Biochemical Evaluation of Genotoxic Biomarkers for 2-Deoxyribonolactone-Mediated Cross-Link Formation with Histones

Abstract

Numerous environmental carcinogens involve radical formation interacting with DNA to produce 2-deoxyribonolactone (dL), a major type of oxidized abasic site, implicated in DNA strand breaks, mutagenesis, and formation of covalent DNA–protein cross-links (DPC). Studies showed major dL-specific DPC occurred due to reactions with DNA polymerase β (Polβ) dependent on native conformation, while other DPC formed involved nonenzymatic reactions of DNA binding proteins with dL lesions. Polβ appeared to play a major role in alleviating the cytotoxic effects of neocarzinostatin, which was used as a dL-producing agent. When a duplex DNA containing a dL at a site-specific position was incubated with purified histones, DPC were formed between dL and each histone protein, including H1, H2A, H2B, H3, and H4. Comparative kinetic analysis of DPC formation with histones and Polβ revealed two distinct mechanisms of dL-mediated DPC formation. The rate of DPC formation with Polβ was approximately two orders of magnitude higher than that with various histone proteins. These results indicate that catalytic activity of Polβ mediates rapid DPC formation between dL and this DNA repair enzyme, whereas nonenzymatic reactions of dL with histones form DPC more slowly. The abundance of histones and their constant interaction with DNA may nevertheless yield significant levels of DPC with dL, as biomarkers of dL-induced cytotoxicity. Overall, data suggest that occurrence of dL-mediated DPC with histones may contribute to the genotoxic effects of dL in DNA.

This work was supported by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD, Basic Research Promotion Fund) (KRF-2006-331-E00031). B. D. is supported in part by U.S. National Institutes of Health (NIH) grant GM40000.