Here, we characterize SPRTN as a specialized DNA-dependent and DNA replication-coupled metalloprotease for DPC repair. SPRTN cleaves various DNA binding substrates during S-phase progression and thus protects proliferative cells from DPC toxicity. Ruijs-Aalfs syndrome (RJALS) patient cells with monogenic and biallelic mutations in SPRTN are hypersensitive to DPC-inducing agents due to a defect in DNA replication fork progression and the inability to eliminate DPCs. We propose that SPRTN protease represents a specialized DNA replication-coupled DPC repair pathway essential for DNA replication progression and genome stability. Defective SPRTN-dependent clearance of DPCs is the molecular mechanism underlying RJALS, and DPCs are contributing to accelerated aging and cancer.

Lee SY, Brem J, Pettinati I, Claridge TDW, Gileadi O, Schofield CJ, McHugh PJ. 2016. Cephalosporins inhibit human metallo β-lactamase fold DNA repair nucleases SNM1A and SNM1B/apollo. Chem Commun (Camb), 52 (40), pp. 6727-6730. | Show Abstract | Read more

Bacterial metallo-β-lactamases (MBLs) are involved in resistance to β-lactam antibiotics including cephalosporins. Human SNM1A and SNM1B are MBL superfamily exonucleases that play a key role in the repair of DNA interstrand cross-links, which are induced by antitumour chemotherapeutics, and are therefore targets for cancer chemosensitization. We report that cephalosporins are competitive inhibitors of SNM1A and SNM1B exonuclease activity; both the intact β-lactam and their hydrolysed products are active. This discovery provides a lead for the development of potent and selective SNM1A and SNM1B inhibitors.

Levy S, Allerston CK, Liveanu V, Habib MR, Gileadi O, Schuster G. 2016. Identification of LACTB2, a metallo-β-lactamase protein, as a human mitochondrial endoribonuclease. Nucleic Acids Res, 44 (4), pp. 1813-1832. | Show Abstract | Read more

Post-transcriptional control of mitochondrial gene expression, including the processing and generation of mature transcripts as well as their degradation, is a key regulatory step in gene expression in human mitochondria. Consequently, identification of the proteins responsible for RNA processing and degradation in this organelle is of great importance. The metallo-β-lactamase (MBL) is a candidate protein family that includes ribo- and deoxyribonucleases. In this study, we discovered a function for LACTB2, an orphan MBL protein found in mammalian mitochondria. Solving its crystal structure revealed almost perfect alignment of the MBL domain with CPSF73, as well as to other ribonucleases of the MBL superfamily. Recombinant human LACTB2 displayed robust endoribonuclease activity on ssRNA with a preference for cleavage after purine-pyrimidine sequences. Mutational analysis identified an extended RNA-binding site. Knockdown of LACTB2 in cultured cells caused a moderate but significant accumulation of many mitochondrial transcripts, and its overexpression led to the opposite effect. Furthermore, manipulation of LACTB2 expression resulted in cellular morphological deformation and cell death. Together, this study discovered that LACTB2 is an endoribonuclease that is involved in the turnover of mitochondrial RNA, and is essential for mitochondrial function in human cells.

Broderick R, Nieminuszczy J, Baddock HT, Deshpande R, Gileadi O, Paull TT, McHugh PJ, Niedzwiedz W. 2016. EXD2 promotes homologous recombination by facilitating DNA end resection. Nat Cell Biol, 18 (3), pp. 271-280. | Show Abstract | Read more

Repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) is critical for survival and genome stability of individual cells and organisms, but also contributes to the genetic diversity of species. A vital step in HR is MRN-CtIP-dependent end resection, which generates the 3' single-stranded DNA overhangs required for the subsequent strand exchange reaction. Here, we identify EXD2 (also known as EXDL2) as an exonuclease essential for DSB resection and efficient HR. EXD2 is recruited to chromatin in a damage-dependent manner and confers resistance to DSB-inducing agents. EXD2 functionally interacts with the MRN complex to accelerate resection through its 3'-5' exonuclease activity, which efficiently processes double-stranded DNA substrates containing nicks. Finally, we establish that EXD2 stimulates both short- and long-range DSB resection, and thus, together with MRE11, is required for efficient HR. This establishes a key role for EXD2 in controlling the initial steps of chromosomal break repair.

Newman JA, Cooper CDO, Roos AK, Aitkenhead H, Oppermann UCT, Cho HJ, Osman R, Gileadi O. 2016. Structures of Two Melanoma-Associated Antigens Suggest Allosteric Regulation of Effector Binding. PLoS One, 11 (2), pp. e0148762. | Show Abstract | Read more

The MAGE (melanoma associated antigen) protein family are tumour-associated proteins normally present only in reproductive tissues such as germ cells of the testis. The human genome encodes over 60 MAGE genes of which one class (containing MAGE-A3 and MAGE-A4) are exclusively expressed in tumours, making them an attractive target for the development of targeted and immunotherapeutic cancer treatments. Some MAGE proteins are thought to play an active role in driving cancer, modulating the activity of E3 ubiquitin ligases on targets related to apoptosis. Here we determined the crystal structures of MAGE-A3 and MAGE-A4. Both proteins crystallized with a terminal peptide bound in a deep cleft between two tandem-arranged winged helix domains. MAGE-A3 (but not MAGE-A4), is predominantly dimeric in solution. Comparison of MAGE-A3 and MAGE-A3 with a structure of an effector-bound MAGE-G1 suggests that a major conformational rearrangement is required for binding, and that this conformational plasticity may be targeted by allosteric binders.

Newman JA, Cooper CDO, Aitkenhead H, Gileadi O. 2015. Structure of the Helicase Domain of DNA Polymerase Theta Reveals a Possible Role in the Microhomology-Mediated End-Joining Pathway. Structure, 23 (12), pp. 2319-2330. | Show Abstract | Read more

DNA polymerase theta (Polθ) has been identified as a crucial alternative non-homologous end-joining factor in mammalian cells. Polθ is upregulated in a range of cancer cell types defective in homologous recombination, and knockdown has been shown to inhibit cell survival in a subset of these, making it an attractive target for cancer treatment. We present crystal structures of the helicase domain of human Polθ in the presence and absence of bound nucleotides, and a characterization of its DNA-binding and DNA-stimulated ATPase activities. Comparisons with related helicases from the Hel308 family identify several unique features. Polθ exists as a tetramer both in the crystals and in solution. We propose a model for DNA binding to the Polθ helicase domain in the context of the Polθ tetramer, which suggests a role for the helicase domain in strand annealing of DNA templates for subsequent processing by the polymerase domain.

Allerston CK, Lee SY, Newman JA, Schofield CJ, McHugh PJ, Gileadi O. 2015. The structures of the SNM1A and SNM1B/Apollo nuclease domains reveal a potential basis for their distinct DNA processing activities. Nucleic Acids Res, 43 (22), pp. 11047-11060. | Show Abstract | Read more

The human SNM1A and SNM1B/Apollo proteins are members of an extended family of eukaryotic nuclease containing a motif related to the prokaryotic metallo-β-lactamase (MBL) fold. SNM1A is a key exonuclease during replication-dependent and transcription-coupled interstrand crosslink repair, while SNM1B/Apollo is required for maintaining telomeric overhangs. Here, we report the crystal structures of SNM1A and SNM1B at 2.16 Å. While both proteins contain a typical MBL-β-CASP domain, a region of positive charge surrounds the active site of SNM1A, which is absent in SNM1B and explains the greater apparent processivity of SNM1A. The structures of both proteins also reveal a putative, wide DNA-binding groove. Extensive mutagenesis of this groove, coupled with detailed biochemical analysis, identified residues that did not impact on SNM1A catalytic activity, but drastically reduced its processivity. Moreover, we identified a key role for this groove for efficient digestion past DNA interstrand crosslinks, facilitating the key DNA repair reaction catalysed by SNM1A. Together, the architecture and dimensions of this groove, coupled to the surrounding region of high positive charge, explain the remarkable ability of SNM1A to accommodate and efficiently digest highly distorted DNA substrates, such as those containing DNA lesions.

Brem J, van Berkel SS, Zollman D, Lee SY, Gileadi O, McHugh PJ, Walsh TR, McDonough MA, Schofield CJ. 2016. Structural Basis of Metallo-β-Lactamase Inhibition by Captopril Stereoisomers. Antimicrob Agents Chemother, 60 (1), pp. 142-150. | Show Abstract | Read more

β-Lactams are the most successful antibacterials, but their effectiveness is threatened by resistance, most importantly by production of serine- and metallo-β-lactamases (MBLs). MBLs are of increasing concern because they catalyze the hydrolysis of almost all β-lactam antibiotics, including recent-generation carbapenems. Clinically useful serine-β-lactamase inhibitors have been developed, but such inhibitors are not available for MBLs. l-Captopril, which is used to treat hypertension via angiotensin-converting enzyme inhibition, has been reported to inhibit MBLs by chelating the active site zinc ions via its thiol(ate). We report systematic studies on B1 MBL inhibition by all four captopril stereoisomers. High-resolution crystal structures of three MBLs (IMP-1, BcII, and VIM-2) in complex with either the l- or d-captopril stereoisomer reveal correlations between the binding mode and inhibition potency. The results will be useful in the design of MBL inhibitors with the breadth of selectivity required for clinical application against carbapenem-resistant Enterobacteriaceae and other organisms causing MBL-mediated resistant infections.

Iyama T, Lee SY, Berquist BR, Gileadi O, Bohr VA, Seidman MM, McHugh PJ, Wilson DM. 2015. CSB interacts with SNM1A and promotes DNA interstrand crosslink processing Nucleic Acids Research, 43 (1), pp. 247-258. | Show Abstract | Read more

© 2015 Published by Oxford University Press on behalf of Nucleic Acids Research. Cockayne syndrome (CS) is a premature aging disorder characterized by photosensitivity, impaired development and multisystem progressive degeneration, and consists of two strict complementation groups, A and B. Using a yeast two-hybrid approach, we identified the 5′-3′ exonuclease SNM1A as one of four strong interacting partners of CSB. This direct interaction was confirmed using purified recombinant proteins-with CSB able to modulate the exonuclease activity of SNM1A on oligonucleotide substrates in vitro-and the two proteins were shown to exist in a common complex in human cell extracts. CSB and SNM1A were also found, using fluorescently tagged proteins in combination with confocal microscopy and laser microirradiation, to be recruited to localized trioxsalen-induced ICL damage in human cells, with accumulation being suppressed by transcription inhibition. Moreover, SNM1A recruitment was significantly reduced in CSB-deficient cells, suggesting coordination between the two proteins in vivo. CSB-deficient neural cells exhibited increased sensitivity to DNA crosslinking agents, particularly, in a non-cycling, differentiated state, as well as delayed ICL processing as revealed by a modified Comet assay and γ-H2AX foci persistence. The results indicate that CSB coordinates the resolution of ICLs, possibly in a transcription-associated repair mechanism involving SNM1A, and that defect s in the process could contribute to the post-mitotic degenerative pathologies associated with CS.

Newman JA, Savitsky P, Allerston CK, Bizard AH, Özer Ö, Sarlós K, Liu Y, Pardon E, Steyaert J, Hickson ID, Gileadi O. 2015. Crystal structure of the Bloom's syndrome helicase indicates a role for the HRDC domain in conformational changes. Nucleic Acids Res, 43 (10), pp. 5221-5235. | Show Abstract | Read more

Bloom's syndrome helicase (BLM) is a member of the RecQ family of DNA helicases, which play key roles in the maintenance of genome integrity in all organism groups. We describe crystal structures of the BLM helicase domain in complex with DNA and with an antibody fragment, as well as SAXS and domain association studies in solution. We show an unexpected nucleotide-dependent interaction of the core helicase domain with the conserved, poorly characterized HRDC domain. The BLM-DNA complex shows an unusual base-flipping mechanism with unique positioning of the DNA duplex relative to the helicase core domains. Comparison with other crystal structures of RecQ helicases permits the definition of structural transitions underlying ATP-driven helicase action, and the identification of a nucleotide-regulated tunnel that may play a role in interactions with complex DNA substrates.

Cooper CDO, Newman JA, Aitkenhead H, Allerston CK, Gileadi O. 2015. Structures of the Ets Protein DNA-binding Domains of Transcription Factors Etv1, Etv4, Etv5, and Fev: DETERMINANTS OF DNA BINDING AND REDOX REGULATION BY DISULFIDE BOND FORMATION. J Biol Chem, 290 (22), pp. 13692-13709. | Show Abstract | Read more

Ets transcription factors, which share the conserved Ets DNA-binding domain, number nearly 30 members in humans and are particularly involved in developmental processes. Their deregulation following changes in expression, transcriptional activity, or by chromosomal translocation plays a critical role in carcinogenesis. Ets DNA binding, selectivity, and regulation have been extensively studied; however, questions still arise regarding binding specificity outside the core GGA recognition sequence and the mode of action of Ets post-translational modifications. Here, we report the crystal structures of Etv1, Etv4, Etv5, and Fev, alone and in complex with DNA. We identify previously unrecognized features of the protein-DNA interface. Interactions with the DNA backbone account for most of the binding affinity. We describe a highly coordinated network of water molecules acting in base selection upstream of the GGAA core and the structural features that may account for discrimination against methylated cytidine residues. Unexpectedly, all proteins crystallized as disulfide-linked dimers, exhibiting a novel interface (distant to the DNA recognition helix). Homodimers of Etv1, Etv4, and Etv5 could be reduced to monomers, leading to a 40-200-fold increase in DNA binding affinity. Hence, we present the first indication of a redox-dependent regulatory mechanism that may control the activity of this subset of oncogenic Ets transcription factors.

Pike ACW, Gomathinayagam S, Swuec P, Berti M, Zhang Y, Schnecke C, Marino F, von Delft F, Renault L, Costa A et al. 2015. Human RECQ1 helicase-driven DNA unwinding, annealing, and branch migration: insights from DNA complex structures. Proc Natl Acad Sci U S A, 112 (14), pp. 4286-4291. | Show Abstract | Read more

RecQ helicases are a widely conserved family of ATP-dependent motors with diverse roles in nearly every aspect of bacterial and eukaryotic genome maintenance. However, the physical mechanisms by which RecQ helicases recognize and process specific DNA replication and repair intermediates are largely unknown. Here, we solved crystal structures of the human RECQ1 helicase in complexes with tailed-duplex DNA and ssDNA. The structures map the interactions of the ssDNA tail and the branch point along the helicase and Zn-binding domains, which, together with reported structures of other helicases, define the catalytic stages of helicase action. We also identify a strand-separating pin, which (uniquely in RECQ1) is buttressed by the protein dimer interface. A duplex DNA-binding surface on the C-terminal domain is shown to play a role in DNA unwinding, strand annealing, and Holliday junction (HJ) branch migration. We have combined EM and analytical ultracentrifugation approaches to show that RECQ1 can form what appears to be a flat, homotetrameric complex and propose that RECQ1 tetramers are involved in HJ recognition. This tetrameric arrangement suggests a platform for coordinated activity at the advancing and receding duplexes of an HJ during branch migration.

