register interest

Professor Catherine Green

Research Area: Cell and Molecular Biology
Technology Exchange: Chromosome mapping, In situ hybridisation, Microscopy (Confocal), Microscopy (Video) and Protein interaction
Scientific Themes: Cancer Biology and Protein Science & Structural Biology
Keywords: DNA replication, Epigenetics and Chromosome Dynamics
Web Links:

The Chromosome Dynamics Group

We are based in the Wellcome Trust Centre for Human Genetics, in the Nuffield Department of Medicine at the University of Oxford (and in the Department of Zoology at the University of Cambridge). We study the processes that occur at replications forks and in replication factories in mammalian cells. These are likely to be physical cellular locations where the genome instability arises that can lead to cancer and other disorders. Cells have to coordinate a large number of activities at each replication fork, and this seems to involve the key replication protein PCNA that acts as a sliding clamp to recruit enzymes and regulators of DNA replication to the right place at the right time. We are particularly interested in processes that ensure the maintenance of genetic and epigenetic stability during DNA replication, but we will be looking also at the origins of chromosomal alterations: copy number variations and translocations, and the relationship between these, replication timing and the three dimensional organisation of the nucleus. 

Projects in the lab include:

  • Determination of the full complement of PCNA interactors, and how this is modified upon replication stress (Simon Cooper – BBSRC funded). This project uses affinity purification and high throughput bimolecular fluorescence complementation (BiFC) screens to identify and characterise novel PCNA interactors.
  • Dynamics of PCNA interactions at replication forks (Shiphali Shetty – CRUK funded). This project uses fluorescence resonance energy transfer (FRET) to monitor protein-protein interactions in real time in living cells.
  • Structure-function of PCNA in replication and repair (Helen Chambers – CRUK funded). This project is investigating how different mutations of PCNA affect its role in diverse cellular processes.
  • PCNA involvement in epigenetic inheritance (Diana Vallejo – funded by the Generalitat Valenciana). This project uses a combination of protein biochemistry and Drosophila genetics to investigate the role of PCNA in coordinating the transmission of epigenetic states during DNA replication.
  • Substrate identification for protein methyltransferases (Lucie Maingot - funded by the Cambridge Cancer Centre (in collaboration with Chris Abell)). This project involves the design and synthesis of S-adenosyl methionine derivatives to label methylated proteins in vitro and in vivo.
  • Physical structure of replication foci and its relationship with genome stability (Michal Gdula – funded by the Wellcome Trust). This project is investigating how the three-dimensional organisation of the nucleus affects the replication program and the formation of chromosome translocations.

Name Department Institution Country
Professor Stephen Kearsey Zoology University of Oxford United Kingdom
Dr Sara Buonomo school of biological sciences University of Edinburgh United Kingdom
Professor Alan Lehmann FRS Genome Damage and Stability centre Sussex University United Kingdom
Professor Andrew Crosby St. George's University London United Kingdom
Professor Michael Olsen Cancer Research UK Beatson Institute Glasgow University United Kingdom
Dr Paola Vagnarelli Brunel University London United Kingdom
Dr Felicity Watts Genome Damage and Stability centre Sussex University United Kingdom
Professor Chris Abell University of Cambridge, Department of Chemistry United Kingdom
Patrick Varga-Weisz The Babraham Institute United Kingdom
Professor Maria Dominguez Instituto de Neurociencias de Alicante Spain
de Castro IJ, Budzak J, Di Giacinto ML, Ligammari L, Gokhan E, Spanos C, Moralli D, Richardson C, de Las Heras JI, Salatino S et al. 2017. Repo-Man/PP1 regulates heterochromatin formation in interphase. Nat Commun, 8 pp. 14048. | Show Abstract | Read more

Repo-Man is a protein phosphatase 1 (PP1) targeting subunit that regulates mitotic progression and chromatin remodelling. After mitosis, Repo-Man/PP1 remains associated with chromatin but its function in interphase is not known. Here we show that Repo-Man, via Nup153, is enriched on condensed chromatin at the nuclear periphery and at the edge of the nucleopore basket. Repo-Man/PP1 regulates the formation of heterochromatin, dephosphorylates H3S28 and it is necessary and sufficient for heterochromatin protein 1 binding and H3K27me3 recruitment. Using a novel proteogenomic approach, we show that Repo-Man is enriched at subtelomeric regions together with H2AZ and H3.3 and that depletion of Repo-Man alters the peripheral localization of a subset of these regions and alleviates repression of some polycomb telomeric genes. This study shows a role for a mitotic phosphatase in the regulation of the epigenetic landscape and gene expression in interphase.

Wilson RHC, Biasutto AJ, Wang L, Fischer R, Baple EL, Crosby AH, Mancini EJ, Green CM. 2017. PCNA dependent cellular activities tolerate dramatic perturbations in PCNA client interactions. DNA Repair (Amst), 50 pp. 22-35. | Show Abstract | Read more

Proliferating cell nuclear antigen (PCNA) is an essential cofactor for DNA replication and repair, recruiting multiple proteins to their sites of action. We examined the effects of the PCNAS228I mutation that causes PCNA-associated DNA repair disorder (PARD). Cells from individuals affected by PARD are sensitive to the PCNA inhibitors T3 and T2AA, showing that the S228I mutation has consequences for undamaged cells. Analysis of the binding between PCNA and PCNA-interacting proteins (PIPs) shows that the S228I change dramatically impairs the majority of these interactions, including that of Cdt1, DNMT1, PolD3p66 and PolD4p12. In contrast p21 largely retains the ability to bind PCNAS228I. This property is conferred by the p21 PIP box sequence itself, which is both necessary and sufficient for PCNAS228I binding. Ubiquitination of PCNA is unaffected by the S228I change, which indirectly alters the structure of the inter-domain connecting loop. Despite the dramatic in vitro effects of the PARD mutation on PIP-degron binding, there are only minor alterations to the stability of p21 and Cdt1 in cells from affected individuals. Overall our data suggests that reduced affinity of PCNAS228I for specific clients causes subtle cellular defects in undamaged cells which likely contribute to the etiology of PARD.

Singh MS, Balmer J, Barnard AR, Aslam SA, Moralli D, Green CM, Barnea-Cramer A, Duncan I, MacLaren RE. 2016. Transplanted photoreceptor precursors transfer proteins to host photoreceptors by a mechanism of cytoplasmic fusion. Nat Commun, 7 pp. 13537. | Show Abstract | Read more

Photoreceptor transplantation is a potential future treatment for blindness caused by retinal degeneration. Photoreceptor transplantation restores visual responses in end-stage retinal degeneration, but has also been assessed in non-degenerate retinas. In the latter scenario, subretinal transplantation places donor cells beneath an intact host outer nuclear layer (ONL) containing host photoreceptors. Here we show that host cells are labelled with the donor marker through cytoplasmic transfer-94±4.1% of apparently well-integrated donor cells containing both donor and host markers. We detect the occurrence of Cre-Lox recombination between donor and host photoreceptors, and we confirm the findings through FISH analysis of X and Y chromosomes in sex-discordant transplants. We do not find evidence of nuclear fusion of donor and host cells. The artefactual appearance of integrated donor cells in host retinas following transplantation is most commonly due to material transfer from donor cells. Understanding this novel mechanism may provide alternate therapeutic strategies at earlier stages of retinal degeneration.

