INDUCTION OF DNA-DAMAGE IN GAMMA-IRRADIATED NUCLEI STRIPPED OF NUCLEAR-PROTEIN CLASSES - DIFFERENTIAL MODULATION OF DOUBLE-STRAND BREAK ANDDNA-PROTEIN CROSS-LINK FORMATION

The influence of chromatin proteins on the induction of DNA double-str and breaks (dsb) and DNA-protein crosslinks (dpc) by gamma-radiation w as investigated. Low molecular weight non-histone proteins and classes of histones were extracted with increasing concentrations of NaCl, wh ereas nuclear matrix proteins were not extractable even by 2.0 M NaCl. The yield of dsb increased with progressive removal of proteins from chromatin. Whilst removal of low molecular weight nonhistone proteins and histone H1 resulted in small increases in the production of dsb, r emoval of histones H2A/H2B, all histones, or all proteins led to 18.4, 46.4 and 55.5-fold increases in the yield of dsb, respectively, relat ive to irradiated cells. Therefore, both histones and non-histone prot eins contribute to the radioprotection of DNA, core histones being the major radioprotectors. In contrast, depletion of chromatin proteins c aused little or no effect on the induction of dpc until the chromatin was extracted with greater than or equal to 1.4 M NaCl. However, our s tudies indicated no direct, quantitative correlation between the remov al of histones and the induction of dpc. The data support our previous conclusion that nuclear matrix protein rather than the majority of th e histones are the predominant substrates for dpc production, although the involvement of a subset of tightly bound histones (H3 and H4) has not been excluded. This finding demonstrates that chromatin proteins can differentially modify the yield of two types of radiation-induced DNA lesions.