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Enzyme mechanism-based, oxidative DNA-protein cross-links formed with DNA polymerase ? in vivo.

ABSTRACT: Free radical attack on the C1' position of DNA deoxyribose generates the oxidized abasic (AP) site 2-deoxyribonolactone (dL). Upon encountering dL, AP lyase enzymes such as DNA polymerase ? (Pol?) form dead-end, covalent intermediates in vitro during attempted DNA repair. However, the conditions that lead to the in vivo formation of such DNA-protein cross-links (DPC), and their impact on cellular functions, have remained unknown. We adapted an immuno-slot blot approach to detect oxidative Pol?-DPC in vivo. Treatment of mammalian cells with genotoxic oxidants that generate dL in DNA led to the formation of Pol?-DPC in vivo. In a dose-dependent fashion, Pol?-DPC were detected in MDA-MB-231 human cells treated with the antitumor drug tirapazamine (TPZ; much more Pol?-DPC under 1% O2 than under 21% O2) and even more robustly with the "chemical nuclease" 1,10-copper-ortho-phenanthroline, Cu(OP)2. Mouse embryonic fibroblasts challenged with TPZ or Cu(OP)2 also incurred Pol?-DPC. Nonoxidative agents did not generate Pol?-DPC. The cross-linking in vivo was clearly a result of the base excision DNA repair pathway: oxidative Pol?-DPC depended on the Ape1 AP endonuclease, which generates the Pol? lyase substrate, and they required the essential lysine-72 in the Pol? lyase active site. Oxidative Pol?-DPC had an unexpectedly short half-life (? 30 min) in both human and mouse cells, and their removal was dependent on the proteasome. Proteasome inhibition under Cu(OP)2 treatment was significantly more cytotoxic to cells expressing wild-type Pol? than to cells with the lyase-defective form. That observation underscores the genotoxic potential of oxidative Pol?-DPC and the biological pressure to repair them.

SUBMITTER: Quinones JL

PROVIDER: S-EPMC4507217 | biostudies-literature | 2015 Jul

REPOSITORIES: biostudies-literature

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Enzyme mechanism-based, oxidative DNA-protein cross-links formed with DNA polymerase β in vivo.

Quiñones Jason L JL Thapar Upasna U Yu Kefei K Fang Qingming Q Sobol Robert William RW Demple Bruce B

Proceedings of the National Academy of Sciences of the United States of America 20150629 28

Free radical attack on the C1' position of DNA deoxyribose generates the oxidized abasic (AP) site 2-deoxyribonolactone (dL). Upon encountering dL, AP lyase enzymes such as DNA polymerase β (Polβ) form dead-end, covalent intermediates in vitro during attempted DNA repair. However, the conditions that lead to the in vivo formation of such DNA-protein cross-links (DPC), and their impact on cellular functions, have remained unknown. We adapted an immuno-slot blot approach to detect oxidative Polβ-D ...[more]

PMID: 26124145

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Project description:Free radical attack on C1' of deoxyribose forms the oxidized abasic (AP) site 2-deoxyribonolactone (dL). In vitro, dL traps the major base excision DNA repair enzyme DNA polymerase beta (Pol?) in covalent DNA-protein crosslinks (DPC) via the enzyme's N-terminal lyase activity acting on 5'-deoxyribose-5-phosphate residues. We previously demonstrated formation of Pol?-DPC in cells challenged with oxidants generating significant levels of dL. Proteasome inhibition under 1,10-copper-ortho-phenanthroline (CuOP) treatment significantly increased Pol?-DPC accumulation and trapped ubiquitin in the DPC, with Pol? accounting for 60-70 % of the total ubiquitin signal. However, the identity of the remaining oxidative ubiquityl-DPC remained unknown. In this report, we surveyed whether additional AP lyases are trapped in oxidative DPC in mammalian cells in culture. Poly(ADP-ribose) polymerase 1 (PARP1), Ku proteins, DNA polymerase ? (Pol?), and the bifunctional 8-oxoguanine DNA glycosylase 1 (OGG1), were all trapped in oxidative DPC in mammalian cells. We also observed significant trapping of Pol?, PARP1, and OGG1 in cells treated with the alkylating agent methylmethane sulfonate (MMS), in addition to dL-inducing agents. Ku proteins, in contrast, followed a pattern of trapping similar to that for Pol?: MMS failed to produce Ku-DPC, while treatment with CuOP or (less effectively) H2O2 gave rise to significant Ku-DPC. Unexpectedly, NEIL1 and NEIL3 were trapped following H2O2 treatment, but not detectably in cells exposed to CuOP. The half-life of all the AP lyase-DPC ranged from 15-60 min, consistent with their active repair. Accordingly, CuOP treatment under proteasome inhibition significantly increased the observed levels of DPC in cultured mammalian cells containing PARP1, Ku protein, Pol?, and OGG1 proteins. As seen for Pol?, blocking the proteasome led to the accumulation of DPC containing ubiquitin. Thus, the ubiquitin-dependent proteolytic mechanisms that control Pol?-DPC removal may also apply to a broad array of oxidative AP lyase-DPC, preventing their toxic accumulation in cells.

Dataset Information

Enzyme mechanism-based, oxidative DNA-protein cross-links formed with DNA polymerase ? in vivo.

Publications

Enzyme mechanism-based, oxidative DNA-protein cross-links formed with DNA polymerase β in vivo.

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