Davies B, Hatton E, Altemose N, Hussin JG, Pratto F, Zhang G, Hinch AG, Moralli D, Biggs D, Diaz R et al. 2016. Re-engineering the zinc fingers of PRDM9 reverses hybrid sterility in mice. Nature, 530 (7589), pp. 171-176. | Show Abstract | Read more

The DNA-binding protein PRDM9 directs positioning of the double-strand breaks (DSBs) that initiate meiotic recombination in mice and humans. Prdm9 is the only mammalian speciation gene yet identified and is responsible for sterility phenotypes in male hybrids of certain mouse subspecies. To investigate PRDM9 binding and its role in fertility and meiotic recombination, we humanized the DNA-binding domain of PRDM9 in C57BL/6 mice. This change repositions DSB hotspots and completely restores fertility in male hybrids. Here we show that alteration of one Prdm9 allele impacts the behaviour of DSBs controlled by the other allele at chromosome-wide scales. These effects correlate strongly with the degree to which each PRDM9 variant binds both homologues at the DSB sites it controls. Furthermore, higher genome-wide levels of such 'symmetric' PRDM9 binding associate with increasing fertility measures, and comparisons of individual hotspots suggest binding symmetry plays a downstream role in the recombination process. These findings reveal that subspecies-specific degradation of PRDM9 binding sites by meiotic drive, which steadily increases asymmetric PRDM9 binding, has impacts beyond simply changing hotspot positions, and strongly support a direct involvement in hybrid infertility. Because such meiotic drive occurs across mammals, PRDM9 may play a wider, yet transient, role in the early stages of speciation.

Moralli D, Nudel R, Chan MTM, Green CM, Volpi EV, Benítez-Burraco A, Newbury DF, García-Bellido P. 2015. Language impairment in a case of a complex chromosomal rearrangement with a breakpoint downstream of FOXP2 Molecular Cytogenetics, 8 (1), | Show Abstract | Read more

© 2015 Moralli et al. Background: We report on a young female, who presents with a severe speech and language disorder and a balanced de novo complex chromosomal rearrangement, likely to have resulted from a chromosome 7 pericentromeric inversion, followed by a chromosome 7 and 11 translocation. Results: Using molecular cytogenetics, we mapped the four breakpoints to 7p21.1-15.3 (chromosome position: 20,954,043-21,001,537, hg19), 7q31 (chromosome position: 114,528,369-114,556,605, hg19), 7q21.3 (chromosome position: 93,884,065-93,933,453, hg19) and 11p12 (chromosome position: 38,601,145-38,621,572, hg19). These regions contain only non-coding transcripts (ENSG00000232790 on 7p21.1 and TCONS-00013886, TCONS-00013887, TCONS-00014353, TCONS-00013888 on 7q21) indicating that no coding sequences are directly disrupted. The breakpoint on 7q31 mapped 200 kb downstream of FOXP2, a well-known language gene. No splice site or non-synonymous coding variants were found in the FOXP2 coding sequence. We were unable to detect any changes in the expression level of FOXP2 in fibroblast cells derived from the proband, although this may be the result of the low expression level of FOXP2 in these cells. Conclusions: We conclude that the phenotype observed in this patient either arises from a subtle change in FOXP2 regulation due to the disruption of a downstream element controlling its expression, or from the direct disruption of non-coding RNAs.

Choudhry H, Albukhari A, Morotti M, Haider S, Moralli D, Smythies J, Schödel J, Green CM, Camps C, Buffa F et al. 2015. Tumor hypoxia induces nuclear paraspeckle formation through HIF-2α dependent transcriptional activation of NEAT1 leading to cancer cell survival. Oncogene, 34 (34), pp. 4482-4490. | Show Abstract | Read more

Activation of cellular transcriptional responses, mediated by hypoxia-inducible factor (HIF), is common in many types of cancer, and generally confers a poor prognosis. Known to induce many hundreds of protein-coding genes, HIF has also recently been shown to be a key regulator of the non-coding transcriptional response. Here, we show that NEAT1 long non-coding RNA (lncRNA) is a direct transcriptional target of HIF in many breast cancer cell lines and in solid tumors. Unlike previously described lncRNAs, NEAT1 is regulated principally by HIF-2 rather than by HIF-1. NEAT1 is a nuclear lncRNA that is an essential structural component of paraspeckles and the hypoxic induction of NEAT1 induces paraspeckle formation in a manner that is dependent upon both NEAT1 and on HIF-2. Paraspeckles are multifunction nuclear structures that sequester transcriptionally active proteins as well as RNA transcripts that have been subjected to adenosine-to-inosine (A-to-I) editing. We show that the nuclear retention of one such transcript, F11R (also known as junctional adhesion molecule 1, JAM1), in hypoxia is dependent upon the hypoxic increase in NEAT1, thereby conferring a novel mechanism of HIF-dependent gene regulation. Induction of NEAT1 in hypoxia also leads to accelerated cellular proliferation, improved clonogenic survival and reduced apoptosis, all of which are hallmarks of increased tumorigenesis. Furthermore, in patients with breast cancer, high tumor NEAT1 expression correlates with poor survival. Taken together, these results indicate a new role for HIF transcriptional pathways in the regulation of nuclear structure and that this contributes to the pro-tumorigenic hypoxia-phenotype in breast cancer.

Cooper SE, Hodimont E, Green CM. 2015. A fluorescent bimolecular complementation screen reveals MAF1, RNF7 and SETD3 as PCNA-associated proteins in human cells. Cell Cycle, 14 (15), pp. 2509-2519. | Show Abstract | Read more

The proliferating cell nuclear antigen (PCNA) is a conserved component of DNA replication factories, and interactions with PCNA mediate the recruitment of many essential DNA replication enzymes to these sites of DNA synthesis. A complete description of the structure and composition of these factories remains elusive, and a better knowledge of them will improve our understanding of how the maintenance of genome and epigenetic stability is achieved. To fully characterize the set of proteins that interact with PCNA we developed a bimolecular fluorescence complementation (BiFC) screen for PCNA-interactors in human cells. This 2-hybrid type screen for interactors from a human cDNA library is rapid and efficient. The fluorescent read-out for protein interaction enables facile selection of interacting clones, and we combined this with next generation sequencing to identify the cDNAs encoding the interacting proteins. This method was able to reproducibly identify previously characterized PCNA-interactors but importantly also identified RNF7, Maf1 and SetD3 as PCNA-interacting proteins. We validated these interactions by co-immunoprecipitation from human cell extracts and by interaction analyses using recombinant proteins. These results show that the BiFC screen is a valuable method for the identification of protein-protein interactions in living mammalian cells. This approach has potentially wide application as it is high throughput and readily automated. We suggest that, given this interaction with PCNA, Maf1, RNF7, and SetD3 are potentially involved in DNA replication, DNA repair, or associated processes.

