A wide variety of human genetic diseases are caused by mutations in genes that function in DNA repair pathways. As an important defense mechanism to maintain genome stability, DNA repair also contributes to the normal development and health of organisms. Bulky DNA lesions resulting from ultraviolet (UV) radiation, cigarette smoke and other endogenous and exogenous agents are repaired by the conserved nucleotide excision repair (NER) pathway. The assembly of DNA into chromatin in eukaryotic cells interferes with the NER process. How NER operates in the context of chromatin is largely unknown. Our previous studies in Saccharomyces cerevisiae first linked the prototype ATP-dependent chromatin remodeling complex SWI/SNF to NER. Very recent findings from our group and others have established a connection between SWI/SNF and NER in human cells. However, the process by which SWI/SNF remodels chromatin to facilitate NER remains undefined. Also unknown is the SWI/SNF recruitment mechanism in NER facilitation. The central objective of this proposal is to elucidate the mechanism by which SWI/SNF facilitates damage detection and repair in S. cerevisiae and cultured mammalian cells. Additionally, in vitro biochemical studies will be undertaken to validate cellular findings and provide detailed biochemical mechanisms on the coordination of chromatin remodeling and NER. We hypothesize that DNA damage recognition factors recruit SWI/SNF to sites of DNA damage via protein-protein interactions. We will also test an alternative mechanism that histone modifications are involved in SWI/SNF recruitment/or retention at sites of UV damage.
In Aim I, we will determine the role of chromatin modifying activities required for efficient global genome NER (GG-NER) in yeast. We will investigate SWI/SNF remodeling at the Sir complex coated HML locus during NER.
Aim II will discern the role of chromatin modification and remodeling during NER in mammalian cells. We will investigate the consequence of SWI/SNF inactivation on the dynamic NER process.
In Aim III, we will ascertain the mechanisms of damage recognition and NER of nucleosomes in vitro. A purified system will be used to examine how a DNA lesion 'buried'in a mononucleosome is detected by DDB2 and XPC and how SWI/SNF remodels the mononucleosome to facilitate damage detection.
Nucleotide excision repair is the major DNA repair pathway that removes bulky DNA damage induced by ultraviolet (UV) radiation to protect genome stability and prevent skin cancers. Our proposed experiments will help understand how bulky DNA lesions are repaired in human cells in the context of chromatin and reveal molecular mechanisms important to overcome the hazardous health effects of environmental exposures. Knowledge gained from these efforts could be exploited to develop strategies for the prevention and/or treatment of cancers induced by genotoxic environmental exposures.
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