Failure of the mammalian cellular defense system, to promptly revert the critical genomic alterations of toxicant exposures, is causal to many deleterious biological consequences. During the previous grant period, it was shown that mammalian cells display unique selectivity of DNA damage processing at different levels of genomic organization. This continuation grant will extend the scope of these studies along a similar overall theme. The proposal is based on the premise that fine analysis of damage processing, as opposed to the customary studies of evaluating DNA damage and repair heterogeneity as an average of the potential component events, will reveal hitherto unknown mechanistic insights that will considerably reshape and advance our knowledge of genetic toxicology. The specific hypotheses addressed are: (i) diverse genotoxins target individual nucleotide positions with different affinity and the capacity and efficiency of cellular repair complex is dictated by lesion flanking sequence and target strand, (ii) presence or absence of certain forms of key regulatory gene products determine the repair efficiency for different DNA strands and gene sites, and (iii) susceptible mutations result from the failure of repair of certain damage subtypes and in some refractory sequence contexts.
The specific aims will focus on: (1) delineating the influence of wild type, mutant and absence of p53 protein on the global, strand-specific and context-sensitive nucleotide specific repair, (2) resolving the role of p53 protein in facilitating the in vivo repair of specific lesions within defined exonic sites of targeted genes, (3) ascertaining the mechanism of repair modulation through the interaction of upstream p53 effectors, and (4) identifying potential contribution of p53 responsive downstream elements on the modulation of specific DNA repair events. The studies will concentrate on sequence modifications induced by a representative physical (UV radiation) and a chemical (anti-BPDE) genotoxic carcinogen. The base specific in vivo mapping of frequency of damage induction and repair events, in the genome of cells of known regulatory protein status, will be performed by the sensitive ligase mediated PCR of select exonic p53 and hprt gene sequences. Discerning the inherent factors of genomic instability has important implications to risk assessment and prevention of biological consequences due to environmental genotoxic human exposures.
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