Green CM, Baple EL, Crosby AH. 2014. PCNA mutation affects DNA repair not replication. Cell Cycle, 13 (20), pp. 3157-3158. | Read more

Cited:

29

Scopus

Baple EL, Chambers H, Cross HE, Fawcett H, Nakazawa Y, Chioza BA, Harlalka GV, Mansour S, Sreekantan-Nair A, Patton MA et al. 2014. Hypomorphic PCNA mutation underlies a human DNA repair disorder Journal of Clinical Investigation, 124 (7), pp. 3137-3146. | Show Abstract | Read more

Numerous human disorders, including Cockayne syndrome, UV-sensitive syndrome, xeroderma pigmentosum, and trichothiodystrophy, result from the mutation of genes encoding molecules important for nucleotide excision repair. Here, we describe a syndrome in which the cardinal clinical features include short stature, hearing loss, premature aging, telangiectasia, neurodegeneration, and photosensitivity, resulting from a homozygous missense (p.Ser228Ile) sequence alteration of the proliferating cell nuclear antigen (PCNA). PCNA is a highly conserved sliding clamp protein essential for DNA replication and repair. Due to this fundamental role, mutations in PCNA that profoundly impair protein function would be incompatible with life. Interestingly, while the p.Ser228Ile alteration appeared to have no effect on protein levels or DNA replication, patient cells exhibited marked abnormalities in response to UV irradiation, displaying substantial reductions in both UV survival and RNA synthesis recovery. The p.Ser228Ile change also profoundly altered PCNA's interaction with Flap endonuclease 1 and DNA Ligase 1, DNA metabolism enzymes. Together, our findings detail a mutation of PCNA in humans associated with a neurodegenerative phenotype, displaying clinical and molecular features common to other DNA repair disorders, which we showed to be attributable to a hypomorphic amino acid alteration.

Cerase A, Smeets D, Tang YA, Gdula M, Kraus F, Spivakov M, Moindrot B, Leleu M, Tattermusch A, Demmerle J et al. 2014. Spatial separation of Xist RNA and polycomb proteins revealed by superresolution microscopy. Proc Natl Acad Sci U S A, 111 (6), pp. 2235-2240. | Show Abstract | Read more

In female mammals, one of the two X chromosomes is transcriptionally silenced to equalize X-linked gene dosage relative to XY males, a process termed X chromosome inactivation. Mechanistically, this is thought to occur via directed recruitment of chromatin modifying factors by the master regulator, X-inactive specific transcript (Xist) RNA, which localizes in cis along the entire length of the chromosome. A well-studied example is the recruitment of polycomb repressive complex 2 (PRC2), for which there is evidence of a direct interaction involving the PRC2 proteins Enhancer of zeste 2 (Ezh2) and Supressor of zeste 12 (Suz12) and the A-repeat region located at the 5' end of Xist RNA. In this study, we have analyzed Xist-mediated recruitment of PRC2 using two approaches, microarray-based epigenomic mapping and superresolution 3D structured illumination microscopy. Making use of an ES cell line carrying an inducible Xist transgene located on mouse chromosome 17, we show that 24 h after synchronous induction of Xist expression, acquired PRC2 binding sites map predominantly to gene-rich regions, notably within gene bodies. Paradoxically, these new sites of PRC2 deposition do not correlate with Xist-mediated gene silencing. The 3D structured illumination microscopy was performed to assess the relative localization of PRC2 proteins and Xist RNA. Unexpectedly, we observed significant spatial separation and absence of colocalization both in the inducible Xist transgene ES cell line and in normal XX somatic cells. Our observations argue against direct interaction between Xist RNA and PRC2 proteins and, as such, prompt a reappraisal of the mechanism for PRC2 recruitment in X chromosome inactivation.

Lehmann AR, Sabbioneda S, Goehler T, Niimi A, Green CM, Bienko M, Dikic I. 2012. Regulation of translesion synthesis in human cells MUTAGENESIS, 27 (1), pp. 106-106.

Rowbotham SP, Barki L, Neves-Costa A, Santos F, Dean W, Hawkes N, Choudhary P, Will WR, Webster J, Oxley D et al. 2011. Maintenance of silent chromatin through replication requires SWI/SNF-like chromatin remodeler SMARCAD1. Mol Cell, 42 (3), pp. 285-296. | Show Abstract | Read more

Epigenetic marks such as posttranslational histone modifications specify the functional states of underlying DNA sequences, though how they are maintained after their disruption during DNA replication remains a critical question. We identify the mammalian SWI/SNF-like protein SMARCAD1 as a key factor required for the re-establishment of repressive chromatin. The ATPase activity of SMARCAD1 is necessary for global deacetylation of histones H3/H4. In this way, SMARCAD1 promotes methylation of H3K9, the establishment of heterochromatin, and faithful chromosome segregation. SMARCAD1 associates with transcriptional repressors including KAP1, histone deacetylases HDAC1/2 and the histone methyltransferase G9a/GLP and modulates the interaction of HDAC1 and KAP1 with heterochromatin. SMARCAD1 directly interacts with PCNA, a central component of the replication machinery, and is recruited to sites of DNA replication. Our findings suggest that chromatin remodeling by SMARCAD1 ensures that silenced loci, such as pericentric heterochromatin, are correctly perpetuated.

Göhler T, Sabbioneda S, Green CM, Lehmann AR. 2011. ATR-mediated phosphorylation of DNA polymerase η is needed for efficient recovery from UV damage. J Cell Biol, 192 (2), pp. 219-227. | Show Abstract | Read more

DNA polymerase η (polη) belongs to the Y-family of DNA polymerases and facilitates translesion synthesis past UV damage. We show that, after UV irradiation, polη becomes phosphorylated at Ser601 by the ataxia-telangiectasia mutated and Rad3-related (ATR) kinase. DNA damage-induced phosphorylation of polη depends on its physical interaction with Rad18 but is independent of PCNA monoubiquitination. It requires the ubiquitin-binding domain of polη but not its PCNA-interacting motif. ATR-dependent phosphorylation of polη is necessary to restore normal survival and postreplication repair after ultraviolet irradiation in xeroderma pigmentosum variant fibroblasts, and is involved in the checkpoint response to UV damage. Taken together, our results provide evidence for a link between DNA damage-induced checkpoint activation and translesion synthesis in mammalian cells.

Ramasubramanyan S, Coulon S, Fuchs RP, Lehmann AR, Green CM. 2010. Ubiquitin-PCNA fusion as a mimic for mono-ubiquitinated PCNA in Schizosaccharomyces pombe. DNA Repair (Amst), 9 (7), pp. 777-784. | Show Abstract | Read more

Translesion synthesis is a major mechanism with which eukaryotic cells deal with DNA damage during replication. Mono-ubiquitinated PCNA is a key regulator of this process. We have investigated whether a ubiquitin-PCNA fusion can mimic ubiquitinated PCNA, by transforming plasmids expressing this fusion protein into different mutants of Schizosaccharomyces pombe. We show that the fusion protein is able to form PCNA trimers and that it can reduce the UV sensitivity and increase translesion synthesis in mutants in which PCNA cannot be ubiquitinated (pcn1-K164R and rhp18), but not of the rad8 mutant in which PCNA can be mono-ubiquitinated but not poly-ubiquitinated. We conclude that the fusion protein is a mimic of mono-ubiquitinated PCNA but it cannot be poly-ubiquitinated. Expression of the fusion protein at levels similar to that of endogenous unmodified protein has little effect on the spontaneous mutation rate of S. pombe. Replacement of the pcn1 locus with PCNA N-terminally tagged with different epitopes resulted in lethality, probably because the tagged proteins were expressed at substantially reduced levels.

