DNA-protein interactions and chromosome banding

Abstract

Pancreatic DNase I was used as a probe to study DNA-protein interactions in condensed and extended chromatin fractions isolated from Chinese hamster liver, and in human lymphocyte and mouse L cell metaphase chromosomes in situ. By studying the rate of digestion of chromatin DNA by DNase, we have previously shown that DNA in extended chromatin is more sensitive to DNase digestion than that in condensed chromatin. In the current investigation, we have examined whether this differential sensitivity of the chromatin fractions to DNase is due to differences in protein binding to DNA or differences in the degree of chromatin condensation. By “decondensing” the condensed chromatin and comparing its rate of digestion to that of untreated condensed and extended chromatin, it was found that differences in the degree of binding of proteins to DNA rather than the degree of condensation of the chromatin primarily determines the sensitivity of each fraction to DNase. Extraction of the various classes of chromosomal proteins, followed by DNase digestion of the residual chromatin revealed that both the histone and non-histone proteins protect the DNA in the chromatin fractions from DNase attack; however, the more tightly associated non-histones appear to be specifically responsible for the differential sensitivity of the chromatin fractions to DNase digestion. These non-histones may be more tightly associated with the DNA in condensed than in extended chromatin, thereby protecting the DNA in condensed chromatin against DNase attack to a greater extent than that in extended chromatin. When metaphase chromosomes were briefly digested with DNase in situ and subsequently stained with Feulgen reagent, incontrovertible C-banding and some G-banding was obtained. This DNaseinduced banding demonstrates that the DNA in C-band and possibly G-band regions is less accessible to DNase than that in the interband regions, and our biochemical data suggest that this differential accessibility is caused by differential DNA-protein binding such that the non-histones are more tightly coupled to the DNA in the G- and C-band regions than they are in the interbands. Differences in the binding of non-histones to DNA in different segments of the metaphase chromosome may be involved in the mechanism of G- and C-banding.