PREFERENTIAL LOCALIZATION OF DNA-DAMAGE INDUCED BY DEPURINATION AND BLEOMYCIN IN A PLASMID CONTAINING A SCAFFOLD-ASSOCIATED REGION

Recent evidence suggests that DNA damage of various origins is not ran domly distributed in the genome but appears to be clustered in unident ified hypersensitive regions of the chromatin. A model was proposed th at stipulates that unpaired DNA stretches, such as those round in scaf fold- (or matrix)-associated regions (SARs) under torsional strain, ar e candidate regions of hypersensitivity to DNA damage in vivo. In this study, we assessed in vitro the relative susceptibility of supercoile d plasmids containing a SAR or chromatin loop DNA segment to DNA damag e induced by acid-catalyzed depurination or Fe-III-bleomycin. Single-s trand specific S1 nuclease was used in combination with 3'-end-labelin g to detect single-strand breaks or gaps, after cleavage of abasic sit es or removal of 3'-phosphoglycolates by Escherichia coli endonuclease IV. The optimal conditions of DNA cleavage specificity by S1 nuclease were determined. Using these conditions, the DNA cleavage patterns ob tained showed (i) a preferential localization of S1 hypersensitive sit es in the SAR DNA as compared with plasmid or chromatin loop DNA and ( ii) a strikingly similar localization of DNA damage with the two clast ogenic treatments.