The maintenance of genomic stability in eukaryotes is relentlessly challenged by DNA damage. In order to understand the basis of damage tolerance and avoidance of deleterious effects, it is important to grasp not only the nature of such DNA damage and its processing pathways in cells, but also to have a clear understanding of how cells manage the lesion repair in the highly compact and tightly regulated chromatin milieu of the eukaryotic nucleus. This proposal is based on the premise that the specific biochemical pathways, relying on wide-ranging inter-molecular cross-talk between the pathway components, operate at a higher efficiency under conditions of macromolecular crowding and sequentially recruit specific factors for access a s well as repair of genotoxic lesions within mammalian genome. The proposal builds on our recent observations that support a strong link between DNA repair and p53 mediated and other related regulatory pathways. The overall experiments are designed to address the specific hypothesis that cellular DNA damage recognition and transcription factors, congregate at DNA lesions sites, open the chromatin via histone acetylation and initiate the key pre-incision events of nucleotide excision repair (NER). The proposed work will utilize the PI's established expertise in relevant, cellular, molecular, biochemical, and immunological technologies to undertake the following specific aims. (1) To assess the nature and extent of contribution of damaged DNA binding protein, DDB, in NER through quantitative assessment of NER sub-pathways in human cells of defined protein status. (2) To demonstrate the role of DDB for sequential recruitment of initial recognition and downstream core NER factors through spatial and temporal co-localization in situ. (3) To determine the participation of histone acetyl transferases in damage recognition and excision via qualitative protein cataloging and targeted recruitment and physical association of interacting factors. (4) To establish the histones acetylation as a direct and key consequence of DNA damage by placement of acetylation events exclusively at the lesion sites in vivo and in vitro. These studies will provide important insights regarding the dynamics of DNA damage recognition and processing through identification of cross-talking events and critical factors that influence the maintenance of genomic stability so important to overcome the hazardous health effects of genotoxic environmental exposures.
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