RESUMEN
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 ß (Polß) 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 Polß-DPC in vivo. Treatment of mammalian cells with genotoxic oxidants that generate dL in DNA led to the formation of Polß-DPC in vivo. In a dose-dependent fashion, Polß-DPC were detected in MDA-MB-231 human cells treated with the antitumor drug tirapazamine (TPZ; much more Polß-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 Polß-DPC. Nonoxidative agents did not generate Polß-DPC. The cross-linking in vivo was clearly a result of the base excision DNA repair pathway: oxidative Polß-DPC depended on the Ape1 AP endonuclease, which generates the Polß lyase substrate, and they required the essential lysine-72 in the Polß lyase active site. Oxidative Polß-DPC had an unexpectedly short half-life (â¼ 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 Polß than to cells with the lyase-defective form. That observation underscores the genotoxic potential of oxidative Polß-DPC and the biological pressure to repair them.