Genotoxic stress, integral to an organisms life, is clearly implicated in many hazardous biological effects. However, mammalian cells have evolved intricate defense systems that orchestrate a cascade of cellular responses to genomic injury. During the previous grant period, the investigator has developed several sensitive, specific and unique approaches to monitor the induction and repair of genotoxic lesions within discrete DNA fragments. This continuation grant builds further and extends the scope of this work along a similar overall theme. The proposal is based on the premise that the origin, nature, and spatial distribution of genotoxic lesions in conjunction with the characteristic processing heterogeneity, specificity and competence of host repair system determine the cell's fate through a hierarchical order of downstream effectors controlling recovery. The specific hypotheses addressed are: (i) genomic stability and cellular recovery directly depend upon the extent of specific damage and corresponding repair mechanisms, (ii) the failure of repair of deleterious lesions at key gene sequences engenders crucial hotspot mutations and (iii) the p53 gene product actively participates in overall, gene- and site-specific repair.
The specific aims will focus on: (1) demonstrating that inherent repair heterogeneity, resulting in selective elimination of critical genotoxic damage from vital gene sequences, is adduct and target specific and occurs in a manner compatible with normal cell survival, (2) delineating the relationship of the spectrum and strand bias of induced mutations with the predominant persistent genotoxic lesions and strand specificity of repair at cognate genomic sites and (3) understanding the potential modulatory role of the p53 gene product in repair stimulation, mutation avoidance and elimination of mutant cell precursors by apoptosis. One representative subtle miscoding (O4-alkylthymine) and one bulky (anti-BPDE-guanine) adduct will be analyzed in human ras, p53 and factor IX gene targets. The p53 mediated post-damage cellular responses will be studied concomitantly with damage processing ability and mutagenesis in phenotypically and genotypically defined human cells. The integrated data of these systematic studies will provide crucial mechanistic insights pertinent to mutagenesis, oncogenesis and risk assessment of environmental hazards confronting human health.
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