Edwards AM, Arrowsmith CH, Bountra C, Bunnage ME, Feldmann M, Knight JC, Patel DD, Prinos P, Taylor MD, Sundström M, SGC Open Source Target-Discovery Partnership. 2015. Preclinical target validation using patient-derived cells. Nat Rev Drug Discov, 14 (3), pp. 149-150. | Show Abstract | Read more

The Structural Genomics Consortium (SGC) and its clinical, industry and disease-foundation partners are launching open-source preclinical translational medicine studies.

Newman JA, Cooper CDO, Aitkenhead H, Gileadi O. 2015. Structural insights into the autoregulation and cooperativity of the human transcription factor Ets-2. J Biol Chem, 290 (13), pp. 8539-8549. | Show Abstract | Read more

Ets-2, like its closely related homologue Ets-1, is a member of the Ets family of DNA binding transcription factors. Both proteins are subject to multiple levels of regulation of their DNA binding and transactivation properties. One such regulatory mechanism is the presence of an autoinhibitory module, which in Ets-1 allosterically inhibits the DNA binding activity. This inhibition can be relieved by interaction with protein partners or cooperative binding to closely separated Ets binding sites in a palindromic arrangement. In this study we describe the 2.5 Å resolution crystal structure of a DNA complex of the Ets-2 Ets domain. The Ets domain crystallized with two distinct species in the asymmetric unit, which closely resemble the autoinhibited and DNA bound forms of Ets-1. This discovery prompted us to re-evaluate the current model for the autoinhibitory mechanism and the structural basis for cooperative DNA binding. In contrast to Ets-1, in which the autoinhibition is caused by a combination of allosteric and steric mechanisms, we were unable to find clear evidence for the allosteric mechanism in Ets-2. We also demonstrated two possibly distinct types of cooperative binding to substrates with Ets binding motifs separated by four and six base pairs and suggest possible molecular mechanisms for this behavior.

Iyama T, Lee SY, Berquist BR, Gileadi O, Bohr VA, Seidman MM, McHugh PJ, Wilson DM. 2015. CSB interacts with SNM1A and promotes DNA interstrand crosslink processing. Nucleic Acids Res, 43 (1), pp. 247-258. | Show Abstract | Read more

Cockayne syndrome (CS) is a premature aging disorder characterized by photosensitivity, impaired development and multisystem progressive degeneration, and consists of two strict complementation groups, A and B. Using a yeast two-hybrid approach, we identified the 5'-3' exonuclease SNM1A as one of four strong interacting partners of CSB. This direct interaction was confirmed using purified recombinant proteins-with CSB able to modulate the exonuclease activity of SNM1A on oligonucleotide substrates in vitro-and the two proteins were shown to exist in a common complex in human cell extracts. CSB and SNM1A were also found, using fluorescently tagged proteins in combination with confocal microscopy and laser microirradiation, to be recruited to localized trioxsalen-induced ICL damage in human cells, with accumulation being suppressed by transcription inhibition. Moreover, SNM1A recruitment was significantly reduced in CSB-deficient cells, suggesting coordination between the two proteins in vivo. CSB-deficient neural cells exhibited increased sensitivity to DNA crosslinking agents, particularly, in a non-cycling, differentiated state, as well as delayed ICL processing as revealed by a modified Comet assay and γ-H2AX foci persistence. The results indicate that CSB coordinates the resolution of ICLs, possibly in a transcription-associated repair mechanism involving SNM1A, and that defects in the process could contribute to the post-mitotic degenerative pathologies associated with CS.

Cooper CDO, Newman JA, Gileadi O. 2014. Recent advances in the structural molecular biology of Ets transcription factors: interactions, interfaces and inhibition. Biochem Soc Trans, 42 (1), pp. 130-138. | Show Abstract | Read more

The Ets family of eukaryotic transcription factors is based around the conserved Ets DNA-binding domain. Although their DNA-binding selectivity is biochemically and structurally well characterized, structures of homodimeric and ternary complexes point to Ets domains functioning as versatile protein-interaction modules. In the present paper, we review the progress made over the last decade to elucidate the structural mechanisms involved in modulation of DNA binding and protein partner selection during dimerization. We see that Ets domains, although conserved around a core architecture, have evolved to utilize a variety of interaction surfaces and binding mechanisms, reflecting Ets domains as dynamic interfaces for both DNA and protein interaction. Furthermore, we discuss recent advances in drug development for inhibition of Ets factors, and the roles structural biology can play in their future.

Gileadi O. 2014. Structures of soluble guanylate cyclase: implications for regulatory mechanisms and drug development. Biochem Soc Trans, 42 (1), pp. 108-113. | Show Abstract | Read more

Activation of cGMP synthesis leads to vasodilation, and is an important mechanism in clinical treatment of angina, heart failure, and severe peripheral and pulmonary hypertension. The nitric oxide-responsive sGC (soluble guanylate cyclase) has been the target of recent drug discovery efforts. The present review surveys recent data on the structure and regulation of sGC, and the prospects of new avenues for therapeutic intervention.

Chatterjee S, Zagelbaum J, Savitsky P, Sturzenegger A, Huttner D, Janscak P, Hickson ID, Gileadi O, Rothenberg E. 2014. Mechanistic insight into the interaction of BLM helicase with intra-strand G-quadruplex structures. Nat Commun, 5 pp. 5556. | Show Abstract | Read more

Bloom syndrome is an autosomal recessive disorder caused by mutations in the RecQ family helicase BLM that is associated with growth retardation and predisposition to cancer. BLM helicase has a high specificity for non-canonical G-quadruplex (G4) DNA structures, which are formed by G-rich DNA strands and play an important role in the maintenance of genomic integrity. Here we used single-molecule FRET to define the mechanism of interaction of BLM helicase with intra-stranded G4 structures. We show that the activity of BLM is substrate dependent, and highly regulated by a short-strand DNA (ssDNA) segment that separates the G4 motif from double-stranded DNA. We demonstrate cooperativity between the RQC and HRDC domains of BLM during binding and unfolding of the G4 structure, where the RQC domain interaction with G4 is stabilized by HRDC binding to ssDNA. We present a model that proposes a unique role for G4 structures in modulating the activity of DNA processing enzymes.

Mahajan P, Strain-Damerell C, Gileadi O, Burgess-Brown NA. 2014. Medium-throughput production of recombinant human proteins: protein production in insect cells. Methods Mol Biol, 1091 pp. 95-121. | Show Abstract | Read more

This chapter describes the step-by-step methods employed by the Structural Genomics Consortium (SGC) for screening and producing proteins in the baculovirus expression vector system (BEVS). This eukaryotic expression system was selected and a screening process established in 2007 as a measure to tackle the more challenging kinase, RNA-DNA processing and integral membrane protein families on our target list. Here, we discuss our platform for identifying soluble proteins from 3 ml of insect cell culture and describe the procedures involved in producing protein from liter-scale cultures. Although not discussed in this chapter, the same process can also be applied to integral membrane proteins (IMPs) with slight adaptations to the purification procedure.

Burgess-Brown NA, Mahajan P, Strain-Damerell C, Gileadi O, Gräslund S. 2014. Medium-throughput production of recombinant human proteins: protein production in E. coli. Methods Mol Biol, 1091 pp. 73-94. | Show Abstract | Read more

In Chapter 4 we described the SGC process for generating multiple constructs of truncated versions of each protein using LIC. In this chapter we provide a step-by-step procedure of our E. coli system for test expressing intracellular (soluble) proteins in a 96-well format that enables us to identify which proteins or truncated versions are expressed in a soluble and stable form suitable for structural studies. In addition, we detail the process for scaling up cultures for large-scale protein purification. This level of production is required to obtain sufficient quantities (i.e., milligram amounts) of protein for further characterization and/or crystallization experiments. Our standard process is purification by immobilized metal affinity chromatography (IMAC) using nickel resin followed by size exclusion chromatography (SEC), with additional procedures arising from the complexity of the protein itself.

Strain-Damerell C, Mahajan P, Gileadi O, Burgess-Brown NA. 2014. Medium-throughput production of recombinant human proteins: ligation-independent cloning. Methods Mol Biol, 1091 pp. 55-72. | Show Abstract | Read more

Structural genomics groups have identified the need to generate multiple truncated versions of each target to improve their success in producing a well-expressed, soluble, and stable protein and one that crystallizes and diffracts to a sufficient resolution for structural determination. At the SGC, we opted for the Ligation-Independent Cloning (LIC) method which provides the medium throughput we desire to produce and screen many proteins in a parallel process. Here, we describe our LIC protocol for generating constructs in a 96-well format and provide a choice of vectors suitable for expressing proteins in both E. coli and the baculovirus expression vector system (BEVS).

Strain-Damerell C, Mahajan P, Gileadi O, Burgess-Brown NA. 2014. Medium-throughput production of recombinant human proteins: Ligation-independent cloning Methods in Molecular Biology, 1091 pp. 55-72. | Show Abstract | Read more

Structural genomics groups have identified the need to generate multiple truncated versions of each target to improve their success in producing a well-expressed, soluble, and stable protein and one that crystallizes and diffracts to a sufficient resolution for structural determination. At the SGC, we opted for the Ligation-Independent Cloning (LIC) method which provides the medium throughput we desire to produce and screen many proteins in a parallel process. Here, we describe our LIC protocol for generating constructs in a 96-well format and provide a choice of vectors suitable for expressing proteins in both E. coli and the baculovirus expression vector system (BEVS). © 2014 Springer Science+Business Media, LLC.

Burgess-Brown NA, Mahajan P, Strain-Damerell C, Gileadi O, Gräslund S. 2014. Medium-throughput production of recombinant human proteins: Protein production in E. Coli Methods in Molecular Biology, 1091 pp. 73-94. | Show Abstract | Read more

In Chapter 4 we described the SGC process for generating multiple constructs of truncated versions of each protein using LIC. In this chapter we provide a step-by-step procedure of our E. coli system for test expressing intracellular (soluble) proteins in a 96-well format that enables us to identify which proteins or truncated versions are expressed in a soluble and stable form suitable for structural studies. In addition, we detail the process for scaling up cultures for large-scale protein purification. This level of production is required to obtain sufficient quantities (i.e., milligram amounts) of protein for further characterization and/or crystallization experiments. Our standard process is purification by immobilized metal affinity chromatography (IMAC) using nickel resin followed by size exclusion chromatography (SEC), with additional procedures arising from the complexity of the protein itself. © 2014 Springer Science+Business Media, LLC.

Mahajan P, Strain-Damerell C, Gileadi O, Burgess-Brown NA. 2014. Medium-throughput production of recombinant human proteins: Protein production in insect cells Methods in Molecular Biology, 1091 pp. 95-121. | Show Abstract | Read more

This chapter describes the step-by-step methods employed by the Structural Genomics Consortium (SGC) for screening and producing proteins in the baculovirus expression vector system (BEVS). This eukaryotic expression system was selected and a screening process established in 2007 as a measure to tackle the more challenging kinase, RNA-DNA processing and integral membrane protein families on our target list. Here, we discuss our platform for identifying soluble proteins from 3 ml of insect cell culture and describe the procedures involved in producing protein from liter-scale cultures. Although not discussed in this chapter, the same process can also be applied to integral membrane proteins (IMPs) with slight adaptations to the purification procedure. © 2014 Springer Science+Business Media, LLC.

Rosenthal AS, Dexheimer TS, Gileadi O, Nguyen GH, Chu WK, Hickson ID, Jadhav A, Simeonov A, Maloney DJ. 2013. Synthesis and SAR studies of 5-(pyridin-4-yl)-1,3,4-thiadiazol-2-amine derivatives as potent inhibitors of Bloom helicase Bioorganic and Medicinal Chemistry Letters, 23 (20), pp. 5660-5666. | Show Abstract | Read more

Human cells utilize a variety of complex DNA repair mechanisms in order to combat constant mutagenic and cytotoxic threats from both exogenous and endogenous sources. The RecQ family of DNA helicases, which includes Bloom helicase (BLM), plays an important function in DNA repair by unwinding complementary strands of duplex DNA as well as atypical DNA structures such as Holliday junctions. Mutations of the BLM gene can result in Bloom syndrome, an autosomal recessive disorder associated with cancer predisposition. BLM-deficient cells exhibit increased sensitivity to DNA damaging agents indicating that a selective BLM inhibitor could be useful in potentiating the anticancer activity of these agents. In this work, we describe the medicinal chemistry optimization of the hit molecule following a quantitative high-throughput screen of > 355,000 compounds. These efforts lead to the identification of ML216 and related analogs, which possess potent BLM inhibition and exhibit selectivity over related helicases. Moreover, these compounds demonstrated cellular activity by inducing sister chromatid exchanges, a hallmark of Bloom syndrome. © 2013 Elsevier Ltd. All rights reserved.

Canning P, Cooper CD, Krojer T, Murray JW, Pike AC, Chaikuad A, Keates T, Thangaratnarajah C, Hojzan V, Ayinampudi V et al. 2013. Structural basis for Cul3 protein assembly with the BTB-Kelch family of E3 ubiquitin ligases. J Biol Chem, 288 (39), pp. 28304. | Read more

Froese DS, Forouhar F, Tran TH, Vollmar M, Kim YS, Lew S, Neely H, Seetharaman J, Shen Y, Xiao R et al. 2013. Crystal structures of malonyl-coenzyme A decarboxylase provide insights into its catalytic mechanism and disease-causing mutations. Structure, 21 (7), pp. 1182-1192. | Show Abstract | Read more

Malonyl-coenzyme A decarboxylase (MCD) is found from bacteria to humans, has important roles in regulating fatty acid metabolism and food intake, and is an attractive target for drug discovery. We report here four crystal structures of MCD from human, Rhodopseudomonas palustris, Agrobacterium vitis, and Cupriavidus metallidurans at up to 2.3 Å resolution. The MCD monomer contains an N-terminal helical domain involved in oligomerization and a C-terminal catalytic domain. The four structures exhibit substantial differences in the organization of the helical domains and, consequently, the oligomeric states and intersubunit interfaces. Unexpectedly, the MCD catalytic domain is structurally homologous to those of the GCN5-related N-acetyltransferase superfamily, especially the curacin A polyketide synthase catalytic module, with a conserved His-Ser/Thr dyad important for catalysis. Our structures, along with mutagenesis and kinetic studies, provide a molecular basis for understanding pathogenic mutations and catalysis, as well as a template for structure-based drug design.

