Genomic integrity is essential for cell survival and is maintained by DNA repair mechanisms in cells. It is thought that up to 90 percent of human cancer results from DNA damage. Nucleotide excision repair (NER) is a major repair pathway to remove DNA damage resulting from exposure to UV irradiation from the sun and environmental carcinogens. NER is the only known human repair system for removing bulky lesions that are caused by the types of damage mentioned above. Although much work has been done to define the mechanism of NER in humans, very little is known about the first and rate-limiting step, DNA damage recognition. Proteins known to play important roles in the recognition process include XPA, RPA, and XPC-hHR23B. It is has been proposed that these molecules recognize damage by binding helical distortions in the DNA, thereby initiating the repair process. The long-term objective of this application is to determine the molecular mechanisms of DNA damage recognition and repair of genome-destabilizing DNA structures by human NER. The mutagenic potential of DNA helical distortions induced by unusual DNA structures formed at repeat sequences, the biochemical details of the protein-nucleic acid interactions in human NER, and the role of helical distortions in DNA damage recognition will be ascertained by addressing the following questions: Are unusual DNA structures recognized as damage by the DNA repair machinery? Are structural alterations in the DNA helix recognized by the repair proteins? Which of the putative damage recognition complexes act first at the site of DNA damage and what is the role of each in damage recognition? Is there a correlation between the extent of helical distortion induced by a lesion and the efficiency with which it is recognized and repaired, and do these properties determine the mutagenic capacity? Specifically, we propose to determine the ability of unusual DNA structures with helical distortions and complex lesions directed by triplex-forming oligonucleotides to induce genomic instability and mutagenesis, the role of NER in the processing of these genome-destabilizing helical distortions, and the molecular details of damage recognition by human XPA-RPA and XPC-hHR23B damage recognition complexes.
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