The broad objective of this proposal is to understand the molecular details of an important defense mechanism (DNA excision repair) against mutations and phenotypic changes in human cells. Such changes are important etiological factors in cell survival and cancer. Bulky DNA lesions result from a plethora of environmental agents, such as ultraviolet (UV) radiation and chemical carcinogens, and repair of these lesions occurs by nucleotide excision repair (NER). As the same repair process is used for removing the majority of these bulky lesions, UV radiation will be used as a prototype environmental agent for these studies. We will study histone modifications during NER at damaged DNA sites in human chromatin by tagging nascent and mature repair sites with nucleotide analogues for isolating these regions as protein-DNA complexes, by chromatin immunoprecipitation (ChIP) with specific modified histone antibodies, and by examining the effect of histone deacetylase inhibition on NER and maturation of nascent repair sites in human cells. Secondly, we will examine NER of single UV damaged sites strategically located in a transcription factor complex by analyzing the effect of single UV photoproducts (CPDs) at strategic sites on complex formation, by examining NER of CPDs at strategic sites in this complex, and by examining displacement of the transcription factor during NER. Thirdly, we will examine NER at UV damaged sites in oligonucleosomes in a cell free repair system by analyzing the effect of packaging DNA into oligonucleosomes on repair of CPDs, by analyzing the effect of NER of CPDs on the packaging of DNA into oligonucleosomes, and by analyzing the effect of histone acetylation in oligonucleosomes on repair of CPDs. Finally, we will explore the potential of chromatin remodeling activity having a role in NER at UV damaged sites by analyzing the accessibility of CPDs in model chromatin substrates following reaction with nucleosome unfolding complexes in vitro, by measuring mitotic viability and metabolic competence in two classes of UV irradiated yeast mutants with deficiencies in chromatin remodeling, and by examining NER in two classes of yeast mutants with deficiencies in chromatin remodeling. Thus, we will use a 'multifaceted"""""""" approach to examine the role of chromatin structure in DNA repair with the ultimate goal of understanding this process in human cells. Since DNA lesions may alter the expression of specific genes required for establishing the neoplastic phenotype, these studies should provide valuable insight into the cell's defense mechanism for resisting neoplastic transformation by environmental carcinogens.
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