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

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 (Pol beta) 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 beta-DPC in vivo. Treatment of mammalian cells with genotoxic oxidants that generate dL in DNA led to the formation of Pol beta-DPC in vivo. In a dose-dependent fashion, Pol beta-DPC were detected in MDA-MB-231 human cells treated with the antitumor drug tirapazamine (TPZ; much more Pol beta-DPC under 1% O-2 than under 21% O-2) 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 beta-DPC. Nonoxidative agents did not generate Pol beta-DPC. The cross-linking in vivo was clearly a result of the base excision DNA repair pathway: oxidative Pol beta-DPC depended on the Ape1 AP endonuclease, which generates the Pol beta lyase substrate, and they required the essential lysine-72 in the Pol beta lyase active site. Oxidative Pol beta-DPC had an unexpectedly short half-life (similar to 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 beta than to cells with the lyase-defective form. That observation underscores the genotoxic potential of oxidative Pol beta-DPC and the biological pressure to repair them.