Exposure of cellular DNA, to endogenous and exogenous genotoxins, produce critical genomic alterations. Highly competent defense mechanisms counteract the hazardous consequences of DNA damage by rapid repair and restoration of the primary genetic information. Recent studies indicate that DNA repair in mammalian cells displays a degree of selectivity. Thus, the differential repair among genes and between separate domains of the same gene need an in-depth fine analysis. This proposal is based on the hypothesis that the heterogenous damage processing, at subgenomic and nucleotide levels, is dependent upon the nature of DNA modification and the specificity and complexity of repair system.
The aim i s to characterize the selective processing of diverse DNA modifications in individually targeted domains of the genes relevant to carcinogenesis. The influence of DNA repair deficiency and host cell specific effects on damage processing will be determined in well characterized human chromosomal sequences. The studies will focus on premutagenic and procarcinogenic modifications that provoke base mispairing and/or major helical distortions. Damage specific antibodies and restriction endonuclease recognition site protection will be used, along with a newly established sensitive and quantitative amplification system for damage identification and repair assessment in target sequences. The patterns of damage induction and repair will be fine mapped in the individual exonic, intronic, upstream and downstream coding regions of the ras gene. The sequence alterations induced by different modifications, under various host repair backgrounds, will be determined at exactly the same sites that are used for the DNA damage analysis. Once established, this novel approach will be extended to divergent DNA lesions, sequences and sites of mammalian genome. The data will provide new insights regarding the target preferences of mammalian repair systems as well as the role of repair deficiency in mutational predisposition of unrepaired sequences. Discerning this relationship has important implications to our understanding of molecular mechanisms of mutagenesis and carcinogenesis and its application to risk assessment and prevention of biological consequences due to environmental genotoxic human exposures.
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