Structural changes in simian virus 40 chromatin as probed by restriction endonucleases

Publisher Website
Google Scholar
Abstract
The structure of SV-40 chromatin was probed by treatment with single- and multiple-site bacterial restriction endonucleases. Approximately the same fraction of the chromatin DNA was cleaved by each of 3 different single-site endonucleases, indicating that the nucleosomes do not have unique positions regarding specific nucleotide sequences within the population of chromatin molecules. The extent of digestion was strongly influenced by salt concentration. At 100 mM NaCl-5 mM MgCl2, only about 20% of the SV-40 DNA I in chromatin was converted to linear SV-40 DNA III. At lower concentrations of NaCl (0.05 or 0.01 M), an additional 20-30% of the DNA was cleaved. At 100 mM NaCl only the DNA between nucleosomes apparently was accessible to the restriction enzymes, whereas at the lower salt concentrations, DNA within the nucleosome regions apparently became available for cleavage. When SV-40 chromatin was digested with multiple-site restriction enzymes, less than 2% of the DNA was digested to limit digest fragments, whereas only a small fraction (9-15%) received 2 or more cuts. The principal digest fragment was full-length linear SV-40 DNA III. The failure to generate limit digest fragments was not a consequence of reduced enzyme activity in the reaction mixtures or of histone exchange. When the position of the principal cleavage site was mapped after Hpa-I digestion, this site was not unique. All sites were not cleaved with equal probability. SV-40 chromatin containing nicked-circular DNA II produced by random nicking of DNA I was resistant to digestion by restriction enzymes. The initial cut which causes relaxation of topological constraint in SV-40 chromatin DNA may impart resistance to further digestion by restriction enzymes. This may be accomplished by winding of the internucleosomal DNA into the body of the nucleosome or by successive right-hand rotation of nucleosomes.