Identification of human satellite DNA sequences associated with chemically resistant nonhistone polypeptide adducts.
Pfütz M., Gileadi O., Werner D.
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.