SWI/SNF chromatin remodeling in nucleotide excision repair

描述（由申请人提供）：多种人类遗传疾病是由在DNA修复途径中起作用的基因突变引起的。DNA修复作为维持基因组稳定的重要防御机制，对生物体的正常发育和健康也有重要作用。由紫外线（UV）辐射、香烟烟雾和其他内源性和外源性物质引起的大体积DNA损伤可通过保守核苷酸切除修复（NER）途径修复。真核细胞中DNA组装成染色质会干扰NER过程。在染色质的背景下，NER如何运作在很大程度上是未知的。我们之前在酿酒酵母中的研究首先将原型atp依赖性染色质重塑复合体SWI/SNF与NER联系起来。我们小组和其他人最近的发现已经在人类细胞中建立了SWI/SNF和NER之间的联系。然而，SWI/SNF重塑染色质以促进NER的过程仍不清楚。在NER促进中SWI/SNF的招募机制也是未知的。本研究的主要目的是阐明SWI/SNF促进酿酒酵母和培养的哺乳动物细胞损伤检测和修复的机制。此外，将进行体外生化研究来验证细胞研究结果，并提供染色质重塑和NER协调的详细生化机制。我们假设DNA损伤识别因子通过蛋白质相互作用将SWI/SNF招募到DNA损伤位点。我们还将测试另一种机制，即组蛋白修饰参与了SWI/SNF在紫外线损伤部位的募集或保留。在Aim I中，我们将确定酵母中有效的全球基因组NER （GG-NER）所需的染色质修饰活性的作用。我们将研究NER过程中Sir复合物包覆HML位点的SWI/SNF重构。目的II将辨别染色质修饰和重塑在哺乳动物细胞NER中的作用。我们将研究SWI/SNF失活对动态NER过程的影响。在第三部分中，我们将在体外确定核小体损伤识别和NER的机制。一个纯化的系统将用于检测DDB2和XPC如何检测“埋藏”在单核小体中的DNA损伤，以及SWI/SNF如何重塑单核小体以促进损伤检测。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: 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.