Formation of DNA-protein Cross-links Mediated by C1'-oxidized Abasic Lesion in Mouse Embryonic Fibroblast Cell-free Extracts

摘要

Oxidized abasic residues arise as a major class of DNA damage by a variety of agents involving free radical attack and oxidation of deoxyribose sugar components. 2-deoxyribonolactone (dL) is a C1'-oxidized abasic lesion implicated in DNA strand scission, mutagenesis, and covalent DNA-protein cross-link (DPC). We show here that mammalian cell-free extract give rise to stable DPC formation that is specifically mediated by dL residue. When a duplex DNA containing dL at the site-specific position was incubated with cell-free extracts of Pol p-proficfent and -deficient mouse embryonic fibroblast cells, the formation of major dL-mediated DPC was dependent on the presence of DNA polymerase (Pol) β. Formation of dL-specific DPC was also observed with histones and FEN1 nuclease, although the reactivity in forming dL-mediated DPC was significantly higher with Pol (3 than with histones or FEN1. DNA repair assay with a defined DPC revealed that the dL lesion once cross-linked with Pol p was resistant to nucleotide excision repair activity of cell-free extract. Analysis of nucleotide excision repair utilizing a model DNA substrate containing a (6-4) photoproduct suggested that excision process for DPC was inhibited because of DNA single-strand incision at 5'of the lesion. Consequently DPC mediated by dL lesion may not be readily repaired by DNA excision repair pathway but instead function as unusual DNA damage causing a prolonged DNA strand break and trapping of the major base excision repair enzyme.