Shintre CA, Pike ACW, Li Q, Kim J-I, Barr AJ, Goubin S, Shrestha L, Yang J, Berridge G, Ross J et al. 2013. Structures of ABCB10, a human ATP-binding cassette transporter in apo- and nucleotide-bound states. Proc Natl Acad Sci U S A, 110 (24), pp. 9710-9715. | Show Abstract | Read more

ABCB10 is one of the three ATP-binding cassette (ABC) transporters found in the inner membrane of mitochondria. In mammals ABCB10 is essential for erythropoiesis, and for protection of mitochondria against oxidative stress. ABCB10 is therefore a potential therapeutic target for diseases in which increased mitochondrial reactive oxygen species production and oxidative stress play a major role. The crystal structure of apo-ABCB10 shows a classic exporter fold ABC transporter structure, in an open-inwards conformation, ready to bind the substrate or nucleotide from the inner mitochondrial matrix or membrane. Unexpectedly, however, ABCB10 adopts an open-inwards conformation when complexed with nonhydrolysable ATP analogs, in contrast to other transporter structures which adopt an open-outwards conformation in complex with ATP. The three complexes of ABCB10/ATP analogs reported here showed varying degrees of opening of the transport substrate binding site, indicating that in this conformation there is some flexibility between the two halves of the protein. These structures suggest that the observed plasticity, together with a portal between two helices in the transmembrane region of ABCB10, assist transport substrate entry into the substrate binding cavity. These structures indicate that ABC transporters may exist in an open-inwards conformation when nucleotide is bound. We discuss ways in which this observation can be aligned with the current views on mechanisms of ABC transporters.

Babbs C, Roberts NA, Sanchez-Pulido L, McGowan SJ, Ahmed MR, Brown JM, Sabry MA, WGS500 Consortium, Bentley DR, McVean GA et al. 2013. Homozygous mutations in a predicted endonuclease are a novel cause of congenital dyserythropoietic anemia type I. Haematologica, 98 (9), pp. 1383-1387. | Show Abstract | Read more

The congenital dyserythropoietic anemias are a heterogeneous group of rare disorders primarily affecting erythropoiesis with characteristic morphological abnormalities and a block in erythroid maturation. Mutations in the CDAN1 gene, which encodes Codanin-1, underlie the majority of congenital dyserythropoietic anemia type I cases. However, no likely pathogenic CDAN1 mutation has been detected in approximately 20% of cases, suggesting the presence of at least one other locus. We used whole genome sequencing and segregation analysis to identify a homozygous T to A transversion (c.533T>A), predicted to lead to a p.L178Q missense substitution in C15ORF41, a gene of unknown function, in a consanguineous pedigree of Middle-Eastern origin. Sequencing C15ORF41 in other CDAN1 mutation-negative congenital dyserythropoietic anemia type I pedigrees identified a homozygous transition (c.281A>G), predicted to lead to a p.Y94C substitution, in two further pedigrees of SouthEast Asian origin. The haplotype surrounding the c.281A>G change suggests a founder effect for this mutation in Pakistan. Detailed sequence similarity searches indicate that C15ORF41 encodes a novel restriction endonuclease that is a member of the Holliday junction resolvase family of proteins.

Nguyen GH, Dexheimer TS, Rosenthal AS, Chu WK, Singh DK, Mosedale G, Bachrati CZ, Schultz L, Sakurai M, Savitsky P et al. 2013. A small molecule inhibitor of the BLM helicase modulates chromosome stability in human cells. Chem Biol, 20 (1), pp. 55-62. | Show Abstract | Read more

The Bloom's syndrome protein, BLM, is a member of the conserved RecQ helicase family. Although cell lines lacking BLM exist, these exhibit progressive genomic instability that makes distinguishing primary from secondary effects of BLM loss problematic. In order to be able to acutely disable BLM function in cells, we undertook a high throughput screen of a chemical compound library for small molecule inhibitors of BLM. We present ML216, a potent inhibitor of the DNA unwinding activity of BLM. ML216 shows cell-based activity and can induce sister chromatid exchanges, enhance the toxicity of aphidicolin, and exert antiproliferative activity in cells expressing BLM, but not those lacking BLM. These data indicate that ML216 shows strong selectivity for BLM in cultured cells. We discuss the potential utility of such a BLM-targeting compound as an anticancer agent.

Canning P, Cooper CDO, Krojer T, Murray JW, Pike ACW, Chaikuad A, Keates T, Thangaratnarajah C, Hojzan V, Ayinampudi V et al. 2013. Structural basis for Cul3 protein assembly with the BTB-Kelch family of E3 ubiquitin ligases. J Biol Chem, 288 (11), pp. 7803-7814. | Show Abstract | Read more

Cullin-RING ligases are multisubunit E3 ubiquitin ligases that recruit substrate-specific adaptors to catalyze protein ubiquitylation. Cul3-based Cullin-RING ligases are uniquely associated with BTB adaptors that incorporate homodimerization, Cul3 assembly, and substrate recognition into a single multidomain protein, of which the best known are BTB-BACK-Kelch domain proteins, including KEAP1. Cul3 assembly requires a BTB protein "3-box" motif, analogous to the F-box and SOCS box motifs of other Cullin-based E3s. To define the molecular basis for this assembly and the overall architecture of the E3, we determined the crystal structures of the BTB-BACK domains of KLHL11 both alone and in complex with Cul3, along with the Kelch domain structures of KLHL2 (Mayven), KLHL7, KLHL12, and KBTBD5. We show that Cul3 interaction is dependent on a unique N-terminal extension sequence that packs against the 3-box in a hydrophobic groove centrally located between the BTB and BACK domains. Deletion of this N-terminal region results in a 30-fold loss in affinity. The presented data offer a model for the quaternary assembly of this E3 class that supports the bivalent capture of Nrf2 and reveals potential new sites for E3 inhibitor design.

Rosenthal AS, Dexheimer TS, Gileadi O, Nguyen GH, Chu WK, Hickson ID, Jadhav A, Simeonov A, Maloney DJ. 2013. Synthesis and SAR studies of 5-(pyridin-4-yl)-1,3,4-thiadiazol-2-amine derivatives as potent inhibitors of Bloom helicase. Bioorg Med Chem Lett, 23 (20), pp. 5660-5666. | Show Abstract | Read more

Human cells utilize a variety of complex DNA repair mechanisms in order to combat constant mutagenic and cytotoxic threats from both exogenous and endogenous sources. The RecQ family of DNA helicases, which includes Bloom helicase (BLM), plays an important function in DNA repair by unwinding complementary strands of duplex DNA as well as atypical DNA structures such as Holliday junctions. Mutations of the BLM gene can result in Bloom syndrome, an autosomal recessive disorder associated with cancer predisposition. BLM-deficient cells exhibit increased sensitivity to DNA damaging agents indicating that a selective BLM inhibitor could be useful in potentiating the anticancer activity of these agents. In this work, we describe the medicinal chemistry optimization of the hit molecule following a quantitative high-throughput screen of >355,000 compounds. These efforts lead to the identification of ML216 and related analogs, which possess potent BLM inhibition and exhibit selectivity over related helicases. Moreover, these compounds demonstrated cellular activity by inducing sister chromatid exchanges, a hallmark of Bloom syndrome.

Allerston CK, von Delft F, Gileadi O. 2013. Crystal structures of the catalytic domain of human soluble guanylate cyclase. PLoS One, 8 (3), pp. e57644. | Show Abstract | Read more

Soluble guanylate cyclase (sGC) catalyses the synthesis of cyclic GMP in response to nitric oxide. The enzyme is a heterodimer of homologous α and β subunits, each of which is composed of multiple domains. We present here crystal structures of a heterodimer of the catalytic domains of the α and β subunits, as well as an inactive homodimer of β subunits. This first structure of a metazoan, heteromeric cyclase provides several observations. First, the structures resemble known structures of adenylate cyclases and other guanylate cyclases in overall fold and in the arrangement of conserved active-site residues, which are contributed by both subunits at the interface. Second, the subunit interaction surface is promiscuous, allowing both homodimeric and heteromeric association; the preference of the full-length enzyme for heterodimer formation must derive from the combined contribution of other interaction interfaces. Third, the heterodimeric structure is in an inactive conformation, but can be superposed onto an active conformation of adenylate cyclase by a structural transition involving a 26° rigid-body rotation of the α subunit. In the modelled active conformation, most active site residues in the subunit interface are precisely aligned with those of adenylate cyclase. Finally, the modelled active conformation also reveals a cavity related to the active site by pseudo-symmetry. The pseudosymmetric site lacks key active site residues, but may bind allosteric regulators in a manner analogous to the binding of forskolin to adenylate cyclase. This indicates the possibility of developing a new class of small-molecule modulators of guanylate cyclase activity targeting the catalytic domain.

Twigg SRF, Lloyd D, Jenkins D, Elçioglu NE, Cooper CDO, Al-Sannaa N, Annagür A, Gillessen-Kaesbach G, Hüning I, Knight SJL et al. 2012. Mutations in multidomain protein MEGF8 identify a Carpenter syndrome subtype associated with defective lateralization. Am J Hum Genet, 91 (5), pp. 897-905. | Show Abstract | Read more

Carpenter syndrome is an autosomal-recessive multiple-congenital-malformation disorder characterized by multisuture craniosynostosis and polysyndactyly of the hands and feet; many other clinical features occur, and the most frequent include obesity, umbilical hernia, cryptorchidism, and congenital heart disease. Mutations of RAB23, encoding a small GTPase that regulates vesicular transport, are present in the majority of cases. Here, we describe a disorder caused by mutations in multiple epidermal-growth-factor-like-domains 8 (MEGF8), which exhibits substantial clinical overlap with Carpenter syndrome but is frequently associated with abnormal left-right patterning. We describe five affected individuals with similar dysmorphic facies, and three of them had either complete situs inversus, dextrocardia, or transposition of the great arteries; similar cardiac abnormalities were previously identified in a mouse mutant for the orthologous Megf8. The mutant alleles comprise one nonsense, three missense, and two splice-site mutations; we demonstrate in zebrafish that, in contrast to the wild-type protein, the proteins containing all three missense alterations provide only weak rescue of an early gastrulation phenotype induced by Megf8 knockdown. We conclude that mutations in MEGF8 cause a Carpenter syndrome subtype frequently associated with defective left-right patterning, probably through perturbation of signaling by hedgehog and nodal family members. We did not observe any subject with biallelic loss-of function mutations, suggesting that some residual MEGF8 function might be necessary for survival and might influence the phenotypes observed.

Sengerová B, Allerston CK, Abu M, Lee SY, Hartley J, Kiakos K, Schofield CJ, Hartley JA, Gileadi O, McHugh PJ. 2012. Characterization of the human SNM1A and SNM1B/Apollo DNA repair exonucleases. J Biol Chem, 287 (31), pp. 26254-26267. | Show Abstract | Read more

Human SNM1A and SNM1B/Apollo have both been implicated in the repair of DNA interstrand cross-links (ICLs) by cellular studies, and SNM1B is also required for telomere protection. Here, we describe studies on the biochemical characterization of the SNM1A and SNM1B proteins. The results reveal some fundamental differences in the mechanisms of the two proteins. Both SNM1A and SNM1B digest double-stranded and single-stranded DNA with a 5'-to-3' directionality in a reaction that is stimulated by divalent cations, and both nucleases are inhibited by the zinc chelator o-phenanthroline. We find that SNM1A has greater affinity for single-stranded DNA over double-stranded DNA that is not observed with SNM1B. Although both proteins demonstrate a low level of processivity on low molecular weight DNA oligonucleotide substrates, when presented with high molecular weight DNA, SNM1A alone is rendered much more active, being capable of digesting kilobase-long stretches of DNA. Both proteins can digest past ICLs induced by the non-distorting minor groove cross-linking agent SJG-136, albeit with SNM1A showing a greater capacity to achieve this. This is consistent with the proposal that SNM1A and SNM1B might exhibit some redundancy in ICL repair. Together, our work establishes differences in the substrate selectivities of SNM1A and SNM1B that are likely to be relevant to their in vivo roles and which might be exploited in the development of selective inhibitors.

Watson AA, Mahajan P, Mertens HDT, Deery MJ, Zhang W, Pham P, Du X, Bartke T, Zhang W, Edlich C et al. 2012. The PHD and chromo domains regulate the ATPase activity of the human chromatin remodeler CHD4. J Mol Biol, 422 (1), pp. 3-17. | Show Abstract | Read more

The NuRD (nucleosome remodeling and deacetylase) complex serves as a crucial epigenetic regulator of cell differentiation, proliferation, and hematopoietic development by coupling the deacetylation and demethylation of histones, nucleosome mobilization, and the recruitment of transcription factors. The core nucleosome remodeling function of the mammalian NuRD complex is executed by the helicase-domain-containing ATPase CHD4 (Mi-2β) subunit, which also contains N-terminal plant homeodomain (PHD) and chromo domains. The mode of regulation of chromatin remodeling by CHD4 is not well understood, nor is the role of its PHD and chromo domains. Here, we use small-angle X-ray scattering, nucleosome binding ATPase and remodeling assays, limited proteolysis, cross-linking, and tandem mass spectrometry to propose a three-dimensional structural model describing the overall shape and domain interactions of CHD4 and discuss the relevance of these for regulating the remodeling of chromatin by the NuRD complex.

Cited:

35

Scopus

Watson AA, Mahajan P, Mertens HDT, Deery MJ, Zhang W, Pham P, Du X, Bartke T, Zhang W, Edlich C et al. 2012. The PHD and chromo domains regulate the atpase activity of the human chromatin remodeler CHD4 Journal of Molecular Biology, 422 (1), pp. 3-17. | Show Abstract | Read more

The NuRD (nucleosome remodeling and deacetylase) complex serves as a crucial epigenetic regulator of cell differentiation, proliferation, and hematopoietic development by coupling the deacetylation and demethylation of histones, nucleosome mobilization, and the recruitment of transcription factors. The core nucleosome remodeling function of the mammalian NuRD complex is executed by the helicase-domain-containing ATPase CHD4 (Mi-2β) subunit, which also contains N-terminal plant homeodomain (PHD) and chromo domains. The mode of regulation of chromatin remodeling by CHD4 is not well understood, nor is the role of its PHD and chromo domains. Here, we use small-angle X-ray scattering, nucleosome binding ATPase and remodeling assays, limited proteolysis, cross-linking, and tandem mass spectrometry to propose a three-dimensional structural model describing the overall shape and domain interactions of CHD4 and discuss the relevance of these for regulating the remodeling of chromatin by the NuRD complex. © 2012 Elsevier Ltd. All rights reserved.

Keates T, Cooper CDO, Savitsky P, Allerston CK, Phillips C, Hammarström M, Daga N, Berridge G, Mahajan P, Burgess-Brown NA et al. 2012. Expressing the human proteome for affinity proteomics: Optimising expression of soluble protein domains and in vivo biotinylation New Biotechnology, 29 (5), pp. 515-525. | Show Abstract | Read more

The generation of affinity reagents to large numbers of human proteins depends on the ability to express the target proteins as high-quality antigens. The Structural Genomics Consortium (SGC) focuses on the production and structure determination of human proteins. In a 7-year period, the SGC has deposited crystal structures of > 800 human protein domains, and has additionally expressed and purified a similar number of protein domains that have not yet been crystallised. The targets include a diversity of protein domains, with an attempt to provide high coverage of protein families. The family approach provides an excellent basis for characterising the selectivity of affinity reagents. We present a summary of the approaches used to generate purified human proteins or protein domains, a test case demonstrating the ability to rapidly generate new proteins, and an optimisation study on the modification of > 70 proteins by biotinylation in vivo. These results provide a unique synergy between large-scale structural projects and the recent efforts to produce a wide coverage of affinity reagents to the human proteome. © 2011 Elsevier B.V.