Bienko M, Green CM, Sabbioneda S, Crosetto N, Matic I, Hibbert RG, Begovic T, Niimi A, Mann M, Lehmann AR, Dikic I. 2010. Regulation of translesion synthesis DNA polymerase eta by monoubiquitination. Mol Cell, 37 (3), pp. 396-407. | Show Abstract | Read more

DNA polymerase eta is a Y family polymerase involved in translesion synthesis (TLS). Its action is initiated by simultaneous interaction between the PIP box in pol eta and PCNA and between the UBZ in pol eta and monoubiquitin attached to PCNA. Whereas monoubiquitination of PCNA is required for its interaction with pol eta during TLS, we now show that monoubiquitination of pol eta inhibits this interaction, preventing its functions in undamaged cells. Identification of monoubiquitination sites within pol eta nuclear localization signal (NLS) led to the discovery that pol eta NLS directly contacts PCNA, forming an extended pol eta-PCNA interaction surface. We name this the PCNA-interacting region (PIR) and show that its monoubiquitination is downregulated by various DNA-damaging agents. We propose that this mechanism ensures optimal availability of nonubiquitinated, TLS-competent pol eta after DNA damage. Our work shows how monoubiquitination can either positively or negatively regulate the assembly of a protein complex, depending on which substrates are targeted by ubiquitin.

Sabbioneda S, Green CM, Bienko M, Kannouche P, Dikic I, Lehmann AR. 2009. Ubiquitin-binding motif of human DNA polymerase eta is required for correct localization. Proc Natl Acad Sci U S A, 106 (8), pp. E20. | Read more

Cseresnyes Z, Schwarz U, Green CM. 2009. Analysis of replication factories in human cells by super-resolution light microscopy. BMC Cell Biol, 10 (1), pp. 88. | Show Abstract | Read more

BACKGROUND: DNA replication in human cells is performed in discrete sub-nuclear locations known as replication foci or factories. These factories form in the nucleus during S phase and are sites of DNA synthesis and high local concentrations of enzymes required for chromatin replication. Why these structures are required, and how they are organised internally has yet to be identified. It has been difficult to analyse the structure of these factories as they are small in size and thus below the resolution limit of the standard confocal microscope. We have used stimulated emission depletion (STED) microscopy, which improves on the resolving power of the confocal microscope, to probe the structure of these factories at sub-diffraction limit resolution. RESULTS: Using immunofluorescent imaging of PCNA (proliferating cell nuclear antigen) and RPA (replication protein A) we show that factories are smaller in size (approximately 150 nm diameter), and greater in number (up to 1400 in an early S- phase nucleus), than is determined by confocal imaging. The replication inhibitor hydroxyurea caused an approximately 40% reduction in number and a 30% increase in diameter of replication factories, changes that were not clearly identified by standard confocal imaging. CONCLUSIONS: These measurements for replication factory size now approach the dimensions suggested by electron microscopy. This agreement between these two methods, that use very different sample preparation and imaging conditions, suggests that we have arrived at a true measurement for the size of these structures. The number of individual factories present in a single nucleus that we measure using this system is greater than has been previously reported. This analysis therefore suggests that each replication factory contains fewer active replication forks than previously envisaged.

Sabbioneda S, Gourdin AM, Green CM, Zotter A, Giglia-Mari G, Houtsmuller A, Vermeulen W, Lehmann AR. 2008. Effect of proliferating cell nuclear antigen ubiquitination and chromatin structure on the dynamic properties of the Y-family DNA polymerases. Mol Biol Cell, 19 (12), pp. 5193-5202. | Show Abstract | Read more

Y-family DNA polymerases carry out translesion synthesis past damaged DNA. DNA polymerases (pol) eta and iota are usually uniformly distributed through the nucleus but accumulate in replication foci during S phase. DNA-damaging treatments result in an increase in S phase cells containing polymerase foci. Using photobleaching techniques, we show that poleta is highly mobile in human fibroblasts. Even when localized in replication foci, it is only transiently immobilized. Although ubiquitination of proliferating cell nuclear antigen (PCNA) is not required for the localization of poleta in foci, it results in an increased residence time in foci. poliota is even more mobile than poleta, both when uniformly distributed and when localized in foci. Kinetic modeling suggests that both poleta and poliota diffuse through the cell but that they are transiently immobilized for approximately 150 ms, with a larger proportion of poleta than poliota immobilized at any time. Treatment of cells with DRAQ5, which results in temporary opening of the chromatin structure, causes a dramatic immobilization of poleta but not poliota. Our data are consistent with a model in which the polymerases are transiently probing the DNA/chromatin. When DNA is exposed at replication forks, the polymerase residence times increase, and this is further facilitated by the ubiquitination of PCNA.

Niimi A, Brown S, Sabbioneda S, Kannouche PL, Scott A, Yasui A, Green CM, Lehmann AR. 2008. Regulation of proliferating cell nuclear antigen ubiquitination in mammalian cells. Proc Natl Acad Sci U S A, 105 (42), pp. 16125-16130. | Show Abstract | Read more

After exposure to DNA-damaging agents that block the progress of the replication fork, monoubiquitination of proliferating cell nuclear antigen (PCNA) mediates the switch from replicative to translesion synthesis DNA polymerases. We show that in human cells, PCNA is monoubiquitinated in response to methyl methanesulfonate and mitomycin C, as well as UV light, albeit with different kinetics, but not in response to bleomycin or camptothecin. Cyclobutane pyrimidine dimers are responsible for most of the PCNA ubiquitination events after UV-irradiation. Failure to ubiquitinate PCNA results in substantial sensitivity to UV and methyl methanesulfonate, but not to camptothecin or bleomycin. PCNA ubiquitination depends on Replication Protein A (RPA), but is independent of ATR-mediated checkpoint activation. After UV-irradiation, there is a temporal correlation between the disappearance of the deubiquitinating enzyme USP1 and the presence of PCNA ubiquitination, but this correlation was not found after chemical mutagen treatment. By using cells expressing photolyases, we are able to remove the UV lesions, and we show that PCNA ubiquitination persists for many hours after the damage has been removed. We present a model of translesion synthesis behind the replication fork to explain the persistence of ubiquitinated PCNA.

Lehmann AR, Niimi A, Ogi T, Brown S, Sabbioneda S, Wing JF, Kannouche PL, Green CM. 2007. Translesion synthesis: Y-family polymerases and the polymerase switch. DNA Repair (Amst), 6 (7), pp. 891-899. | Show Abstract | Read more

Replicative DNA polymerases are blocked at DNA lesions. Synthesis past DNA damage requires the replacement of the replicative polymerase by one of a group of specialised translesion synthesis (TLS) polymerases, most of which belong to the Y-family. Each of these has different substrate specificities for different types of damage. In eukaryotes mono-ubiquitination of PCNA plays a crucial role in the switch from replicative to TLS polymerases at stalled forks. All the Y-family polymerases have ubiquitin binding sites that increase their binding affinity for ubiquitinated PCNA at the sites of stalled forks.

Green CM. 2006. One ring to rule them all? Another cellular responsibility for PCNA. Trends Mol Med, 12 (10), pp. 455-458. | Show Abstract | Read more

To prevent duplication or loss of genomic regions during DNA replication, it is essential that the entire genome is copied precisely once every S phase. Cells achieve this by mutually exclusive regulation of origin firing and licensing. A crucial protein that is involved in origin licensing is chromatin licensing and DNA replication factor 1 (CDT1) and, therefore, activity of this protein must be strictly controlled. Four recent articles have demonstrated that proliferating cell nuclear antigen (PCNA), an essential sliding clamp used in replication and DNA repair, has a crucial role in this process by mediating the proteasomal degradation of CDT1.

