Analysis of products formed during bleomycin-mediated DNA degradation

Murugesan, N.; Xu, Cheng; Ehrenfeld, G. M.; Sugiyama, Hiroshi; Kilkuskie, Robert E.; Rodriguez, Luis O.; Li, Chang; Hecht, Sidney M.

doi:10.1021/bi00342a008

Cited by 73 publications

(35 citation statements)

References 30 publications

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“…One possible explanation is that in the repair of ends generated by bleomycin in A-T cells, other pathways predominate over microhomology-mediated endjoining. Bleomycin induces oxidative damage and is believed to produce DSBs that resemble those induced by ionizing radiation [31]. By virtue of their chemistry (3′-phosphoglycolate and 5′-phosphate termini), such ends may be resistant to the degradation process we observed in our assays.…”

Section: Discussionmentioning

confidence: 90%

ATM mediates repression of DNA end-degradation in an ATP-dependent manner

Rahal

Henricksen

et al. 2008

DNA Repair

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Ataxia telangiectasia mutated (ATM) is a PI3-kinase-like kinase (PIKK) associated with DNA double-strand break (DSB) repair and cell cycle control. We have previously reported comparable efficiencies of DSB repair in nuclear extracts from both ATM deficient (A-T) and control (ATM+) cells; however, the repair products from the A-T nuclear extracts contained deletions encompassing longer stretches of DNA compared to controls. These deletions appeared to result from end-joining at sites of microhomology. These data suggest that ATM hinders error-prone repair pathways that depend on degradation of DNA ends at a break. Such degradation may account for the longer deletions we formerly observed in A-T cell extracts. To address this possibility we assessed the degradation of DNA duplex substrates in A-T and control nuclear extracts under DSB repair conditions. We observed a marked shift in signal intensity from full-length products to shorter products in A-T nuclear extracts, and addition of purified ATM to A-T nuclear extracts restored full-length product detection. This repression of degradation by ATM was both ATP-dependent and inhibited by the PIKK inhibitors wortmannin and caffeine. Addition of pre-phosphorylated ATM to an A-T nuclear extract in the presence of PIKK inhibitors was insufficient in repressing degradation, indicating that kinase activities are required. These results demonstrate a role for ATM in preventing the degradation of DNA ends possibly through repressing nucleases implicated in microhomology-mediated endjoining.

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Section: Discussionmentioning

confidence: 90%

ATM mediates repression of DNA end-degradation in an ATP-dependent manner

Rahal

Henricksen

et al. 2008

DNA Repair

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“…DNA strand breaks produced by either ionizing radiation or bleomycin can have phosphoglycolate termini as the cleavage site 3'-end group. For DNA treated under aqueous aerobic conditions in vitro, with either ionizing radiation (2) or bleomycin (3,4), 3 '-phosphoglycolates (3'-PG) are present at approximately 50 or 100 percent, respectively, of the strand break sites. The 5'-end group of cleavage for both agents is a phosphate (5'-P).…”

Section: Introductionmentioning

confidence: 99%

Human HeLa cell enzymes that remove phosphoglycolate 3′-end groups from DNA

Winters¹,

Weinfeld²,

Jorgensen³

1992

Nucl Acids Res

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We have purified three chromatographically distinct human enzyme activities from HeLa cells, that are capable of converting bleomycin-treated DNA into a substrate for E. coli DNA polymerase I. The bleomycin-treated DNA substrate used in this study has been characterized via a 32P-postlabeling assay and shown to contain strand breaks with 3'-phosphoglycolate termini as greater than 95% of the detectable dose-dependent lesions. The purified HeLa cell enzymes were shown to be capable of removing 3'-phosphoglycolates from this substrate. Also 3'-phosphoglycolate removal and nucleotide incorporation were enzyme dependent. In addition, all three Hela cell enzymes have been determined to possess Class II AP endonuclease activity. The enzymes lack 3'----5' exonuclease activity and are, therefore, dissimilar to exonuclease III--an E. coli enzyme that can remove 3'-phosphoglycolate.

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“…In general, however, the structure of the resulting oligomers has not been verified but rather inferred from earlier work. Moreover, even the studies identifying the 3Ј-PG terminus of bleomycin-induced breaks showed only that the PG end was a major product, not that it accounted for all of or even a majority of the fragments formed (10,11).…”

Section: Discussionmentioning

confidence: 99%

“…In most cases, however, the structure of the substrates was not chemically verified. Rather, identification was based on inference from their electrophoretic mobility, and on previous studies demonstrating that 3Ј-PG-terminated breaks are predominant products of bleomycin-mediated cleavage (10,11). Moreover, none of the earlier studies provided any estimate of the degree to which the putative 3Ј-PG fragments might have been contaminated with other cleavage products.…”

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confidence: 99%