Cited:

21

Scopus

Twigg SRF, Lloyd D, Jenkins D, Elçioglu NE, Cooper CDO, Al-Sannaa N, Annagür A, Gillessen-Kaesbach G, Hüning I, Knight SJL et al. 2012. Mutations in multidomain protein MEGF8 identify a carpenter syndrome subtype associated with defective lateralization American Journal of Human Genetics, 91 (5), pp. 897-905. | Show Abstract | Read more

Carpenter syndrome is an autosomal-recessive multiple-congenital- malformation disorder characterized by multisuture craniosynostosis and polysyndactyly of the hands and feet; many other clinical features occur, and the most frequent include obesity, umbilical hernia, cryptorchidism, and congenital heart disease. Mutations of RAB23, encoding a small GTPase that regulates vesicular transport, are present in the majority of cases. Here, we describe a disorder caused by mutations in multiple epidermal-growth-factor- like-domains 8 (MEGF8), which exhibits substantial clinical overlap with Carpenter syndrome but is frequently associated with abnormal left-right patterning. We describe five affected individuals with similar dysmorphic facies, and three of them had either complete situs inversus, dextrocardia, or transposition of the great arteries; similar cardiac abnormalities were previously identified in a mouse mutant for the orthologous Megf8. The mutant alleles comprise one nonsense, three missense, and two splice-site mutations; we demonstrate in zebrafish that, in contrast to the wild-type protein, the proteins containing all three missense alterations provide only weak rescue of an early gastrulation phenotype induced by Megf8 knockdown. We conclude that mutations in MEGF8 cause a Carpenter syndrome subtype frequently associated with defective left-right patterning, probably through perturbation of signaling by hedgehog and nodal family members. We did not observe any subject with biallelic loss-of function mutations, suggesting that some residual MEGF8 function might be necessary for survival and might influence the phenotypes observed. © 2012 The American Society of Human Genetics.

Krastanova I, Sannino V, Amenitsch H, Gileadi O, Pisani FM, Onesti S. 2012. Structural and functional insights into the DNA replication factor Cdc45 reveal an evolutionary relationship to the DHH family of phosphoesterases. J Biol Chem, 287 (6), pp. 4121-4128. | Show Abstract | Read more

Cdc45 is an essential protein conserved in all eukaryotes and is involved both in the initiation of DNA replication and the progression of the replication fork. With GINS, Cdc45 is an essential cofactor of the Mcm2-7 replicative helicase complex. Despite its importance, no detailed information is available on either the structure or the biochemistry of the protein. Intriguingly, whereas homologues of both GINS and Mcm proteins have been described in Archaea, no counterpart for Cdc45 is known. Herein we report a bioinformatic analysis that shows a weak but significant relationship among eukaryotic Cdc45 proteins and a large family of phosphoesterases that has been described as the DHH family, including inorganic pyrophosphatases and RecJ ssDNA exonucleases. These enzymes catalyze the hydrolysis of phosphodiester bonds via a mechanism involving two Mn(2+) ions. Only a subset of the amino acids that coordinates Mn(2+) is conserved in Cdc45. We report biochemical and structural data on the recombinant human Cdc45 protein, consistent with the proposed DHH family affiliation. Like the RecJ exonucleases, the human Cdc45 protein is able to bind single-stranded, but not double-stranded DNA. Small angle x-ray scattering data are consistent with a model compatible with the crystallographic structure of the RecJ/DHH family members.

Keates T, Cooper CDO, Savitsky P, Allerston CK, Phillips C, Hammarström M, Daga N, Berridge G, Mahajan P, Burgess-Brown NA et al. 2012. Expressing the human proteome for affinity proteomics: optimising expression of soluble protein domains and in vivo biotinylation. N Biotechnol, 29 (5), pp. 515-525. | Show Abstract | Read more

The generation of affinity reagents to large numbers of human proteins depends on the ability to express the target proteins as high-quality antigens. The Structural Genomics Consortium (SGC) focuses on the production and structure determination of human proteins. In a 7-year period, the SGC has deposited crystal structures of >800 human protein domains, and has additionally expressed and purified a similar number of protein domains that have not yet been crystallised. The targets include a diversity of protein domains, with an attempt to provide high coverage of protein families. The family approach provides an excellent basis for characterising the selectivity of affinity reagents. We present a summary of the approaches used to generate purified human proteins or protein domains, a test case demonstrating the ability to rapidly generate new proteins, and an optimisation study on the modification of >70 proteins by biotinylation in vivo. These results provide a unique synergy between large-scale structural projects and the recent efforts to produce a wide coverage of affinity reagents to the human proteome.

Wang AT, Sengerová B, Cattell E, Inagawa T, Hartley JM, Kiakos K, Burgess-Brown NA, Swift LP, Enzlin JH, Schofield CJ et al. 2011. Human SNM1A and XPF-ERCC1 collaborate to initiate DNA interstrand cross-link repair. Genes Dev, 25 (17), pp. 1859-1870. | Show Abstract | Read more

One of the major DNA interstrand cross-link (ICL) repair pathways in mammalian cells is coupled to replication, but the mechanistic roles of the critical factors involved remain largely elusive. Here, we show that purified human SNM1A (hSNM1A), which exhibits a 5'-3' exonuclease activity, can load from a single DNA nick and digest past an ICL on its substrate strand. hSNM1A-depleted cells are ICL-sensitive and accumulate replication-associated DNA double-strand breaks (DSBs), akin to ERCC1-depleted cells. These DSBs are Mus81-induced, indicating that replication fork cleavage by Mus81 results from the failure of the hSNM1A- and XPF-ERCC1-dependent ICL repair pathway. Our results reveal how collaboration between hSNM1A and XPF-ERCC1 is necessary to initiate ICL repair in replicating human cells.

Picaud S, Kavanagh KL, Yue WW, Lee WH, Muller-Knapp S, Gileadi O, Sacchettini J, Oppermann U. 2011. Structural basis of fumarate hydratase deficiency. J Inherit Metab Dis, 34 (3), pp. 671-676. | Show Abstract | Read more

Fumarate hydratase catalyzes the stereospecific hydration across the olefinic double bond in fumarate leading to L-malate. The enzyme is expressed in mitochondrial and cytosolic compartments, and participates in the Krebs cycle in mitochondria, as well as in regulation of cytosolic fumarate levels. Fumarate hydratase deficiency is an autosomal recessive trait presenting as metabolic disorder with severe encephalopathy, seizures and poor neurological outcome. Heterozygous mutations are associated with a predisposition to cutaneous and uterine leiomyomas and to renal cancer. The crystal structure of human fumarate hydratase shows that mutations can be grouped into two distinct classes either affecting structural integrity of the core enzyme architecture, or are localized around the enzyme active site. An interactive version of this manuscript (which may contain additional mutations appended after acceptance of this manuscript) may be found on the SSIEM website at: http://www.ssiem.org/resources/structures/FH .

Chen D, Vollmar M, Rossi MN, Phillips C, Kraehenbuehl R, Slade D, Mehrotra PV, von Delft F, Crosthwaite SK, Gileadi O et al. 2011. Identification of macrodomain proteins as novel O-acetyl-ADP-ribose deacetylases. J Biol Chem, 286 (15), pp. 13261-13271. | Show Abstract | Read more

Sirtuins are a family of protein lysine deacetylases, which regulate gene silencing, metabolism, life span, and chromatin structure. Sirtuins utilize NAD(+) to deacetylate proteins, yielding O-acetyl-ADP-ribose (OAADPr) as a reaction product. The macrodomain is a ubiquitous protein module known to bind ADP-ribose derivatives, which diverged through evolution to support many different protein functions and pathways. The observation that some sirtuins and macrodomains are physically linked as fusion proteins or genetically coupled through the same operon, provided a clue that their functions might be connected. Indeed, here we demonstrate that the product of the sirtuin reaction OAADPr is a substrate for several related macrodomain proteins: human MacroD1, human MacroD2, Escherichia coli YmdB, and the sirtuin-linked MacroD-like protein from Staphylococcus aureus. In addition, we show that the cell extracts derived from MacroD-deficient Neurospora crassa strain exhibit a major reduction in the ability to hydrolyze OAADPr. Our data support a novel function of macrodomains as OAADPr deacetylases and potential in vivo regulators of cellular OAADPr produced by NAD(+)-dependent deacetylation.

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Wang AT, Sengerová B, Cattell E, Inagawa T, Hartley JM, Kiakos K, Burgess-Brown NA, Swift LP, Enzlin JH, Schofield CJ et al. 2011. Human SNM1a and XPF-ERCC1 collaborate to initiate DNA interstrand cross-link repair Genes and Development, 25 (17), pp. 1859-1870. | Show Abstract | Read more

One of the major DNA interstrand cross-link (ICL) repair pathways in mammalian cells is coupled to replication, but the mechanistic roles of the critical factors involved remain largely elusive. Here, we show that purified human SNM1A (hSNM1A), which exhibits a 59-39 exonuclease activity, can load from a single DNA nick and digest past an ICL on its substrate strand. hSNM1A-depleted cells are ICL-sensitive and accumulate replicationassociated DNA double-strand breaks (DSBs), akin to ERCC1-depleted cells. These DSBs are Mus81-induced, indicating that replication fork cleavage by Mus81 results from the failure of the hSNM1A- and XPF-ERCC1-dependent ICL repair pathway. Our results reveal how collaboration between hSNM1A and XPF-ERCC1 is necessary to initiate ICL repair in replicating human cells. © 2011 by Cold Spring Harbor Laboratory Press.

Berridge G, Chalk R, D'Avanzo N, Dong L, Doyle D, Kim JI, Xia X, Burgess-Brown N, Deriso A, Carpenter EP, Gileadi O. 2011. High-performance liquid chromatography separation and intact mass analysis of detergent-solubilized integral membrane proteins Analytical Biochemistry, 410 (2), pp. 272-280. | Show Abstract | Read more

We have developed a method for intact mass analysis of detergent- solubilized and purified integral membrane proteins using liquid chromatography-mass spectrometry (LC-MS) with methanol as the organic mobile phase. Membrane proteins and detergents are separated chromatographically during the isocratic stage of the gradient profile from a 150-mm C3 reversed-phase column. The mass accuracy is comparable to standard methods employed for soluble proteins; the sensitivity is 10-fold lower, requiring 0.2-5 μg of protein. The method is also compatible with our standard LC-MS method used for intact mass analysis of soluble proteins and may therefore be applied on a multiuser instrument or in a high-throughput environment. © 2010 Elsevier Inc. All rights reserved.

Berridge G, Chalk R, D'Avanzo N, Dong L, Doyle D, Kim J-I, Xia X, Burgess-Brown N, Deriso A, Carpenter EP, Gileadi O. 2011. High-performance liquid chromatography separation and intact mass analysis of detergent-solubilized integral membrane proteins. Anal Biochem, 410 (2), pp. 272-280. | Show Abstract | Read more

We have developed a method for intact mass analysis of detergent-solubilized and purified integral membrane proteins using liquid chromatography-mass spectrometry (LC-MS) with methanol as the organic mobile phase. Membrane proteins and detergents are separated chromatographically during the isocratic stage of the gradient profile from a 150-mm C3 reversed-phase column. The mass accuracy is comparable to standard methods employed for soluble proteins; the sensitivity is 10-fold lower, requiring 0.2-5 μg of protein. The method is also compatible with our standard LC-MS method used for intact mass analysis of soluble proteins and may therefore be applied on a multiuser instrument or in a high-throughput environment.

Lucic B, Zhang Y, King O, Mendoza-Maldonado R, Berti M, Niesen FH, Burgess-Brown NA, Pike ACW, Cooper CDO, Gileadi O, Vindigni A. 2011. A prominent β-hairpin structure in the winged-helix domain of RECQ1 is required for DNA unwinding and oligomer formation. Nucleic Acids Res, 39 (5), pp. 1703-1717. | Show Abstract | Read more

RecQ helicases have attracted considerable interest in recent years due to their role in the suppression of genome instability and human diseases. These atypical helicases exert their function by resolving a number of highly specific DNA structures. The crystal structure of a truncated catalytic core of the human RECQ1 helicase (RECQ1(49-616)) shows a prominent β-hairpin, with an aromatic residue (Y564) at the tip, located in the C-terminal winged-helix domain. Here, we show that the β-hairpin is required for the DNA unwinding and Holliday junction (HJ) resolution activity of full-length RECQ1, confirming that it represents an important determinant for the distinct substrate specificity of the five human RecQ helicases. In addition, we found that the β-hairpin is required for dimer formation in RECQ1(49-616) and tetramer formation in full-length RECQ1. We confirmed the presence of stable RECQ1(49-616) dimers in solution and demonstrated that dimer formation favours DNA unwinding; even though RECQ1 monomers are still active. Tetramers are instead necessary for more specialized activities such as HJ resolution and strand annealing. Interestingly, two independent protein-protein contacts are required for tetramer formation, one involves the β-hairpin and the other the N-terminus of RECQ1, suggesting a non-hierarchical mechanism of tetramer assembly.

Panigrahi GB, Slean MM, Simard JP, Gileadi O, Pearson CE. 2010. Isolated short CTG/CAG DNA slip-outs are repaired efficiently by hMutSbeta, but clustered slip-outs are poorly repaired. Proc Natl Acad Sci U S A, 107 (28), pp. 12593-12598. | Show Abstract | Read more

Expansions of CTG/CAG trinucleotide repeats, thought to involve slipped DNAs at the repeats, cause numerous diseases including myotonic dystrophy and Huntington's disease. By unknown mechanisms, further repeat expansions in transgenic mice carrying expanded CTG/CAG tracts require the mismatch repair (MMR) proteins MSH2 and MSH3, forming the MutSbeta complex. Using an in vitro repair assay, we investigated the effect of slip-out size, with lengths of 1, 3, or 20 excess CTG repeats, as well as the effect of the number of slip-outs per molecule, on the requirement for human MMR. Long slip-outs escaped repair, whereas short slip-outs were repaired efficiently, much greater than a G-T mismatch, but required hMutSbeta. Higher or lower levels of hMutSbeta or its complete absence were detrimental to proper repair of short slip-outs. Surprisingly, clusters of as many as 62 short slip-outs (one to three repeat units each) along a single DNA molecule with (CTG)50*(CAG)50 repeats were refractory to repair, and repair efficiency was reduced further without MMR. Consistent with the MutSbeta requirement for instability, hMutSbeta is required to process isolated short slip-outs; however, multiple adjacent short slip-outs block each other's repair, possibly acting as roadblocks to progression of repair and allowing error-prone repair. Results suggest that expansions can arise by escaped repair of long slip-outs, tandem short slip-outs, or isolated short slip-outs; the latter two types are sensitive to hMutSbeta. Poor repair of clustered DNA lesions has previously been associated only with ionizing radiation damage. Our results extend this interference in repair to neurodegenerative disease-causing mutations in which clustered slip-outs escape proper repair and lead to expansions.

