In vitro detection of endonuclease IVspecific DNA damage formed by bleomycin in vivo. Nucleic Acids Res

Endonuclease IV of Escherichia coli has been impli-cated by genetic studies in the repair of DNA damage caused by the antitumor drug bleomycin, but the lesion(s) recognized by this enzyme in vivo have not been identified. We used the sensitive primer activation assay, which monitors the formation of 3′-OH groups that support in vitro synthesis by E.coli DNA poly-merase I, to determine whether endonuclease IV-specific damage could be detected in the chromosomal DNA of cells lacking the enzyme after in vivo treatment with bleomycin. Chromosomal DNA isolated after a 1 h bleomycin treatment from wild-type, endonuclease IV-deficient (nfo–) and endonuclease IV-overproducing (p-nfo; ∼10-fold) strains all supported modest poly-merase activity. However, in vitro treatment with purified endonuclease IV activated subsequent DNA synthesis 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 still 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 bleomycin-treated nfo – cells with polymerase and dideoxynucleoside triphosphates lowered the endonuclease IV-independent priming activity, but did not affect the amount of activation seen after endo-nuclease IV treatment. Primer activation with DNA from the nfo – strain could also be obtained with purified E.coli exonuclease III in vitro, but a quantitative comparison demonstrated that endonuclease IV was 5-fold more active in this assay. Thus, endonuclease IV-specific damage can be detected after in vivo expo-sure to bleomycin. These may be 2-deoxy-pentos-4-ulose residues, but other possibilities are discussed