IN-VITRO DETECTION OF ENDONUCLEASE IV-SPECIFIC DNA-DAMAGE FORMED BY BLEOMYCIN IN-VIVO

Endonuclease IV of Escherichia coil has been implicated by genetic stu dies in the repair of DNA damage caused by the antitumor drug bleomyci n, but the lesion(s) recognized by this enzyme in vivo have not been i dentified. We used the sensitive primer activation assay, which monito rs the formation of 3'-OH groups that support in vitro synthesis by E. coli DNA polymerase I, to determine whether endonuclease IV-specific damage could be detected in the chromosomal DNA of cells lacking the e nzyme after in vivo treatment with bleomycin. Chromosomal DNA isolated after a 1 h bleomycin treatment from wild-type, endonuclease IV-defic ient (nfo(-)) and endonuclease IV-overproducing (p-nfo; similar to 10- fold) strains all supported modest polymerase activity. However, in vi tro treatment with purified endonuclease IV activated subsequent DNA s ynthesis with samples from the nfo(-) strain (an average of 2.6-fold), to a lesser extent for samples from wild-type cells (2.1-fold), and s till less for the p-nfo samples (1.5-fold). This pattern is consistent with the presence of unrepaired damage that correlates inversely with the in vivo activity of endonuclease IV. Incubation of the DNA from b leomycin-treated nfo(-) cells with polymerase and dideoxynucleoside tr iphosphates lowered the endonuclease IV-independent priming activity, but did not affect the amount of activation seen after endonuclease IV treatment. Primer activation with DNA from the nfo(-) strain could al so be obtained with purified E. coli exonuclease III in vitro, but a q uantitative comparison demonstrated that endonuclease IV was greater t han or equal to 5-fold more active in this assay. Thus, endonuclease I V-specific damage can be detected after in vivo exposure to bleomycin. These may be 2-deoxy-pentos-4-ulose residues, but other possibilities are discussed.