Savitsky P, Bray J, Cooper CDO, Marsden BD, Mahajan P, Burgess-Brown NA, Gileadi O. 2010. High-throughput production of human proteins for crystallization: the SGC experience. J Struct Biol, 172 (1), pp. 3-13. | Show Abstract | Read more

Producing purified human proteins with high yield and purity remains a considerable challenge. We describe the methods utilized in the Structural Genomics Consortium (SGC) in Oxford, resulting in successful purification of 48% of human proteins attempted; of those, the structures of approximately 40% were solved by X-ray crystallography. The main driver has been the parallel processing of multiple (typically 9-20) truncated constructs of each target; modest diversity in vectors and host systems; and standardized purification procedures. We provide method details as well as data on the properties of the constructs leading to crystallized proteins and the impact of methodological variants. These can be used to formulate guidelines for initial approaches to expression of new eukaryotic proteins.

Vindigni A, Marino F, Gileadi O. 2010. Probing the structural basis of RecQ helicase function. Biophys Chem, 149 (3), pp. 67-77. | Show Abstract | Read more

RecQ helicases are a ubiquitous family of DNA unwinding enzymes required to preserve genome integrity, thus preventing premature aging and cancer formation. The five human representatives of this family play non-redundant roles in the suppression of genome instability using a combination of enzymatic activities that specifically characterize each member of the family. These enzymes are in fact not only able to catalyze the transient opening of DNA duplexes, as any other conventional helicase, but can also promote annealing of complementary strands, branch migration of Holliday junctions and, in some cases, excision of ssDNA tails. Remarkably, the balance between these different activities seems to be regulated by protein oligomerization. This review illustrates the recent progress made in the definition of the structural determinants that control the different enzymatic activities of RecQ helicases and speculates on the possible mechanisms that RecQ proteins might use to promote their multiple functions.

Pike ACW, Shrestha B, Popuri V, Burgess-Brown N, Muzzolini L, Costantini S, Vindigni A, Gileadi O. 2009. Structure of the human RECQ1 helicase reveals a putative strand-separation pin. Proc Natl Acad Sci U S A, 106 (4), pp. 1039-1044. | Show Abstract | Read more

RecQ-like helicases, which include 5 members in the human genome, are important in maintaining genome integrity. We present a crystal structure of a truncated form of the human RECQ1 protein with Mg-ADP. The truncated protein is active in DNA fork unwinding but lacks other activities of the full-length enzyme: disruption of Holliday junctions and DNA strand annealing. The structure of human RECQ1 resembles that of Escherichia coli RecQ, with some important differences. All structural domains are conserved, including the 2 RecA-like domains and the RecQ-specific zinc-binding and winged-helix (WH) domains. However, the WH domain is positioned at a different orientation from that of the E. coli enzyme. We identify a prominent beta-hairpin of the WH domain as essential for DNA strand separation, which may be analogous to DNA strand-separation features of other DNA helicases. This hairpin is significantly shorter in the E. coli enzyme and is not required for its helicase activity, suggesting that there are significant differences between the modes of action of RecQ family members.

Burgess-Brown NA, Sharma S, Sobott F, Loenarz C, Oppermann U, Gileadi O. 2008. Codon optimization can improve expression of human genes in Escherichia coli: A multi-gene study. Protein Expr Purif, 59 (1), pp. 94-102. | Show Abstract | Read more

The efficiency of heterologous protein production in Escherichia coli (E. coli) can be diminished by biased codon usage. Approaches normally used to overcome this problem include targeted mutagenesis to remove rare codons or the addition of rare codon tRNAs in specific cell lines. Recently, improvements in technology have enabled cost-effective production of synthetic genes, making this a feasible alternative. To explore this option, the expression patterns in E. coli of 30 human short-chain dehydrogenase/reductase genes (SDRs) were analyzed in three independent experiments, comparing the native and synthetic (codon-optimized) versions of each gene. The constructs were prepared in a pET-derived vector that appends an N-terminal polyhistidine tag to the protein; expression was induced using IPTG and soluble proteins were isolated by Ni-NTA metal-affinity chromatography. Expression of the native and synthetic gene constructs was compared in two isogenic bacterial strains, one of which contained a plasmid (pRARE2) that carries seven tRNAs recognizing rare codons. Although we found some degree of variability between experiments, in normal E. coli synthetic genes could be expressed and purified more readily than the native version. In only one case was native gene expression better. Importantly, in most but not all cases, expression of the native genes in combination with rare codon tRNAs mimicked the behavior of the synthetic genes in the native strain. The trend is that heterologous expression of some proteins in bacteria can be improved by altering codon preference, but that this effect can be generally recapitulated by introducing rare codon tRNAs into the host cell.

Structural Genomics Consortium, China Structural Genomics Consortium, Northeast Structural Genomics Consortium, Gräslund S, Nordlund P, Weigelt J, Hallberg BM, Bray J, Gileadi O, Knapp S et al. 2008. Protein production and purification. Nat Methods, 5 (2), pp. 135-146. | Show Abstract | Read more

In selecting a method to produce a recombinant protein, a researcher is faced with a bewildering array of choices as to where to start. To facilitate decision-making, we describe a consensus 'what to try first' strategy based on our collective analysis of the expression and purification of over 10,000 different proteins. This review presents methods that could be applied at the outset of any project, a prioritized list of alternate strategies and a list of pitfalls that trip many new investigators.

Gileadi O, Burgess-Brown NA, Colebrook SM, Berridge G, Savitsky P, Smee CEA, Loppnau P, Johansson C, Salah E, Pantic NH. 2008. High throughput production of recombinant human proteins for crystallography. Methods Mol Biol, 426 pp. 221-246. | Show Abstract | Read more

This chapter presents in detail the process used in high throughput bacterial production of recombinant human proteins for crystal structure determination. The core principles are: (1) Generating at least 10 truncated constructs from each target gene. (2) Ligation-independent cloning (LIC) into a bacterial expression vector. All proteins are expressed with an N-terminal, TEV protease cleavable fusion peptide. (3) Small-scale test expression to identify constructs producing soluble protein. (4) Liter-scale production in shaker flasks. (5) Purification by Ni-affinity chromatography and gel filtration. (6) Protein characterization and preparation for crystallography. The chapter also briefly presents alternative procedures, to be applied based on specific knowledge of protein families or when the core protocol is unsatisfactory. This scheme has been applied to more than 550 human proteins (>10,000 constructs) and has resulted in the deposition of 112 unique structures. The methods presented do not depend on specialized equipment or robotics; hence, they provide an effective approach for handling individual proteins in a regular research lab.

Shrestha B, Smee C, Gileadi O. 2008. Baculovirus expression vector system: an emerging host for high-throughput eukaryotic protein expression. Methods Mol Biol, 439 pp. 269-289. | Show Abstract | Read more

The increasing demand for production and characterization of diverse groups of recombinant proteins necessitates the analysis of several constructs and fusion tags in a variety of expression systems. The challenge is to screen multiple clones quickly for the desired properties. When using a eukaryotic system, such as baculovirus-mediated expression in insect cells, the total time required and the volume of culture needed to obtain reasonable results are limiting factors. This chapter focuses on addressing these issues by describing rapid small-scale expression as a mode of screening. The method allows the rapid identification of the best clone before scaling-up and the production of heterologous protein.

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Shrestha B, Smee C, Gileadi O. 2008. Baculovirus expression vector system: An emerging host for high-throughput eukaryotic protein expression Methods in Molecular Biology, 439 pp. 269-289. | Show Abstract | Read more

The increasing demand for production and characterization of diverse groups of recombinant proteins necessitates the analysis of several constructs and fusion tags in a variety of expression systems. The challenge is to screen multiple clones quickly for the desired properties. When using a eukaryotic system, such as baculovirus-mediated expression in insect cells, the total time required and the volume of culture needed to obtain reasonable results are limiting factors. This chapter focuses on addressing these issues by describing rapid small-scale expression as a mode of screening. The method allows the rapid identification of the best clone before scaling-up and the production of heter-ologous protein. © 2008 Humana Press.

Gileadi O, Knapp S, Lee WH, Marsden BD, Müller S, Niesen FH, Kavanagh KL, Ball LJ, von Delft F, Doyle DA et al. 2007. The scientific impact of the Structural Genomics Consortium: a protein family and ligand-centered approach to medically-relevant human proteins. J Struct Funct Genomics, 8 (2-3), pp. 107-119. | Show Abstract | Read more

As many of the structural genomics centers have ended their first phase of operation, it is a good point to evaluate the scientific impact of this endeavour. The Structural Genomics Consortium (SGC), operating from three centers across the Atlantic, investigates human proteins involved in disease processes and proteins from Plasmodium falciparum and related organisms. We present here some of the scientific output of the Oxford node of the SGC, where the target areas include protein kinases, phosphatases, oxidoreductases and other metabolic enzymes, as well as signal transduction proteins. The SGC has aimed to achieve extensive coverage of human gene families with a focus on protein-ligand interactions. The methods employed for effective protein expression, crystallization and structure determination by X-ray crystallography are summarized. In addition to the cumulative impact of accelerated delivery of protein structures, we demonstrate how family coverage, generic screening methodology, and the availability of abundant purified protein samples, allow a level of discovery that is difficult to achieve otherwise. The contribution of NMR to structure determination and protein characterization is discussed. To make this information available to a wide scientific audience, a new tool for disseminating annotated structural information was created that also represents an interactive platform allowing for a continuous update of the annotation by the scientific community.

Ng SS, Kavanagh KL, McDonough MA, Butler D, Pilka ES, Lienard BMR, Bray JE, Savitsky P, Gileadi O, von Delft F et al. 2007. Crystal structures of histone demethylase JMJD2A reveal basis for substrate specificity. Nature, 448 (7149), pp. 87-91. | Show Abstract | Read more

Post-translational histone modification has a fundamental role in chromatin biology and is proposed to constitute a 'histone code' in epigenetic regulation. Differential methylation of histone H3 and H4 lysyl residues regulates processes including heterochromatin formation, X-chromosome inactivation, genome imprinting, DNA repair and transcriptional regulation. The discovery of lysyl demethylases using flavin (amine oxidases) or Fe(II) and 2-oxoglutarate as cofactors (2OG oxygenases) has changed the view of methylation as a stable epigenetic marker. However, little is known about how the demethylases are selective for particular lysyl-containing sequences in specific methylation states, a key to understanding their functions. Here we reveal how human JMJD2A (jumonji domain containing 2A), which is selective towards tri- and dimethylated histone H3 lysyl residues 9 and 36 (H3K9me3/me2 and H3K36me3/me2), discriminates between methylation states and achieves sequence selectivity for H3K9. We report structures of JMJD2A-Ni(II)-Zn(II) inhibitor complexes bound to tri-, di- and monomethyl forms of H3K9 and the trimethyl form of H3K36. The structures reveal a lysyl-binding pocket in which substrates are bound in distinct bent conformations involving the Zn-binding site. We propose a mechanism for achieving methylation state selectivity involving the orientation of the substrate methyl groups towards a ferryl intermediate. The results suggest distinct recognition mechanisms in different demethylase subfamilies and provide a starting point to develop chemical tools for drug discovery and to study and dissect the complexity of reversible histone methylation and its role in chromatin biology.

Johansson C, Kavanagh KL, Gileadi O, Oppermann U. 2007. Reversible sequestration of active site cysteines in a 2Fe-2S-bridged dimer provides a mechanism for glutaredoxin 2 regulation in human mitochondria. J Biol Chem, 282 (5), pp. 3077-3082. | Show Abstract | Read more

Human mitochondrial glutaredoxin 2 (GLRX2), which controls intracellular redox balance and apoptosis, exists in a dynamic equilibrium of enzymatically active monomers and quiescent dimers. Crystal structures of both monomeric and dimeric forms of human GLRX2 reveal a distinct glutathione binding mode and show a 2Fe-2S-bridged dimer. The iron-sulfur cluster is coordinated through the N-terminal active site cysteine, Cys-37, and reduced glutathione. The structures indicate that the enzyme can be inhibited by a high GSH/GSSG ratio either by forming a 2Fe-2S-bridged dimer that locks away the N-terminal active site cysteine or by binding non-covalently and blocking the active site as seen in the monomer. The properties that permit GLRX2, and not other glutaredoxins, to form an iron-sulfur-containing dimer are likely due to the proline-to-serine substitution in the active site motif, allowing the main chain more flexibility in this area and providing polar interaction with the stabilizing glutathione. This appears to be a novel use of an iron-sulfur cluster in which binding of the cluster inactivates the protein by sequestering active site residues and where loss of the cluster through changes in subcellular redox status creates a catalytically active protein. Under oxidizing conditions, the dimers would readily separate into iron-free active monomers, providing a structural explanation for glutaredoxin activation under oxidative stress.

Amit R, Gileadi O, Stavans J. 2006. Direct observation of RuvAB-catalyzed branch migration of single Holliday junctions (vol 101, pg 11605, 2004) PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 103 (33), pp. 12654-12654.

Amit R, Gileadi O, Stavans J. 2006. Corrections for Amit et al., Direct observation of RuvAB-catalyzed branch migration of single Holliday junctions and Dawid et al., Single-molecule study of RuvAB-mediated Holliday-junction migration Proceedings of the National Academy of Sciences, 103 (33), pp. 12654-12654. | Read more

Amit R, Gileadi O, Stavans J. 2004. Direct observation of RuvAB-catalyzed branch migration of single Holliday junctions. Proc Natl Acad Sci U S A, 101 (32), pp. 11605-11610. | Show Abstract | Read more

Holliday junctions form during DNA repair and homologous recombination processes. These processes entail branch migration, whereby the length of two arms of a cruciform increases at the expense of the two others. Branch migration is carried out in prokaryotic cells by the RuvAB motor complex. We study RuvAB-catalyzed branch migration by following the motion of a small paramagnetic bead tethered to a surface by two opposing arms of a single cruciform. The bead, pulled under the action of magnetic tweezers, exerts tension on the cruciform, which in turn transmits the force to a single RuvAB complex bound at the crossover point. This setup provides a unique means of measuring several kinetic parameters of interest such as the translocation rate, the processivity, and the force on the substrate against which the RuvAB complex cannot effect translocation. RuvAB-catalyzed branch migration proceeds with a small, discrete number of rates, supporting the view that the monomers comprising the RuvB hexameric rings are not functionally homogeneous and that dimers or trimers constitute the active subunits. The most frequently encountered rate, 98 +/- 3 bp/sec, is approximately five times faster than previously estimated. The apparent processivity of branch migration between pauses of inactivity is approximately 7,000 bp. Branch migration persists against opposing forces up to 23 pN.