Frampton J, Irmisch A, Green CM, Neiss A, Trickey M, Ulrich HD, Furuya K, Watts FZ, Carr AM, Lehmann AR. 2006. Postreplication repair and PCNA modification in Schizosaccharomyces pombe. Mol Biol Cell, 17 (7), pp. 2976-2985. | Show Abstract | Read more

Ubiquitination of proliferating cell nuclear antigen (PCNA) plays a crucial role in regulating replication past DNA damage in eukaryotes, but the detailed mechanisms appear to vary in different organisms. We have examined the modification of PCNA in Schizosaccharomyces pombe. We find that, in response to UV irradiation, PCNA is mono- and poly-ubiquitinated in a manner similar to that in Saccharomyces cerevisiae. However in undamaged Schizosaccharomyces pombe cells, PCNA is ubiquitinated in S phase, whereas in S. cerevisiae it is sumoylated. Furthermore we find that, unlike in S. cerevisiae, mutants defective in ubiquitination of PCNA are also sensitive to ionizing radiation, and PCNA is ubiquitinated after exposure of cells to ionizing radiation, in a manner similar to the response to UV-irradiation. We show that PCNA modification and cell cycle checkpoints represent two independent signals in response to DNA damage. Finally, we unexpectedly find that PCNA is ubiquitinated in response to DNA damage when cells are arrested in G2.

Bienko M, Green CM, Crosetto N, Rudolf F, Zapart G, Coull B, Kannouche P, Wider G, Peter M, Lehmann AR et al. 2005. Ubiquitin-binding domains in Y-family polymerases regulate translesion synthesis. Science, 310 (5755), pp. 1821-1824. | Show Abstract | Read more

Translesion synthesis (TLS) is the major pathway by which mammalian cells replicate across DNA lesions. Upon DNA damage, ubiquitination of proliferating cell nuclear antigen (PCNA) induces bypass of the lesion by directing the replication machinery into the TLS pathway. Yet, how this modification is recognized and interpreted in the cell remains unclear. Here we describe the identification of two ubiquitin (Ub)-binding domains (UBM and UBZ), which are evolutionarily conserved in all Y-family TLS polymerases (pols). These domains are required for binding of poleta and poliota to ubiquitin, their accumulation in replication factories, and their interaction with monoubiquitinated PCNA. Moreover, the UBZ domain of poleta is essential to efficiently restore a normal response to ultraviolet irradiation in xeroderma pigmentosum variant (XP-V) fibroblasts. Our results indicate that Ub-binding domains of Y-family polymerases play crucial regulatory roles in TLS.

Albertella MR, Green CM, Lehmann AR, O'Connor MJ. 2005. A role for polymerase eta in the cellular tolerance to cisplatin-induced damage. Cancer Res, 65 (21), pp. 9799-9806. | Show Abstract | Read more

Mutation of the POLH gene encoding DNA polymerase eta (pol eta) causes the UV-sensitivity syndrome xeroderma pigmentosum-variant (XP-V) which is linked to the ability of pol eta to accurately bypass UV-induced cyclobutane pyrimidine dimers during a process termed translesion synthesis. Pol eta can also bypass other DNA damage adducts in vitro, including cisplatin-induced intrastrand adducts, although the physiological relevance of this is unknown. Here, we show that independent XP-V cell lines are dramatically more sensitive to cisplatin than the same cells complemented with functional pol eta. Similar results were obtained with the chemotherapeutic agents, carboplatin and oxaliplatin, thus revealing a general requirement for pol eta expression in providing tolerance to these platinum-based drugs. The level of sensitization observed was comparable to that of XP-A cells deficient in nucleotide excision repair, a recognized and important mechanism for repair of cisplatin adducts. However, unlike in XP-A cells, the absence of pol eta expression resulted in a reduced ability to overcome cisplatin-induced S phase arrest, suggesting that pol eta is involved in translesion synthesis past these replication-blocking adducts. Subcellular localization studies also highlighted an accumulation of nuclei with pol eta foci that correlated with the formation of monoubiquitinated proliferating cell nuclear antigen following treatment with cisplatin, reminiscent of the response to UV irradiation and further indicating a role for pol eta in dealing with cisplatin-induced damage. Together, these data show that pol eta represents an important determinant of cellular responses to cisplatin, which could have implications for acquired or intrinsic resistance to this key chemotherapeutic agent.

Green CM, Lehmann AR. 2005. Translesion synthesis and error-prone polymerases. Adv Exp Med Biol, 570 pp. 199-223. | Read more

Green CM, Lowndes NF. 2004. Purification and analysis of checkpoint protein complexes from Saccharomyces cerevisiae. Methods Mol Biol, 280 pp. 291-306. | Show Abstract | Read more

The DNA damage-dependent checkpoint of Saccharomyces cerevisiae is a paradigm for eukaryotic checkpoint pathways that regulate cell cycle progression in the presence of insults to the genetic material. In order to better understand this pathway, we undertook a biochemical study of the proteins implicated in its functioning. Analysis of the hydrodynamic properties of a protein in a crude mixture can give insights into possible tertiary organization such as participation in high-molecular-mass protein complexes. We here describe the determination of Stokes radius and sedimentation coefficients for the Rad24 protein, which enabled us to predict that this protein was a component of a protein complex in crude yeast extracts. This led us to develop a protocol to purify this complex to homogeneity in order to determine the component proteins. The methods described here should be applicable to the hydrodynamic analysis and subsequent purification of any soluble protein from organisms amenable to genetic manipulation, such as yeast, as long as the function of that protein is not perturbed by the addition of an epitope tag.

Gontijo AMDMC, Green CM, Almouzni G. 2003. Repairing DNA damage in chromatin. Biochimie, 85 (11), pp. 1133-1147. | Show Abstract | Read more

Understanding how DNA repair processes occur in vivo when access to DNA is hindered by chromatin structural organisation is a current challenge. In general terms, the following sequence of events has to be considered within a chromatin environment: (i) finding a lesion (ii) repairing this lesion, and (iii) full restoration of a functional chromatin locus. In this review, basic principles concerning nucleosome dynamics, both intrinsic properties and those dependent on accessory factors, will be used to discuss the issue of lesion accessibility to damage-detecting proteins within chromatin. In addition, opportunities for damage detection due to chromatin alterations directly linked with transcription and replication processes will be considered. After damage detection, additional processes to enhance accessibility within chromatin may be needed to accommodate downstream repair factors or to allow DNA synthesis, resulting in interdependency between repair and accessibility mechanisms in chromatin. Finally, we will comment on the way in which chromatin assembly factors can participate in the maintenance of chromatin structures during DNA repair.

Green CM, Almouzni G. 2003. Local action of the chromatin assembly factor CAF-1 at sites of nucleotide excision repair in vivo. EMBO J, 22 (19), pp. 5163-5174. | Show Abstract | Read more

DNA damage and its repair can cause both local and global rearrangements of chromatin structure. In each case, the epigenetic information contained within this structure must be maintained. Using the recently developed method for the localized UV irradiation of cells, we analysed responses that occur locally to damage sites and global events triggered by local damage recognition. We thus demonstrate that, within a single cell, the recruitment of chromatin assembly factor 1 (CAF-1) to UV-induced DNA damage is a strictly local phenomenon, restricted to damage sites. Concomitantly, proliferating cell nuclear antigen (PCNA) locates to the same sites. This localized recruitment suggests that CAF-1 participates directly in chromatin structural rearrangements that occur in the vicinity of the damage. Use of nucleotide excision repair (NER)-deficient cells shows that the NER pathway--specifically dual incision--is required for recruitment of CAF-1 and PCNA. This in vivo demonstration of the local role of CAF-1, depending directly on NER, supports the hypothesis that CAF-1 ensures the maintenance of epigenetic information by acting locally at repair sites.

