Structural and Biochemical Investigation of the Molecular Mechanisms of DNA Response and Repair in Humans and \u3cem\u3eEscherichia coli\u3c/em\u3e.

The genomes of all living cells are under constant attack from both endogenous and exogenous agents that damage DNA. In order to maintain genetic integrity a variety of response pathways have evolved to recognize and eliminate DNA damage. Replication protein A (RPA), the eukaryotic single-stranded DNA (ssDNA) binding protein, is a required factor for all major DNA metabolisms. Although much work has been done to elucidate the nature of the interaction between RPA and ssDNA currently there is no structural information on how the full-length protein binds to ssDNA. This study presents a novel examination of the full nucleoprotein complex formed between RPA and ssDNA. We identified three previously unknown contacts between ssDNA and lysine residues in DNA binding domain C located in the p70 subunit. This represents the first single amino-acid resolution determination of how full-length RPA contacts ssDNA. The Ataxia-Telangiectasia Mutated and RAD3-Related (ATR) mediated DNA damage checkpoint and nucleotide excision repair (NER) pathway are primarily responsible for repair of UV-C-induced photolesions in DNA. However, it is unclear how these two pathways are coordinated. We found the ATR-dependent checkpoint induces a rapid nuclear accumulation of the required NER factor Xeroderma pigmentosum group A (XPA) in both a dose- and time-dependent fashion. Also, using surface topology mapping we have defined an α-helix motif on XPA required for XPA-ATR complex formation necessary for XPA phosphorylation. In addition, we have determined that XPA phosphorylation promotes repair of persistent DNA lesions, such as cyclobutane pyrimidine dimers. The basis for initial damage recognition in NER is structural distortion of duplex DNA; however, the effects of adduct structure and sequence on strand opening and recognition are unclear. Using the E. coli NER system we determined that the identity of the adduct dictates the size of the strand opening generated by the UvrA2B complex. In addition we found that the sequence immediately surrounding the damaged nucleotide affects damage recognition by influencing the amount of helical distortion induced by the adduct. These effects are a result of the equilibrium conformation the adduct adopts in addition to the amount of hydrogen bonding available to maintain the structure