Nyska A, Cummings CA, Vainshtein A, Nadler J, Ezov N, Grunfeld Y, Gileadi O, Behar V. 2004. Electron microscopy of wet tissues: a case study in renal pathology. Toxicol Pathol, 32 (3), pp. 357-363. | Show Abstract | Read more

In this report we introduce wet-tissue scanning electron microscopy, a novel technique for direct imaging of wet tissue samples using backscattered electrons. Samples placed in sealed capsules are imaged through a resilient, electron-transparent membrane. The contrast of the imaged samples may be enhanced by chemical staining. The samples several millimeters thick and imaged without sectioning, makes this technique suitable for rapid analysis of tissue specimens. We applied this technique to D-limonene-induced nephropathy where accumulation of hyaline protein droplets is induced in proximal and distal convoluted tubules of the kidney. Images obtained by scanning electron microscopy of hydrated kidney specimens exhibited superior resolution, contrast, and magnification compared with those obtained by conventional light microscopy of paraffin sections. The electron micrographs can be obtained within an hour of tissue removal, whereas preparation for light microscopy requires at least 1 day. These advantages of the wet scanning electron microscopy technique indicate its potential utility in a wide range of applications in histopathology and toxicology.

Thiberge S, Nechushtan A, Sprinzak D, Gileadi O, Behar V, Zik O, Chowers Y, Michaeli S, Schlessinger J, Moses E. 2004. Scanning electron microscopy of cells and tissues under fully hydrated conditions. Proc Natl Acad Sci U S A, 101 (10), pp. 3346-3351. | Show Abstract | Read more

A capability for scanning electron microscopy of wet biological specimens is presented. A membrane that is transparent to electrons protects the fully hydrated sample from the vacuum. The result is a hybrid technique combining the ease of use and ability to see into cells of optical microscopy with the higher resolution of electron microscopy. The resolution of low-contrast materials is approximately 100 nm, whereas in high-contrast materials the resolution can reach 10 nm. Standard immunogold techniques and heavy-metal stains can be applied and viewed in the fluid to improve the contrast. Images present a striking combination of whole-cell morphology with a wealth of internal details. A possibility for direct inspection of tissue slices transpires, imaging only the external layer of cells. Simultaneous imaging with photons excited by the electrons incorporates data on material distribution, indicating a potential for multilabeling and specific scintillating markers.

Barshack I, Kopolovic J, Chowers Y, Gileadi O, Vainshtein A, Zik O, Behar V. 2004. A novel method for "Wet" SEM. Ultrastruct Pathol, 28 (1), pp. 29-31. | Show Abstract | Read more

Progress in the processing of wet tissues, without the need of fixation and complex preparation procedures, may facilitate the microscopic examination of tissues and cells. Microscopic examination of tissues is a central tool in clinical diagnosis as well as in diverse areas of research. The authors present the application of Wet SEM, a technology for imaging fully hydrated samples at atmospheric pressure in a scanning electron microscope (SEM). The technique is based on 2 principles. First, samples are imaged in sealed specimen capsules and are separated from the evacuated interior of the electron microscope by a thin, electron-transparent partition membrane that is strong enough to sustain a 1-atm pressure difference. Second, imaging is done in a SEM, based on detection of backscattered electrons, which penetrate a few microns into the specimen and thus give information on the cellular level.

Gileadi O, Sabban A. 2003. Squid sperm to clam eggs: imaging wet samples in a scanning electron microscope. Biol Bull, 205 (2), pp. 177-179. | Read more

Gileadi O, Behar V, Ezer S, Horowitz M, Nechushtan A, Sabban A, Sprinzak D, Vainshtein A, Zik O. 2003. Scanning electron microscopy of wet biological specimens Microscopy and Microanalysis, 9 (SUPPL. 3), pp. 486-487.

Jona G, Livi LL, Gileadi O. 2002. Mutations in the RING domain of TFB3, a subunit of yeast transcription factor IIH, reveal a role in cell cycle progression. J Biol Chem, 277 (42), pp. 39409-39416. | Show Abstract | Read more

The RNA polymerase II general transcription factor TFIIH is composed of 9 known subunits and possesses DNA helicase and protein kinase activities. The kinase subunits of TFIIH in animal cells, Cdk7, cyclin H, and MAT1, were independently isolated as an activity termed CAK (Cdk-activating kinase), which phosphorylates and activates cell cycle kinases. However, CAK activity of TFIIH subunits could not be demonstrated in budding yeast. TFB3, the 38-kDa subunit of yeast TFIIH, is the homolog of mammalian MAT1. By random mutagenesis we have isolated a temperature-sensitive mutation in the conserved RING domain. The mutant Tfb3 protein associates less efficiently with the kinase moiety of TFIIH than the wild type protein. In contrast to lethal mutants in other subunits of TFIIH, this mutation does not impair general transcription. Transcription of CLB2, and possibly other genes, is reduced in the mutant. At the restrictive temperature, the cells display a defect in cell cycle progression, which is manifest at more than one phase of the cycle. To conclude, in the present study we bring another demonstration of the multifunctional nature of TFIIH.

Schnurr B, Amit R, Oppenheim AB, Gileadi O, Stavans J. 2002. Compaction of single DNA molecules by histone-like binding protein HU. BIOPHYSICAL JOURNAL, 82 (1), pp. 51A-51A.

Braslavsky I, Amit R, Jaffar Ali BM, Gileadi O, Oppenheim A, Stavans J. 2001. Objective-type dark-field illumination for scattering from microbeads. Appl Opt, 40 (31), pp. 5650-5657. | Show Abstract | Read more

We introduce a method for detecting and tracking small particles in a solution near a surface. The method is based on blocking the backreflected illumination beam in an objective-type total internal reflection microscope, leaving unhindered the light scattered by the particles and resulting in dark-field illumination. Using this method, we tracked the motion of 60-nm polystyrene beads with a signal-to-noise ratio of 6 and detected 20-nm gold particles with a signal-to-noise ratio of 5. We illustrate the method's use by following the Brownian motion of small beads attached by short DNA tethers to a substrate.

Ali BM, Amit R, Braslavsky I, Oppenheim AB, Gileadi O, Stavans J. 2001. Compaction of single DNA molecules induced by binding of integration host factor (IHF). Proc Natl Acad Sci U S A, 98 (19), pp. 10658-10663. | Show Abstract | Read more

We studied the interaction between the integration host factor (IHF), a major nucleoid-associated protein in bacteria, and single DNA molecules. Force-extension measurements of lambda DNA and an analysis of the Brownian motion of small beads tethered to a surface by single short DNA molecules, in equilibrium with an IHF solution, indicate that: (i) the DNA-IHF complex retains a random, although more compact, coiled configuration for zero or small values of the tension, (ii) IHF induces DNA compaction by binding to multiple DNA sites with low specificity, and (iii) with increasing tension on the DNA, the elastic properties of bare DNA are recovered. This behavior is consistent with the predictions of a statistical mechanical model describing how proteins bending DNA are driven off by an applied tension on the DNA molecule. Estimates of the amount of bound IHF in DNA-IHF complexes obtained from the model agree very well with independent measurements of this quantity obtained from the analysis of DNA-IHF crosslinking. Our findings support the long-held view that IHF and other histone-like proteins play an important role in shaping the long-scale structure of the bacterial nucleoid.

Jona G, Wittschieben BO, Svejstrup JQ, Gileadi O. 2001. Involvement of yeast carboxy-terminal domain kinase I (CTDK-I) in transcription elongation in vivo. Gene, 267 (1), pp. 31-36. | Show Abstract | Read more

Yeast cells lacking transcription elongation factor genes such as PPR2 (TFIIS) and ELP (Elongator) are viable and show deleterious phenotypes only when transcription is rendered less effective by RNA polymerase mutations or by decreasing nucleotide pools. Here we demonstrate that deletion of the CTK1 gene, encoding the kinase subunit of RNA polymerase II carboxy-terminal domain kinase I (CTDK-I), is synthetically lethal when combined with deletion of PPR2 or ELP genes. The inviability of ctk1 elp3 double mutants can be rescued by expression of an Elp3 mutant that has retained its ability to form the Elongator complex but has severely diminished histone acetyltransferase activity, suggesting that the functional overlap between CTDK-I and Elongator is in assembly of RNA polymerase II elongation complexes. Our results suggest that CTDK-I plays an important role in transcriptional elongation in vivo, possibly by creating a form of RNA polymerase that is less prone to transcriptional arrest.

Shani G, Henis-Korenblit S, Jona G, Gileadi O, Eisenstein M, Ziv T, Admon A, Kimchi A. 2001. Autophosphorylation restrains the apoptotic activity of DRP-1 kinase by controlling dimerization and calmodulin binding. EMBO J, 20 (5), pp. 1099-1113. | Show Abstract | Read more

DRP-1 is a pro-apoptotic Ca2+/calmodulin (CaM)-regulated serine/threonine kinase, recently isolated as a novel member of the DAP-kinase family of proteins. It contains a short extra-catalytic tail required for homodimerization. Here we identify a novel regulatory mechanism that controls its pro-apoptotic functions. It comprises a single autophosphorylation event mapped to Ser308 within the CaM regulatory domain. A negative charge at this site reduces both the binding to CaM and the formation of DRP-1 homodimers. Conversely, the dephosphorylation of Ser308, which takes place in response to activated Fas or tumour necrosis factor-alpha death receptors, increases the formation of DRP-1 dimers, facilitates the binding to CaM and activates the pro-apoptotic effects of the protein. Thus, the process of enzyme activation is controlled by two unlocking steps that must work in concert, i.e. dephosphorylation, which probably weakens the electrostatic interactions between the CaM regulatory domain and the catalytic cleft, and homodimerization. This mechanism of negative autophosphorylation provides a safety barrier that restrains the killing effects of DRP-1, and a target for efficient activation of the kinase by various apoptotic stimuli.

Busso D, Keriel A, Sandrock B, Poterszman A, Gileadi O, Egly JM. 2000. Distinct regions of MAT1 regulate cdk7 kinase and TFIIH transcription activities. J Biol Chem, 275 (30), pp. 22815-22823. | Show Abstract | Read more

The transcription/DNA repair factor TFIIH may be resolved into at least two subcomplexes: the core TFIIH and the cdk-activating kinase (CAK) complex. The CAK complex, which is also found free in the cell, is composed of cdk7, cyclin H, and MAT1. In the present work, we found that the C terminus of MAT1 binds to the cdk7 x cyclin H complex and activates the cdk7 kinase activity. The median portion of MAT1, which contains a coiled-coil motif, allows the binding of CAK to the TFIIH core through interactions with both XPD and XPB helicases. Furthermore, using recombinant TFIIH complexes, it is demonstrated that the N-terminal RING finger domain of MAT1 is crucial for transcription activation and participates to the phosphorylation of the C-terminal domain of the largest subunit of the RNA polymerase II.

Jona G, Choder M, Gileadi O. 2000. Glucose starvation induces a drastic reduction in the rates of both transcription and degradation of mRNA in yeast. Biochim Biophys Acta, 1491 (1-3), pp. 37-48. | Show Abstract | Read more

Gradual depletion of essential nutrients in yeast cultures induces a complex physiological response, leading initially to induction of pathways required for the utilization of alternative nutrients and, when such sources are depleted, to entry into stationary phase. Abrupt removal of sugar does not allow the proper establishment of stationary phase. Here we report that abrupt removal of glucose from the growth medium elicits a coordinated response in yeast cells that resembles, in some aspects, the gradual passage to stationary phase. Phosphorylation of RNA polymerase II at a subset of sites in the COOH-terminal domain (CTD) is decreased. Transcription by RNA polymerases I and II is shut down almost completely, whereas transcription by RNA polymerase III continues. In parallel, the rate of mRNA degradation is drastically reduced, at a stage preceding poly(A) shortening. This response is suited for conservation of scarce resources while preserving the ability of cells to recover when nutrients become available.

Ostapenko D, Gileadi O. 2000. Rad25p, a DNA helicase subunit of yeast transcription factor TFIIH, is required for promoter escape in vivo. Gene, 245 (1), pp. 109-117. | Show Abstract | Read more

The general transcription factor TFIIH is required for initial DNA unwinding and promoter escape by RNA polymerase II in vitro. We examined whether Rad25p, a DNA helicase subunit of TFIIH, mediates promoter opening and promoter escape in the yeast Saccharomyces cerevisiae. DNA unwinding was probed with an in vivo permanganate reactivity assay, in a temperature-sensitive mutant of RAD25. The consequences of Rad25p inactivation were promoter-specific. Whereas in the TDH2 promoter permanganate reactivity was entirely abolished, the reactivity at the GAL1 and GAL10 promoter regions was only moderately affected. In the GAL genes permanganate reactivity uniformly decreased downstream of the transcription start site, indicating that progression of RNA polymerase II to this region was impaired. Our results suggest that in yeast cells, promoter opening is not sufficient for productive initiation and that Rad25p-mediated promoter escape may be a limiting step in the transcription of some promoters.

Espinoza FH, Farrell A, Nourse JL, Chamberlin HM, Gileadi O, Morgan DO. 2000. Cak1 is required for Kin28 phosphorylation and activation in vivo (vol 18, pg 6365, 1998) MOLECULAR AND CELLULAR BIOLOGY, 20 (5), pp. 1898-1898. | Read more

Feaver WJ, Huang W, Gileadi O, Myers L, Gustafsson CM, Kornberg RD, Friedberg EC. 2000. Subunit interactions in yeast transcription/repair factor TFIIH. Requirement for Tfb3 subunit in nucleotide excision repair. J Biol Chem, 275 (8), pp. 5941-5946. | Show Abstract | Read more

A yeast strain harboring a temperature-sensitive allele of TFB3 (tfb3(ts)), the 38-kDa subunit of the RNA polymerase II transcription/nucleotide excision repair factor TFIIH, was found to be sensitive to ultraviolet (UV) radiation and defective for nucleotide excision repair in vitro. Interestingly, tfb3(ts) failed to grow on medium containing caffeine. A comprehensive pairwise two-hybrid analysis between yeast TFIIH subunits identified novel interactions between Rad3 and Tfb3, Tfb4 and Ssl1, as well as Ssl2 and Tfb2. These interactions have facilitated a more complete model of the structure of TFIIH and the nucleotide excision repairosome.