Gilbert CS, van den Bosch M, Green CM, Vialard JE, Grenon M, Erdjument-Bromage H, Tempst P, Lowndes NF. 2003. The budding yeast Rad9 checkpoint complex: chaperone proteins are required for its function. EMBO Rep, 4 (10), pp. 953-958. | Show Abstract | Read more

Rad9 functions in the DNA-damage checkpoint pathway of Saccharomyces cerevisiae. In whole-cell extracts, Rad9 is found in large, soluble complexes, which have functions in amplifying the checkpoint signal. The two main soluble forms of Rad9 complexes that are found in cells exposed to DNA-damaging treatments were purified to homogeneity. Both of these Rad9 complexes contain the Ssa1 and/or Ssa2 chaperone proteins, suggesting a function for these proteins in checkpoint regulation. Consistent with this possibility, genetic experiments indicate redundant functions for SSA1 and SSA2 in survival, G2/M-checkpoint regulation, and phosphorylation of both Rad9 and Rad53 after irradiation with ultraviolet light. Ssa1 and Ssa2 can now be considered as novel checkpoint proteins that are likely to be required for remodelling Rad9 complexes during checkpoint-pathway activation.

Green CM, Almouzni G. 2002. When repair meets chromatin. First in series on chromatin dynamics. EMBO Rep, 3 (1), pp. 28-33. | Show Abstract | Read more

In eukaryotic cells, the inheritance of both the DNA sequence and its organization into chromatin is critical to maintain genome stability. This maintenance is challenged by DNA damage. To fully understand how the cell can tolerate genotoxic stress, it is necessary to integrate knowledge of the nature of DNA damage, its detection and its repair within the chromatin environment of a eukaryotic nucleus. The multiplicity of the DNA damage and repair processes, as well as the complex nature of chromatin, have made this issue difficult to tackle. Recent progress in each of these areas enables us to address, both at a molecular and a cellular level, the importance of inter-relationships between them. In this review we revisit the 'access, repair, restore' model, which was proposed to explain how the conserved process of nucleotide excision repair operates within chromatin. Recent studies have identified factors potentially involved in this process and permit refinement of the basic model. Drawing on this model, the chromatin alterations likely to be required during other processes of DNA damage repair, particularly double-strand break repair, are discussed and recently identified candidates that might perform such alterations are highlighted.

Gilbert CS, Green CM, Lowndes NF. 2001. Budding yeast Rad9 is an ATP-dependent Rad53 activating machine. Mol Cell, 8 (1), pp. 129-136. | Show Abstract | Read more

We find budding yeast Rad9 in two distinct, large, and soluble complexes in cell extracts. The larger (> or =850 kDa) complex, found in nondamaged cells, contains hypophosphorylated Rad9, whereas the smaller (560 kDa) complex, which forms after DNA damage, contains hyperphosphorylated Rad9 and Rad53. This smaller Rad9 complex is capable of catalyzing phosphorylation and release of active Rad53 kinase, a process requiring the kinase activity of Rad53. However, Mec1 and Tel1 are no longer required once the 560 kDa complex has been formed. We propose a model whereby Mec1/Tel1-dependent hyperphosphorylation of Rad9 results in formation of the smaller Rad9 complex and recruitment of Rad53. This complex then catalyzes activation of Rad53 by acting as a scaffold that brings Rad53 molecules into close proximity, facilitating Rad53 in trans autophosphorylation and subsequent release of activated Rad53.

Green CM, Erdjument-Bromage H, Tempst P, Lowndes NF. 2000. A novel Rad24 checkpoint protein complex closely related to replication factor C. Curr Biol, 10 (1), pp. 39-42. | Show Abstract | Read more

Rad24 functions in the DNA damage checkpoint pathway of Saccharomyces cerevisiae. Here, analysis of Rad24 in whole cell extracts demonstrated that its mass was considerably greater than its predicted molecular weight, suggesting that Rad24 is a component of a protein complex. The Rad24 complex was purified to homogeneity. In addition to Rad24, the complex included polypeptides of 40 kDa and 35 kDa. The 40 kDa species was found by mass spectrometry to contain Rfc2 and Rfc3, subunits of replication factor C (RFC), a five subunit protein that is required for the loading of polymerases onto DNA during replication and repair [3]. We hypothesised that other RFC subunits, all of which share sequence homologles with Rad24, might also be components of the Rad24 complex. Reciprocal co-immunoprecipitation studies were performed using extracts prepared from strains containing epitope-tagged RFC proteins. These experiments showed that the small RFC proteins, Rfc2, Rfc3, Rfc4 and Rfc5, interacted with Rad24, whereas the Rfc1 subunit did not. We suggest that this RFC-like Rad24 complex may function as a structure-specific sensor in the DNA damage checkpoint pathway.

Vialard JE, Gilbert CS, Green CM, Lowndes NF. 1998. The budding yeast Rad9 checkpoint protein is subjected to Mec1/Tel1-dependent hyperphosphorylation and interacts with Rad53 after DNA damage. EMBO J, 17 (19), pp. 5679-5688. | Show Abstract | Read more

The Saccharomyces cerevisiae RAD9 checkpoint gene is required for transient cell-cycle arrests and transcriptional induction of DNA repair genes in response to DNA damage. Polyclonal antibodies raised against the Rad9 protein recognized several polypeptides in asynchronous cultures, and in cells arrested in S or G2/M phases while a single form was observed in G1-arrested cells. Treatment with various DNA damaging agents, i.e. UV, ionizing radiation or methyl methane sulfonate, resulted in the appearance of hypermodified forms of the protein. All modifications detected during a normal cell cycle and after DNA damage were sensitive to phosphatase treatment, indicating that they resulted from phosphorylation. Damage-induced hyperphosphorylation of Rad9 correlated with checkpoint functions (cell-cycle arrest and transcriptional induction) and was cell-cycle stage- and progression-independent. In asynchronous cultures, Rad9 hyperphosphorylation was dependent on MEC1 and TEL1, homologues of the ATR and ATM genes. In G1-arrested cells, damage-dependent hyperphosphorylation required functional MEC1 in addition to RAD17, RAD24, MEC3 and DDC1, demonstrating cell-cycle stage specificity of the checkpoint genes in this response to DNA damage. Analysis of checkpoint protein interactions after DNA damage revealed that Rad9 physically associates with Rad53.

de la Torre-Ruiz MA, Green CM, Lowndes NF. 1998. RAD9 and RAD24 define two additive, interacting branches of the DNA damage checkpoint pathway in budding yeast normally required for Rad53 modification and activation. EMBO J, 17 (9), pp. 2687-2698. | Show Abstract | Read more

In budding yeast, RAD9 and RAD24/RAD17/MEC3 are believed to function upstream of MEC1 and RAD53 in signalling the presence of DNA damage. Deletion of any one of these genes reduces the normal G1/S and G2/M checkpoint delays after UV irradiation, whereas in rad9Delta-rad24Delta cells the G1/S checkpoint is undetectable, although there is a residual G2/M checkpoint. We have shown previously that RAD9 also controls the transcriptional induction of a DNA damage regulon (DDR). We now report that efficient DDR induction requires all the above-mentioned checkpoint genes. Residual induction of the DDR after UV irradiation observed in all single mutants is not detectable in rad9Delta-rad24Delta. We have examined the G2/M checkpoint and UV sensitivity of single mutants after overexpression of the checkpoint proteins. This analysis indicates that RAD9 and the RAD24 epistasis group can be placed onto two separate, additive branches that converge on MEC1 and RAD53. Furthermore, MEC3 appears to function downstream of RAD24/RAD17. The transcriptional response to DNA damage revealed unexpected and specific antagonism between RAD9 and RAD24. Further support for genetic interaction between RAD9 and RAD24 comes from study of the modification and activation of Rad53 after damage. Evidence for bypass of RAD53 function under some conditions is also presented.

