title: Enzyme mechanism-based, oxidative DNA-protein cross-links formed with DNA polymerase β 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 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-DPC in vivo. Treatment of mammalian cells with genotoxic oxidants that generate dL in DNA led to the formation of Polβ-DPC in vivo. In a dose-dependent fashion, Polβ-DPC were detected in MDA-MB-231 human cells treated with the antitumor drug tirapazamine (TPZ; much more Polβ-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 Polβ-DPC. Nonoxidative agents did not generate Polβ-DPC. The cross-linking in vivo was clearly a result of the base excision DNA repair pathway: oxidative Polβ-DPC depended on the Ape1 AP endonuclease, which generates the Polβ lyase substrate, and they required the essential lysine-72 in the Polβ lyase active site. Oxidative Polβ-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 Polβ than to cells with the lyase-defective form. That observation underscores the genotoxic potential of oxidative Polβ-DPC and the biological pressure to repair them.
authors.affiliation: Department of Pharmacological Sciences, Stony Brook University School of Medicine, Stony Brook, NY 11794;
authors.forename: Jason L
authors.initials: JL
authors.lastname: Quiñones
authors.affiliation: Department of Pharmacological Sciences, Stony Brook University School of Medicine, Stony Brook, NY 11794;
authors.forename: Upasna
authors.initials: U
authors.lastname: Thapar
authors.affiliation: Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824;
authors.forename: Kefei
authors.initials: K
authors.lastname: Yu
authors.affiliation: Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213; Hillman Cancer Center, University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213.
authors.forename: Qingming
authors.initials: Q
authors.lastname: Fang
authors.affiliation: Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213; Hillman Cancer Center, University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213.
authors.forename: Robert William
authors.identifier: http://orcid.org/0000-0001-7385-3563
authors.initials: RW
authors.lastname: Sobol
authors.affiliation: Department of Pharmacological Sciences, Stony Brook University School of Medicine, Stony Brook, NY 11794; bruce.demple@stonybrook.edu.
authors.forename: Bruce
authors.initials: B
authors.lastname: Demple
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D004247:DNA / Q000378:metabolism*; D004249:DNA Damage; D019951:DNA Polymerase beta / Q000378:metabolism*; D006801:Humans; D051379:Mice; D010084:Oxidation-Reduction
chemical_list: D004247:DNA; D019951:DNA Polymerase beta
keywords: 2-deoxyribonolactone; AP lyase; abasic site; base excision repair; free radical damage