Formation and Fate of Oxidative DNA-Protein Crosslinks

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DESCRIPTION (provided by applicant): About 1.6 million new cancer cases were reported for 2012 in the U.S.A, of which about one-third were expected to result in death due to complications in treating the disease. The complexity and aggressiveness of cancers depends on the accumulation of mutations. There is a growing body of literature linking genomic instability to carcinogenesis, with much focus on oxidative damage. The genome is constantly under assault by a variety of endogenous and exogenous insults, most notably those that generate reactive oxygen species (ROS). Along with other lesions, exposure to these agents produces oxidized abasic sites. One such example, 2-deoxyribonolactone (dL), which results from C1' oxidation, can instigate the formation of DNA-protein crosslinks (DPCs) with repair enzymes and other proteins. The principal repair mechanism for oxidative DNA damage is the base excision DNA repair pathway (BER). During attempted BER of dL by, DNA polymerase ß (Polß), the enzyme's 5'-deoxyribose-5-phosphate (5'dRp) lyase activity attempts to excise the lesion but becomes trapped leading to DPC formation. Other DNA lyase enzymes (some DNA glycosylases, DNA polymerases γ and λ, and the Ku proteins) also become covalently trapped by dL residues. However, it remains unknown how cells may repair these DPCs. This proposal seeks to understand the induction and removal kinetics of dL-mediated DPCs, and to uncover whether these lesions are actively repaired in mammalian cells. The goals of Aim 1 propose to develop or adapt a sensitive biochemical assay which will enable the study of dL-Polß DPCs in vivo. Aim 2 will identify the conditions required to form Polß DPCs in cells using dL-inducing agents, and their removal kinetics from the genome will be followed. Finally, Aim 3 will investigate whether dL-Polß DPCs are actively removed by repair processes involving homologous recombination, nucleotide excision repair or some combination that may involve proteolysis as a prerequisite for repair. I anticipate that the findings of these studies will provde new information on the contribution of DPCs to DNA damage and open new avenues of investigation to understand their relationship to human carcinogenesis.