Nucleic Acids Research, Vol 24, Issue 5 885-889, Copyright © 1996 by Oxford University Press
JD Levin and B Demple
Endonuclease IV of Escherichia coli has been implicated 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 polymerase 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; approximately 10-fold) strains all supported
modest polymerase 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 endonuclease 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
> or = 5-fold more active in this assay. Thus, endonuclease IV-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.
ARTICLES
In vitro detection of endonuclease IV-specific DNA damage formed by bleomycin in vivo
Department of Molecular and Cellular Toxicology, Harvard School of Public Health, Boston, MA 02115, USA.
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