| First Author | Quiñones JL | Year | 2015 |
| Journal | Proc Natl Acad Sci U S A | Volume | 112 |
| Issue | 28 | Pages | 8602-7 |
| PubMed ID | 26124145 | Mgi Jnum | J:223801 |
| Mgi Id | MGI:5660425 | Doi | 10.1073/pnas.1501101112 |
| Citation | Quinones JL, et al. (2015) Enzyme mechanism-based, oxidative DNA-protein cross-links formed with DNA polymerase beta in vivo. Proc Natl Acad Sci U S A 112(28):8602-7 |
| abstractText | 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 beta (Polbeta) 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 Polbeta-DPC in vivo. Treatment of mammalian cells with genotoxic oxidants that generate dL in DNA led to the formation of Polbeta-DPC in vivo. In a dose-dependent fashion, Polbeta-DPC were detected in MDA-MB-231 human cells treated with the antitumor drug tirapazamine (TPZ; much more Polbeta-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 Polbeta-DPC. Nonoxidative agents did not generate Polbeta-DPC. The cross-linking in vivo was clearly a result of the base excision DNA repair pathway: oxidative Polbeta-DPC depended on the Ape1 AP endonuclease, which generates the Polbeta lyase substrate, and they required the essential lysine-72 in the Polbeta lyase active site. Oxidative Polbeta-DPC had an unexpectedly short half-life ( approximately 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 Polbeta than to cells with the lyase-defective form. That observation underscores the genotoxic potential of oxidative Polbeta-DPC and the biological pressure to repair them. |