Davies B, Hatton E, Altemose N, Hussin JG, Pratto F, Zhang G, Hinch AG, Moralli D, Biggs D, Diaz R et al. 2016. Re-engineering the zinc fingers of PRDM9 reverses hybrid sterility in mice. Nature, 530 (7589), pp. 171-176. | Show Abstract | Read more

The DNA-binding protein PRDM9 directs positioning of the double-strand breaks (DSBs) that initiate meiotic recombination in mice and humans. Prdm9 is the only mammalian speciation gene yet identified and is responsible for sterility phenotypes in male hybrids of certain mouse subspecies. To investigate PRDM9 binding and its role in fertility and meiotic recombination, we humanized the DNA-binding domain of PRDM9 in C57BL/6 mice. This change repositions DSB hotspots and completely restores fertility in male hybrids. Here we show that alteration of one Prdm9 allele impacts the behaviour of DSBs controlled by the other allele at chromosome-wide scales. These effects correlate strongly with the degree to which each PRDM9 variant binds both homologues at the DSB sites it controls. Furthermore, higher genome-wide levels of such 'symmetric' PRDM9 binding associate with increasing fertility measures, and comparisons of individual hotspots suggest binding symmetry plays a downstream role in the recombination process. These findings reveal that subspecies-specific degradation of PRDM9 binding sites by meiotic drive, which steadily increases asymmetric PRDM9 binding, has impacts beyond simply changing hotspot positions, and strongly support a direct involvement in hybrid infertility. Because such meiotic drive occurs across mammals, PRDM9 may play a wider, yet transient, role in the early stages of speciation.

Cooper SE, Hodimont E, Green CM. 2015. A fluorescent bimolecular complementation screen reveals MAF1, RNF7 and SETD3 as PCNA-associated proteins in human cells. Cell Cycle, 14 (15), pp. 2509-2519. | Show Abstract | Read more

The proliferating cell nuclear antigen (PCNA) is a conserved component of DNA replication factories, and interactions with PCNA mediate the recruitment of many essential DNA replication enzymes to these sites of DNA synthesis. A complete description of the structure and composition of these factories remains elusive, and a better knowledge of them will improve our understanding of how the maintenance of genome and epigenetic stability is achieved. To fully characterize the set of proteins that interact with PCNA we developed a bimolecular fluorescence complementation (BiFC) screen for PCNA-interactors in human cells. This 2-hybrid type screen for interactors from a human cDNA library is rapid and efficient. The fluorescent read-out for protein interaction enables facile selection of interacting clones, and we combined this with next generation sequencing to identify the cDNAs encoding the interacting proteins. This method was able to reproducibly identify previously characterized PCNA-interactors but importantly also identified RNF7, Maf1 and SetD3 as PCNA-interacting proteins. We validated these interactions by co-immunoprecipitation from human cell extracts and by interaction analyses using recombinant proteins. These results show that the BiFC screen is a valuable method for the identification of protein-protein interactions in living mammalian cells. This approach has potentially wide application as it is high throughput and readily automated. We suggest that, given this interaction with PCNA, Maf1, RNF7, and SetD3 are potentially involved in DNA replication, DNA repair, or associated processes.

Green CM, Baple EL, Crosby AH. 2014. PCNA mutation affects DNA repair not replication. Cell Cycle, 13 (20), pp. 3157-3158. | Read more

Cited:

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Baple EL, Chambers H, Cross HE, Fawcett H, Nakazawa Y, Chioza BA, Harlalka GV, Mansour S, Sreekantan-Nair A, Patton MA et al. 2014. Hypomorphic PCNA mutation underlies a human DNA repair disorder Journal of Clinical Investigation, 124 (7), pp. 3137-3146. | Show Abstract | Read more

Numerous human disorders, including Cockayne syndrome, UV-sensitive syndrome, xeroderma pigmentosum, and trichothiodystrophy, result from the mutation of genes encoding molecules important for nucleotide excision repair. Here, we describe a syndrome in which the cardinal clinical features include short stature, hearing loss, premature aging, telangiectasia, neurodegeneration, and photosensitivity, resulting from a homozygous missense (p.Ser228Ile) sequence alteration of the proliferating cell nuclear antigen (PCNA). PCNA is a highly conserved sliding clamp protein essential for DNA replication and repair. Due to this fundamental role, mutations in PCNA that profoundly impair protein function would be incompatible with life. Interestingly, while the p.Ser228Ile alteration appeared to have no effect on protein levels or DNA replication, patient cells exhibited marked abnormalities in response to UV irradiation, displaying substantial reductions in both UV survival and RNA synthesis recovery. The p.Ser228Ile change also profoundly altered PCNA's interaction with Flap endonuclease 1 and DNA Ligase 1, DNA metabolism enzymes. Together, our findings detail a mutation of PCNA in humans associated with a neurodegenerative phenotype, displaying clinical and molecular features common to other DNA repair disorders, which we showed to be attributable to a hypomorphic amino acid alteration.

Rowbotham SP, Barki L, Neves-Costa A, Santos F, Dean W, Hawkes N, Choudhary P, Will WR, Webster J, Oxley D et al. 2011. Maintenance of silent chromatin through replication requires SWI/SNF-like chromatin remodeler SMARCAD1. Mol Cell, 42 (3), pp. 285-296. | Show Abstract | Read more

Epigenetic marks such as posttranslational histone modifications specify the functional states of underlying DNA sequences, though how they are maintained after their disruption during DNA replication remains a critical question. We identify the mammalian SWI/SNF-like protein SMARCAD1 as a key factor required for the re-establishment of repressive chromatin. The ATPase activity of SMARCAD1 is necessary for global deacetylation of histones H3/H4. In this way, SMARCAD1 promotes methylation of H3K9, the establishment of heterochromatin, and faithful chromosome segregation. SMARCAD1 associates with transcriptional repressors including KAP1, histone deacetylases HDAC1/2 and the histone methyltransferase G9a/GLP and modulates the interaction of HDAC1 and KAP1 with heterochromatin. SMARCAD1 directly interacts with PCNA, a central component of the replication machinery, and is recruited to sites of DNA replication. Our findings suggest that chromatin remodeling by SMARCAD1 ensures that silenced loci, such as pericentric heterochromatin, are correctly perpetuated.

Göhler T, Sabbioneda S, Green CM, Lehmann AR. 2011. ATR-mediated phosphorylation of DNA polymerase η is needed for efficient recovery from UV damage. J Cell Biol, 192 (2), pp. 219-227. | Show Abstract | Read more

DNA polymerase η (polη) belongs to the Y-family of DNA polymerases and facilitates translesion synthesis past UV damage. We show that, after UV irradiation, polη becomes phosphorylated at Ser601 by the ataxia-telangiectasia mutated and Rad3-related (ATR) kinase. DNA damage-induced phosphorylation of polη depends on its physical interaction with Rad18 but is independent of PCNA monoubiquitination. It requires the ubiquitin-binding domain of polη but not its PCNA-interacting motif. ATR-dependent phosphorylation of polη is necessary to restore normal survival and postreplication repair after ultraviolet irradiation in xeroderma pigmentosum variant fibroblasts, and is involved in the checkpoint response to UV damage. Taken together, our results provide evidence for a link between DNA damage-induced checkpoint activation and translesion synthesis in mammalian cells.