Espinoza FH, Farrell A, Nourse JL, Chamberlin HM, Gileadi O, Morgan DO. 2000. Cak1 Is Required for Kin28 Phosphorylation and Activation In Vivo Molecular and Cellular Biology, 20 (5), pp. 1898-1898. | Read more

Kobor MS, Archambault J, Lester W, Holstege FC, Gileadi O, Jansma DB, Jennings EG, Kouyoumdjian F, Davidson AR, Young RA, Greenblatt J. 1999. An unusual eukaryotic protein phosphatase required for transcription by RNA polymerase II and CTD dephosphorylation in S. cerevisiae. Mol Cell, 4 (1), pp. 55-62. | Show Abstract | Read more

The carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II is phosphorylated soon after transcriptional initiation. We show here that the essential FCP1 gene of S. cerevisiae is linked genetically to RNA polymerase II and encodes a CTD phosphatase essential for dephosphorylation of RNA polymerase II in vivo. Fcp1p contains a phosphatase motif, psi psi psi DXDX(T/V)psi psi, which is novel for eukaryotic protein phosphatases and essential for Fcp1p to function in vivo. This motif is also required for recombinant Fcp1p to dephosphorylate the RNA polymerase II CTD or the artificial substrate p-nitrophenylphosphate in vitro. The effects of fcp1 mutations in global run-on and genome-wide expression studies show that transcription by RNA polymerase II in S. cerevisiae generally requires CTD phosphatase.

Espinoza FH, Farrell A, Nourse JL, Chamberlin HM, Gileadi O, Morgan DO. 1998. Cak1 is required for Kin28 phosphorylation and activation in vivo. Mol Cell Biol, 18 (11), pp. 6365-6373. | Show Abstract | Read more

Complete activation of most cyclin-dependent protein kinases (CDKs) requires phosphorylation by the CDK-activating kinase (CAK). In the budding yeast, Saccharomyces cerevisiae, the major CAK is a 44-kDa protein kinase known as Cak1. Cak1 is required for the phosphorylation and activation of Cdc28, a major CDK involved in cell cycle control. We addressed the possibility that Cak1 is also required for the activation of other yeast CDKs, such as Kin28, Pho85, and Srb10. We generated three new temperature-sensitive cak1 mutant strains, which arrested at the restrictive temperature with nonuniform budding morphology. All three cak1 mutants displayed significant synthetic interactions with loss-of-function mutations in CDC28 and KIN28. Loss of Cak1 function reduced the phosphorylation and activity of both Cdc28 and Kin28 but did not affect the activity of Pho85 or Srb10. In the presence of the Kin28 regulatory subunits Ccl1 and Tfb3, Kin28 was phosphorylated and activated when coexpressed with Cak1 in insect cells. We conclude that Cak1 is required for the activating phosphorylation of Kin28 as well as that of Cdc28.

Fridlender M, Sitrit Y, Shaul O, Gileadi O, Levy AA. 1998. Analysis of the Ac promoter: structure and regulation. Mol Gen Genet, 258 (3), pp. 306-314. | Show Abstract | Read more

The Ac-encoded transposase, a factor that is essential for the mobility of the Ac element, is expressed under the control of a promoter that lacks a conventional TATA box. The regulation of this promoter is poorly understood. We have analyzed Ac promoter structure and activity, both in vitro and in vivo, using transgenic tobacco plants and cell suspensions. A deletion analysis of the Ac 5' region showed that the minimal promoter is located within 70 bp of the major transcription initiation site (at position 334). The minimal promoter includes the sequence TAAGAAATA at position 294 303, i.e., about 30 nucleotides upstream from the transcription start site. This sequence binds specifically to the TATA-binding protein (TBP), suggesting that it is functional as a TATA box. The regulation of the Ac promoter was studied throughout plant development. Levels of Ac mRNA were low in all tissues studied, with higher expression being observed in dividing cells. In order to test whether Ac promoter is regulated during the cell cycle, a tobacco cell suspension transformed with Ac, was grown synchronously. No differences were found in Ac mRNA levels between cells in S, G2, M, or G1 phases; however, expression was lower in the stationary phase. We conclude that Ac promoter is not cell-cycle regulated but is expressed at a higher level in dividing cells. The possible relationship between promoter features and the regulation of Ac element transposition is discussed.

Meisels E, Gileadi O, Corden JL. 1995. Partial truncation of the yeast RNA polymerase II carboxyl-terminal domain preferentially reduces expression of glycolytic genes. J Biol Chem, 270 (52), pp. 31255-31261. | Show Abstract | Read more

The largest subunit of RNA polymerase II contains an essential carboxyl-terminal domain (CTD) that consists of highly conserved heptapeptide repeats with the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. Yeast cells with a partially truncated CTD grow slowly, are temperature- and cold-sensitive, and are unable to fully activate transcription of some genes. Screening a yeast wild-type cDNA library by means of comparative hybridization we find that CTD truncation preferentially reduces transcription of genes encoding glycolytic enzymes. Using a newly developed dual reporter assay we demonstrate that sensitivity to CTD truncation is conferred by the glycolytic gene promoters. Expression driven by glycolytic gene promoters is reduced, on average, about 3-fold in strains with the shortest CTD growing on either fermentable or nonfermentable carbon sources. Sensitivity to CTD truncation is particularly acute for the constitutively expressed ENO1 gene, which is reduced 10-fold in a strain with only eight CTD repeats. The sensitivity of constitutive ENO1 expression argues that CTD truncation can cause defects in uninduced as well as induced transcription.

Feaver WJ, Henry NL, Bushnell DA, Sayre MH, Brickner JH, Gileadi O, Kornberg RD. 1994. Yeast TFIIE. Cloning, expression, and homology to vertebrate proteins. J Biol Chem, 269 (44), pp. 27549-27553. | Show Abstract

Genes encoding both the 66- and the 43-kDa subunits of yeast RNA polymerase II initiation factor a, designated TFA1 and TFA2, have been isolated. Both genes are essential for cell viability. The bacterially expressed gene products could replace factor a in transcription in vitro, and both recombinant subunits were required for activity. The deduced amino acid sequences of the TFA1 and TFA2 gene products were homologous to those of the large and small subunits of human TFIIE, respectively, identifying factor a as the yeast homolog of TFIIE.

KORNBERG RD, CHASMAN DI, DARST SA, DAVID P, EDWARDS AM, FEAVER WJ, FLANAGAN PM, GILEADI O, HENRY NL, KELLEHER RJ et al. 1993. YEAST RNA POLYMERASE-II TRANSCRIPTION - STRUCTURE, MECHANISM AND REGULATION JOURNAL OF CELLULAR BIOCHEMISTRY, pp. 55-55.

FEAVER WJ, GILEADI O, KORNBERG RD. 1993. CHARACTERIZATION OF YEAST RNA POLYMERASE-II TRANSCRIPTION FACTOR-B AND CTD KINASE JOURNAL OF CELLULAR BIOCHEMISTRY, pp. 71-71.

Pfütz M, Gileadi O, Werner D. 1992. Identification of human satellite DNA sequences associated with chemically resistant nonhistone polypeptide adducts. Chromosoma, 101 (10), pp. 609-617. | Show Abstract | Read more

A fraction of DNA fragments of highly purified and completely unfolded eukaryotic DNA inevitably remains associated with chemically resistant nonhistone DNA-polypeptide complexes. This fraction can be isolated by nitrocellulose filtration because the polypeptide-associated DNA fragments are retained on nitrocellulose filters while bulk DNA passes through the filters. The fraction of AluI-fragmented DNA from human placenta retained on filters as a result of the binding factors (R-DNA, approximately 12%) represents a subset of genomic sequences with a sequence complexity different from unfractionated DNA and DNA recovered in the filtrate (F-DNA). DNA sequences prevalent in the retained fraction were detected by differential plaque hybridization of a recombinant lambda gt10 library with radiolabeled F- and R-DNA fractions. Several recombinant phages showing much stronger hybridization signals with the R-DNA probe than with the F-DNA probe were selected, plaque-purified and analyzed. Analysis of the inserts of such clones showed that repetitive DNA sequences of the alphoid dimeric and tetrameric family, satellite III and satellite III-like sequences are highly enriched in the retained fraction, which indicates that these sequences specifically attract the polypeptides involved in the tightly bound and resistant complexes. This property of repetitive sequences is of interest since tandemly repetitive sequences have been suggested to code for locus-specific fixation and stabilization of the chromatin fiber in the cell nucleus.

Gileadi O, Feaver WJ, Kornberg RD. 1992. Cloning of a subunit of yeast RNA polymerase II transcription factor b and CTD kinase. Science, 257 (5075), pp. 1389-1392. | Show Abstract | Read more

Yeast RNA polymerase II initiation factor b copurifies with three polypeptides of 85, 73, and 50 kilodaltons and with a protein kinase that phosphorylates the carboxyl-terminal repeat domain (CTD) of the largest polymerase subunit. The gene that encodes the 73-kilodalton polypeptide, designated TFB1, was cloned and found to be essential for cell growth. The deduced protein sequence exhibits no similarity to those of protein kinases. However, the sequence is similar to that of the 62-kilodalton subunit of the HeLa transcription factor BFT2, suggesting that this factor is the human counterpart of yeast factor b. Immunoprecipitation experiments using antibodies to the TFB1 gene product demonstrate that the transcriptional and CTD kinase activities of factor b are closely associated with an oligomer of the three polypeptides. Photoaffinity labeling with 3'-O-(4-benzoyl)benzoyl-ATP (adenosine triphosphate) identified an ATP-binding site in the 85-kilodalton polypeptide, suggesting that the 85-kilodalton subunit contains the catalytic domain of the kinase.

Rachmilewitz J, Gileadi O, Eldar-Geva T, Schneider T, de-Groot N, Hochberg A. 1992. Transcription of the H19 gene in differentiating cytotrophoblasts from human placenta. Mol Reprod Dev, 32 (3), pp. 196-202. | Show Abstract | Read more

Placental differentiation is closely correlated with the appearance of specific proteins, yet factors regulating cytotrophoblast differentiation are unknown. One strategy employed to search for such factors makes use of differential screening of cDNA libraries. For this purpose, cytotrophoblasts were isolated from human term placentae and cultured for 24 and 120 hr. cDNA libraries were constructed from the cell's RNA, and differential screening resulted in the isolation of three identical clones highly expressed after 120 hr. A DNA sequencing of 139 bp at the 3' end of these clones and a search of the data bank revealed that the sequence was identical to the parallel domain in the human H19 gene. This highly conserved gene is unusual in that it may not encode a protein. In the mouse, its RNA was shown to accumulate to high levels in embryonic tissues of endodermal and mesodermal origin. Our present findings imply that, in humans, the H19 gene is efficiently expressed in placental tissue and differentiated cytotrophoblasts, which are of ectodermal origin. RNA blot hybridization revealed a unique bimodal pattern of expression for the H19 gene in cultured cytotrophoblasts. The modulation in expression of H19 during cytotrophoblast growth was not due to the increase in the number of multinuclear cells. Size fractionation of cytotrophoblasts by centrifugal elutriation revealed that H19 expression is correlated with the stage of cell differentiation. We therefore propose that H19 transcripts might play a regulatory role in the process of cytotrophoblast differentiation.

KORNBERG R, CHASMAN D, DARST S, EDWARDS A, FEAVER W, FLANAGAN P, GILEADI O, HINDS D, KELLEHER R, MEREDITH G et al. 1992. YEAST RNA POLYMERASE-II TRANSCRIPTION - ENZYMATIC-ACTIVITIES OF GENERAL INITIATION-FACTORS, 3-D STRUCTURES OF INITIATION-FACTORS AND RNA-POLYMERASE, AND MECHANISM OF TRANSCRIPTIONAL ACTIVATION FASEB JOURNAL, 6 (1), pp. A274-A274.

Feaver WJ, Gileadi O, Li Y, Kornberg RD. 1991. CTD kinase associated with yeast RNA polymerase II initiation factor b. Cell, 67 (6), pp. 1223-1230. | Show Abstract | Read more

A kinase activity specific for the C-terminal repeat domain (CTD) of RNA polymerase II is associated with nearly homogeneous yeast general initiation factor b by three criteria: cofractionation on the basis of size and charge and coinactivation by mild heat treatment. The kinase phosphorylates the CTD at multiple sites in a processive manner. Factor b may possess a DNA-dependent ATPase activity as well. Both kinase and DNA-dependent ATPase activities exhibit the same nucleotide requirements as previously demonstrated for the initiation of transcription. These results support the idea that phosphorylation of the CTD lies on the pathway of transcription initiation and identify a catalytic activity of a general factor essential for the initiation process.

Feaver WJ, Gileadi O, Kornberg RD. 1991. Purification and characterization of yeast RNA polymerase II transcription factor b. J Biol Chem, 266 (28), pp. 19000-19005. | Show Abstract

Heat treatment of yeast nuclear extracts abolished the capacity to initiate transcription at RNA polymerase II promoters. Activity was restored by the addition of both recombinant yeast TFIID and partially purified factor b, a yeast fraction shown previously to be required for polymerase II transcription. On the basis of this assay with heat-treated extract, factor b was purified to virtual homogeneity. The factor appears to comprise polypeptides of approximately 85, 75, and 50 kDa, since these three polypeptides co-purify with activity, and since a native mass of about 200 kDa is estimated from glycerol gradient sedimentation and gel filtration.

Gileadi O, Lorberboum H, de Groot N, Hochberg AA. 1984. Location of RNase and RNase inhibitor on free cytoplasmic mRNA-protein particles from human placenta. Mol Biol Rep, 9 (4), pp. 241-244. | Show Abstract | Read more

Free cytoplasmic mRNPs were isolated from human placenta. An activity of RNase was associated with these particles but was mostly inhibited by a labile protein inhibitor. Both RNase and RNase inhibitor were extractable from mRNPs by 0.5 M KCl. The nature of the association of the RNase-RNase inhibitor complex with mRNPs makes it suitable as a putative system for control of expression and turnover of mRNPs and therefore of protein synthesis.

GALSKI H, WEINSTEIN D, GILEADI O, LORBERBOUM H, DEGROOT N, FOLMAN R, HOCHBERG AA. 1982. PHOSPHORYLATION OF PROTEINS IN CULTURED HUMAN-PLACENTA BIOCHEMISTRY INTERNATIONAL, 5 (1), pp. 137-143. | Show Abstract

The in vitro phosphorylation of proteins in human first trimester and term placental tissues have been studied. The data show the following: There is a difference in the apparent phosphorylation rate between early and term placentas which cannot be explained by changes in the level and the specific radioactivity of the cellular ATP pool. Some proteins are exclusively phosphorylated in the placenta early in gestation, while others are phosphorylated at the end of gestation and could not be detected in first trimester placenta. In first trimester placenta phosphorylation occurs predominantly on threonine residues; term placenta exhibits a significant relative increase in the transfer of radioactive phosphate to serine residues.

Newman JA, Aitkenhead H, Savitsky P, Gileadi O. 2017. Insights into the RecQ helicase mechanism revealed by the structure of the helicase domain of human RECQL5. Nucleic Acids Res, 45 (7), pp. 4231-4243. | Show Abstract | Read more

RecQ helicases are important maintainers of genome integrity with distinct roles in almost every cellular process requiring access to DNA. RECQL5 is one of five human RecQ proteins and is particularly versatile in this regard, forming protein complexes with a diverse set of cellular partners in order to coordinate its helicase activity to various processes including replication, recombination and DNA repair. In this study, we have determined crystal structures of the core helicase domain of RECQL5 both with and without the nucleotide ADP in two distinctly different ('Open' and 'Closed') conformations. Small angle X-ray scattering studies show that the 'Open' form of the protein predominates in solution and we discuss implications of this with regards to the RECQL5 mechanism and conformational changes. We have measured the ATPase, helicase and DNA binding properties of various RECQL5 constructs and variants and discuss the role of these regions and residues in the various RECQL5 activities. Finally, we have performed a systematic comparison of the RECQL5 structures with other RecQ family structures and based on these comparisons we have constructed a model for the mechano-chemical cycle of the common catalytic core of these helicases.

Levy S, Allerston CK, Liveanu V, Habib MR, Gileadi O, Schuster G. 2016. Identification of LACTB2, a metallo-β-lactamase protein, as a human mitochondrial endoribonuclease. Nucleic Acids Res, 44 (4), pp. 1813-1832. | Show Abstract | Read more

Post-transcriptional control of mitochondrial gene expression, including the processing and generation of mature transcripts as well as their degradation, is a key regulatory step in gene expression in human mitochondria. Consequently, identification of the proteins responsible for RNA processing and degradation in this organelle is of great importance. The metallo-β-lactamase (MBL) is a candidate protein family that includes ribo- and deoxyribonucleases. In this study, we discovered a function for LACTB2, an orphan MBL protein found in mammalian mitochondria. Solving its crystal structure revealed almost perfect alignment of the MBL domain with CPSF73, as well as to other ribonucleases of the MBL superfamily. Recombinant human LACTB2 displayed robust endoribonuclease activity on ssRNA with a preference for cleavage after purine-pyrimidine sequences. Mutational analysis identified an extended RNA-binding site. Knockdown of LACTB2 in cultured cells caused a moderate but significant accumulation of many mitochondrial transcripts, and its overexpression led to the opposite effect. Furthermore, manipulation of LACTB2 expression resulted in cellular morphological deformation and cell death. Together, this study discovered that LACTB2 is an endoribonuclease that is involved in the turnover of mitochondrial RNA, and is essential for mitochondrial function in human cells.

Newman JA, Cooper CDO, Roos AK, Aitkenhead H, Oppermann UCT, Cho HJ, Osman R, Gileadi O. 2016. Structures of Two Melanoma-Associated Antigens Suggest Allosteric Regulation of Effector Binding. PLoS One, 11 (2), pp. e0148762. | Show Abstract | Read more

The MAGE (melanoma associated antigen) protein family are tumour-associated proteins normally present only in reproductive tissues such as germ cells of the testis. The human genome encodes over 60 MAGE genes of which one class (containing MAGE-A3 and MAGE-A4) are exclusively expressed in tumours, making them an attractive target for the development of targeted and immunotherapeutic cancer treatments. Some MAGE proteins are thought to play an active role in driving cancer, modulating the activity of E3 ubiquitin ligases on targets related to apoptosis. Here we determined the crystal structures of MAGE-A3 and MAGE-A4. Both proteins crystallized with a terminal peptide bound in a deep cleft between two tandem-arranged winged helix domains. MAGE-A3 (but not MAGE-A4), is predominantly dimeric in solution. Comparison of MAGE-A3 and MAGE-A3 with a structure of an effector-bound MAGE-G1 suggests that a major conformational rearrangement is required for binding, and that this conformational plasticity may be targeted by allosteric binders.

Newman JA, Cooper CDO, Aitkenhead H, Gileadi O. 2015. Structure of the Helicase Domain of DNA Polymerase Theta Reveals a Possible Role in the Microhomology-Mediated End-Joining Pathway. Structure, 23 (12), pp. 2319-2330. | Show Abstract | Read more

DNA polymerase theta (Polθ) has been identified as a crucial alternative non-homologous end-joining factor in mammalian cells. Polθ is upregulated in a range of cancer cell types defective in homologous recombination, and knockdown has been shown to inhibit cell survival in a subset of these, making it an attractive target for cancer treatment. We present crystal structures of the helicase domain of human Polθ in the presence and absence of bound nucleotides, and a characterization of its DNA-binding and DNA-stimulated ATPase activities. Comparisons with related helicases from the Hel308 family identify several unique features. Polθ exists as a tetramer both in the crystals and in solution. We propose a model for DNA binding to the Polθ helicase domain in the context of the Polθ tetramer, which suggests a role for the helicase domain in strand annealing of DNA templates for subsequent processing by the polymerase domain.

Allerston CK, Lee SY, Newman JA, Schofield CJ, McHugh PJ, Gileadi O. 2015. The structures of the SNM1A and SNM1B/Apollo nuclease domains reveal a potential basis for their distinct DNA processing activities. Nucleic Acids Res, 43 (22), pp. 11047-11060. | Show Abstract | Read more

The human SNM1A and SNM1B/Apollo proteins are members of an extended family of eukaryotic nuclease containing a motif related to the prokaryotic metallo-β-lactamase (MBL) fold. SNM1A is a key exonuclease during replication-dependent and transcription-coupled interstrand crosslink repair, while SNM1B/Apollo is required for maintaining telomeric overhangs. Here, we report the crystal structures of SNM1A and SNM1B at 2.16 Å. While both proteins contain a typical MBL-β-CASP domain, a region of positive charge surrounds the active site of SNM1A, which is absent in SNM1B and explains the greater apparent processivity of SNM1A. The structures of both proteins also reveal a putative, wide DNA-binding groove. Extensive mutagenesis of this groove, coupled with detailed biochemical analysis, identified residues that did not impact on SNM1A catalytic activity, but drastically reduced its processivity. Moreover, we identified a key role for this groove for efficient digestion past DNA interstrand crosslinks, facilitating the key DNA repair reaction catalysed by SNM1A. Together, the architecture and dimensions of this groove, coupled to the surrounding region of high positive charge, explain the remarkable ability of SNM1A to accommodate and efficiently digest highly distorted DNA substrates, such as those containing DNA lesions.

Newman JA, Savitsky P, Allerston CK, Bizard AH, Özer Ö, Sarlós K, Liu Y, Pardon E, Steyaert J, Hickson ID, Gileadi O. 2015. Crystal structure of the Bloom's syndrome helicase indicates a role for the HRDC domain in conformational changes. Nucleic Acids Res, 43 (10), pp. 5221-5235. | Show Abstract | Read more

Bloom's syndrome helicase (BLM) is a member of the RecQ family of DNA helicases, which play key roles in the maintenance of genome integrity in all organism groups. We describe crystal structures of the BLM helicase domain in complex with DNA and with an antibody fragment, as well as SAXS and domain association studies in solution. We show an unexpected nucleotide-dependent interaction of the core helicase domain with the conserved, poorly characterized HRDC domain. The BLM-DNA complex shows an unusual base-flipping mechanism with unique positioning of the DNA duplex relative to the helicase core domains. Comparison with other crystal structures of RecQ helicases permits the definition of structural transitions underlying ATP-driven helicase action, and the identification of a nucleotide-regulated tunnel that may play a role in interactions with complex DNA substrates.

Cooper CDO, Newman JA, Aitkenhead H, Allerston CK, Gileadi O. 2015. Structures of the Ets Protein DNA-binding Domains of Transcription Factors Etv1, Etv4, Etv5, and Fev: DETERMINANTS OF DNA BINDING AND REDOX REGULATION BY DISULFIDE BOND FORMATION. J Biol Chem, 290 (22), pp. 13692-13709. | Show Abstract | Read more

Ets transcription factors, which share the conserved Ets DNA-binding domain, number nearly 30 members in humans and are particularly involved in developmental processes. Their deregulation following changes in expression, transcriptional activity, or by chromosomal translocation plays a critical role in carcinogenesis. Ets DNA binding, selectivity, and regulation have been extensively studied; however, questions still arise regarding binding specificity outside the core GGA recognition sequence and the mode of action of Ets post-translational modifications. Here, we report the crystal structures of Etv1, Etv4, Etv5, and Fev, alone and in complex with DNA. We identify previously unrecognized features of the protein-DNA interface. Interactions with the DNA backbone account for most of the binding affinity. We describe a highly coordinated network of water molecules acting in base selection upstream of the GGAA core and the structural features that may account for discrimination against methylated cytidine residues. Unexpectedly, all proteins crystallized as disulfide-linked dimers, exhibiting a novel interface (distant to the DNA recognition helix). Homodimers of Etv1, Etv4, and Etv5 could be reduced to monomers, leading to a 40-200-fold increase in DNA binding affinity. Hence, we present the first indication of a redox-dependent regulatory mechanism that may control the activity of this subset of oncogenic Ets transcription factors.

Pike ACW, Gomathinayagam S, Swuec P, Berti M, Zhang Y, Schnecke C, Marino F, von Delft F, Renault L, Costa A et al. 2015. Human RECQ1 helicase-driven DNA unwinding, annealing, and branch migration: insights from DNA complex structures. Proc Natl Acad Sci U S A, 112 (14), pp. 4286-4291. | Show Abstract | Read more

RecQ helicases are a widely conserved family of ATP-dependent motors with diverse roles in nearly every aspect of bacterial and eukaryotic genome maintenance. However, the physical mechanisms by which RecQ helicases recognize and process specific DNA replication and repair intermediates are largely unknown. Here, we solved crystal structures of the human RECQ1 helicase in complexes with tailed-duplex DNA and ssDNA. The structures map the interactions of the ssDNA tail and the branch point along the helicase and Zn-binding domains, which, together with reported structures of other helicases, define the catalytic stages of helicase action. We also identify a strand-separating pin, which (uniquely in RECQ1) is buttressed by the protein dimer interface. A duplex DNA-binding surface on the C-terminal domain is shown to play a role in DNA unwinding, strand annealing, and Holliday junction (HJ) branch migration. We have combined EM and analytical ultracentrifugation approaches to show that RECQ1 can form what appears to be a flat, homotetrameric complex and propose that RECQ1 tetramers are involved in HJ recognition. This tetrameric arrangement suggests a platform for coordinated activity at the advancing and receding duplexes of an HJ during branch migration.

Mahajan P, Strain-Damerell C, Gileadi O, Burgess-Brown NA. 2014. Medium-throughput production of recombinant human proteins: protein production in insect cells. Methods Mol Biol, 1091 pp. 95-121. | Show Abstract | Read more

This chapter describes the step-by-step methods employed by the Structural Genomics Consortium (SGC) for screening and producing proteins in the baculovirus expression vector system (BEVS). This eukaryotic expression system was selected and a screening process established in 2007 as a measure to tackle the more challenging kinase, RNA-DNA processing and integral membrane protein families on our target list. Here, we discuss our platform for identifying soluble proteins from 3 ml of insect cell culture and describe the procedures involved in producing protein from liter-scale cultures. Although not discussed in this chapter, the same process can also be applied to integral membrane proteins (IMPs) with slight adaptations to the purification procedure.

Burgess-Brown NA, Mahajan P, Strain-Damerell C, Gileadi O, Gräslund S. 2014. Medium-throughput production of recombinant human proteins: protein production in E. coli. Methods Mol Biol, 1091 pp. 73-94. | Show Abstract | Read more

In Chapter 4 we described the SGC process for generating multiple constructs of truncated versions of each protein using LIC. In this chapter we provide a step-by-step procedure of our E. coli system for test expressing intracellular (soluble) proteins in a 96-well format that enables us to identify which proteins or truncated versions are expressed in a soluble and stable form suitable for structural studies. In addition, we detail the process for scaling up cultures for large-scale protein purification. This level of production is required to obtain sufficient quantities (i.e., milligram amounts) of protein for further characterization and/or crystallization experiments. Our standard process is purification by immobilized metal affinity chromatography (IMAC) using nickel resin followed by size exclusion chromatography (SEC), with additional procedures arising from the complexity of the protein itself.

Strain-Damerell C, Mahajan P, Gileadi O, Burgess-Brown NA. 2014. Medium-throughput production of recombinant human proteins: ligation-independent cloning. Methods Mol Biol, 1091 pp. 55-72. | Show Abstract | Read more

Structural genomics groups have identified the need to generate multiple truncated versions of each target to improve their success in producing a well-expressed, soluble, and stable protein and one that crystallizes and diffracts to a sufficient resolution for structural determination. At the SGC, we opted for the Ligation-Independent Cloning (LIC) method which provides the medium throughput we desire to produce and screen many proteins in a parallel process. Here, we describe our LIC protocol for generating constructs in a 96-well format and provide a choice of vectors suitable for expressing proteins in both E. coli and the baculovirus expression vector system (BEVS).

Rosenthal AS, Dexheimer TS, Gileadi O, Nguyen GH, Chu WK, Hickson ID, Jadhav A, Simeonov A, Maloney DJ. 2013. Synthesis and SAR studies of 5-(pyridin-4-yl)-1,3,4-thiadiazol-2-amine derivatives as potent inhibitors of Bloom helicase Bioorganic and Medicinal Chemistry Letters, 23 (20), pp. 5660-5666. | Show Abstract | Read more

Human cells utilize a variety of complex DNA repair mechanisms in order to combat constant mutagenic and cytotoxic threats from both exogenous and endogenous sources. The RecQ family of DNA helicases, which includes Bloom helicase (BLM), plays an important function in DNA repair by unwinding complementary strands of duplex DNA as well as atypical DNA structures such as Holliday junctions. Mutations of the BLM gene can result in Bloom syndrome, an autosomal recessive disorder associated with cancer predisposition. BLM-deficient cells exhibit increased sensitivity to DNA damaging agents indicating that a selective BLM inhibitor could be useful in potentiating the anticancer activity of these agents. In this work, we describe the medicinal chemistry optimization of the hit molecule following a quantitative high-throughput screen of > 355,000 compounds. These efforts lead to the identification of ML216 and related analogs, which possess potent BLM inhibition and exhibit selectivity over related helicases. Moreover, these compounds demonstrated cellular activity by inducing sister chromatid exchanges, a hallmark of Bloom syndrome. © 2013 Elsevier Ltd. All rights reserved.

Brachyury, a transcription factor driving the rare cancer chordoma: mechanism and therapeutic potential

Chordoma is a rare cancer (1 per million, or ~700 new patients every year in the US and Europe). This cancer is derived from an embryonic structure that is crucial in patterning the spine and the body plan. A nearly universal molecular characteristic of chordoma is the duplication or amplification of the gene encoding Brachyury, a T-box transcription factor that is normally expressed only during early embryogenesis and regulates the development of the notochord as well as the general epithelial ...

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Controlling DNA repair by small-molecule inhibitors of human DNA helicases.

Background: DNA in our chromosomes is continuously exposed to chemical damage: from chemicals or radiation absorbed from the environment, but more commonly from byproducts of our own metabolism. The process of DNA replication is itself capable of damaging DNA. Our cells cope with DNA damage in a variety of ways, including direct reversal of the damage (e.g. re-connecting a broken DNA strand) and removal of chemically modified bases followed by re-synthesis of the proper DNA strand. Restoring ...

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