Ramasubramanyan S, Coulon S, Fuchs RP, Lehmann AR, Green CM. 2010. Ubiquitin-PCNA fusion as a mimic for mono-ubiquitinated PCNA in Schizosaccharomyces pombe. DNA Repair (Amst), 9 (7), pp. 777-784. | Show Abstract | Read more

Translesion synthesis is a major mechanism with which eukaryotic cells deal with DNA damage during replication. Mono-ubiquitinated PCNA is a key regulator of this process. We have investigated whether a ubiquitin-PCNA fusion can mimic ubiquitinated PCNA, by transforming plasmids expressing this fusion protein into different mutants of Schizosaccharomyces pombe. We show that the fusion protein is able to form PCNA trimers and that it can reduce the UV sensitivity and increase translesion synthesis in mutants in which PCNA cannot be ubiquitinated (pcn1-K164R and rhp18), but not of the rad8 mutant in which PCNA can be mono-ubiquitinated but not poly-ubiquitinated. We conclude that the fusion protein is a mimic of mono-ubiquitinated PCNA but it cannot be poly-ubiquitinated. Expression of the fusion protein at levels similar to that of endogenous unmodified protein has little effect on the spontaneous mutation rate of S. pombe. Replacement of the pcn1 locus with PCNA N-terminally tagged with different epitopes resulted in lethality, probably because the tagged proteins were expressed at substantially reduced levels.

Bienko M, Green CM, Sabbioneda S, Crosetto N, Matic I, Hibbert RG, Begovic T, Niimi A, Mann M, Lehmann AR, Dikic I. 2010. Regulation of translesion synthesis DNA polymerase eta by monoubiquitination. Mol Cell, 37 (3), pp. 396-407. | Show Abstract | Read more

DNA polymerase eta is a Y family polymerase involved in translesion synthesis (TLS). Its action is initiated by simultaneous interaction between the PIP box in pol eta and PCNA and between the UBZ in pol eta and monoubiquitin attached to PCNA. Whereas monoubiquitination of PCNA is required for its interaction with pol eta during TLS, we now show that monoubiquitination of pol eta inhibits this interaction, preventing its functions in undamaged cells. Identification of monoubiquitination sites within pol eta nuclear localization signal (NLS) led to the discovery that pol eta NLS directly contacts PCNA, forming an extended pol eta-PCNA interaction surface. We name this the PCNA-interacting region (PIR) and show that its monoubiquitination is downregulated by various DNA-damaging agents. We propose that this mechanism ensures optimal availability of nonubiquitinated, TLS-competent pol eta after DNA damage. Our work shows how monoubiquitination can either positively or negatively regulate the assembly of a protein complex, depending on which substrates are targeted by ubiquitin.

Sabbioneda S, Green CM, Bienko M, Kannouche P, Dikic I, Lehmann AR. 2009. Ubiquitin-binding motif of human DNA polymerase eta is required for correct localization. Proc Natl Acad Sci U S A, 106 (8), pp. E20. | Read more

Cseresnyes Z, Schwarz U, Green CM. 2009. Analysis of replication factories in human cells by super-resolution light microscopy. BMC Cell Biol, 10 (1), pp. 88. | Show Abstract | Read more

BACKGROUND: DNA replication in human cells is performed in discrete sub-nuclear locations known as replication foci or factories. These factories form in the nucleus during S phase and are sites of DNA synthesis and high local concentrations of enzymes required for chromatin replication. Why these structures are required, and how they are organised internally has yet to be identified. It has been difficult to analyse the structure of these factories as they are small in size and thus below the resolution limit of the standard confocal microscope. We have used stimulated emission depletion (STED) microscopy, which improves on the resolving power of the confocal microscope, to probe the structure of these factories at sub-diffraction limit resolution. RESULTS: Using immunofluorescent imaging of PCNA (proliferating cell nuclear antigen) and RPA (replication protein A) we show that factories are smaller in size (approximately 150 nm diameter), and greater in number (up to 1400 in an early S- phase nucleus), than is determined by confocal imaging. The replication inhibitor hydroxyurea caused an approximately 40% reduction in number and a 30% increase in diameter of replication factories, changes that were not clearly identified by standard confocal imaging. CONCLUSIONS: These measurements for replication factory size now approach the dimensions suggested by electron microscopy. This agreement between these two methods, that use very different sample preparation and imaging conditions, suggests that we have arrived at a true measurement for the size of these structures. The number of individual factories present in a single nucleus that we measure using this system is greater than has been previously reported. This analysis therefore suggests that each replication factory contains fewer active replication forks than previously envisaged.

Bienko M, Green CM, Crosetto N, Rudolf F, Zapart G, Coull B, Kannouche P, Wider G, Peter M, Lehmann AR et al. 2005. Ubiquitin-binding domains in Y-family polymerases regulate translesion synthesis. Science, 310 (5755), pp. 1821-1824. | Show Abstract | Read more

Translesion synthesis (TLS) is the major pathway by which mammalian cells replicate across DNA lesions. Upon DNA damage, ubiquitination of proliferating cell nuclear antigen (PCNA) induces bypass of the lesion by directing the replication machinery into the TLS pathway. Yet, how this modification is recognized and interpreted in the cell remains unclear. Here we describe the identification of two ubiquitin (Ub)-binding domains (UBM and UBZ), which are evolutionarily conserved in all Y-family TLS polymerases (pols). These domains are required for binding of poleta and poliota to ubiquitin, their accumulation in replication factories, and their interaction with monoubiquitinated PCNA. Moreover, the UBZ domain of poleta is essential to efficiently restore a normal response to ultraviolet irradiation in xeroderma pigmentosum variant (XP-V) fibroblasts. Our results indicate that Ub-binding domains of Y-family polymerases play crucial regulatory roles in TLS.

Green CM, Almouzni G. 2003. Local action of the chromatin assembly factor CAF-1 at sites of nucleotide excision repair in vivo. EMBO J, 22 (19), pp. 5163-5174. | Show Abstract | Read more

DNA damage and its repair can cause both local and global rearrangements of chromatin structure. In each case, the epigenetic information contained within this structure must be maintained. Using the recently developed method for the localized UV irradiation of cells, we analysed responses that occur locally to damage sites and global events triggered by local damage recognition. We thus demonstrate that, within a single cell, the recruitment of chromatin assembly factor 1 (CAF-1) to UV-induced DNA damage is a strictly local phenomenon, restricted to damage sites. Concomitantly, proliferating cell nuclear antigen (PCNA) locates to the same sites. This localized recruitment suggests that CAF-1 participates directly in chromatin structural rearrangements that occur in the vicinity of the damage. Use of nucleotide excision repair (NER)-deficient cells shows that the NER pathway--specifically dual incision--is required for recruitment of CAF-1 and PCNA. This in vivo demonstration of the local role of CAF-1, depending directly on NER, supports the hypothesis that CAF-1 ensures the maintenance of epigenetic information by acting locally at repair sites.

A novel method to study the three dimensional organisation of the genome during DNA replication

Double strand breaks in DNA are the initiating lesion for the translocation events that underlie the genome instability that causes cancer. However, the three dimensional organisation of the genetic material within the nucleus also influences the outcome of translocations because proximity of DNA strands increases the risk of their inappropriate joining. DNA replication has a dramatic effect both on break formation and on 3D nuclear organisation, but its roles in oncogenic translocations are ...

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