Chromatin Modulation Associated with DNA Breakage and Repair in Human Cells

描述（由申请人提供）：细胞对DNA损伤和其他应激的反应是细胞活力和诱变的重要决定因素，并影响多种人类疾病的发展。特别是DNA损伤反应是癌症发展和癌症治疗结果的主要决定因素。信号转导途径的诱导是细胞对这些应激反应的一个重要方面，近年来在阐明这些信号转导途径中的生物化学步骤方面取得了重大进展。澄清这些步骤有助于调节这些反应，这可以加强研究，并可能导致产生新的药物来预防和治疗这些疾病。本申请描述了Kastan实验室开发的一种新技术的增强，其中在人类基因组中的限定位点引入DNA双链断裂（DSB）。引入定点DNA损伤允许研究在DSB处和周围发生的分子事件，并直接评估DNA断裂的修复（重新连接）。该技术的改进允许更好地控制DSB的引入，并允许评估DSB修复的动力学和完整性。这种增强的方法将用于本申请中提出的实验中，以阐明在DSB处和周围发生的蛋白质移动/修饰的动态变化，并探索可能决定DNA断裂修复效率的因素。一个主要的焦点是探索在DSB网站的核小体破坏的分子控制，并阐明这种核小体破坏的功能重要性。众所周知，在损伤/修复过程中，DSB周围的染色质相关蛋白发生了许多修饰;然而，这些修饰中的许多修饰，特别是与DNA修复相关的修饰的功能作用仍有待澄清。实验提出，将使用这种新的系统，以探索这些染色质的变化DSB周围的细胞修复（重新连接）DNA断裂的能力的潜在影响。此外，乳腺癌易感基因产物Brca 1对这一过程的机制和影响将被研究。拟议的研究将为人类细胞中DNA DSB修复过程相关的分子机制提供新的见解。

DESCRIPTION (provided by applicant): Cellular responses to DNA damage and other stresses are important determinants of cell viability and mutagenesis and impact the development of a wide range of human diseases. DNA damage responses, in particular, are major determinants of both cancer development and outcomes of cancer therapies. Induction of signal transduction pathways is a critical aspect of cellular responses to these stresses and significant advances have been made in recent years elucidating the biochemical steps in such signaling pathways. Clarification of such steps enables modulation of these responses, which can enhance research studies and can lead to the generation of new medicines to prevent and treat these diseases. This application describes an enhancement of a novel technique developed in the Kastan laboratory in which DNA double strand breaks (DSBs) are introduced at defined sites in the human genome. Introduction of site-directed DNA damage permits studies of molecular events occurring at and around the DSB and direct assessment of repair (re- ligation) of DNA breaks. The enhancements of this technique permit greater temporal control of introduction of the DSBs and allow assessment of the kinetics and completeness of DSB repair. This enhanced approach will be used in the experiments proposed in this application to elucidate the dynamic changes in protein movements/modifications occurring at and around the DSB and explore factors which may determine efficiency of repair of the DNA breaks. One major focus is exploration of the molecular controls of nucleosome disruption at the DSB site and elucidating the functional importance of this nucleosomal disruption. It is well established that numerous modifications occur in chromatin-associated proteins surrounding DSBs during the damage/repair process; however, the functional role(s) of many of these modifications, especially related to DNA repair, remain to be clarified. Experiments are proposed that will use this novel system to explore the potential impact of these chromatin changes surrounding the DSBs on the ability of the cell to repair (re-ligate) DNA breaks. Further, the mechanisms and impact of the breast cancer susceptibility gene product, Brca1, on this process will be investigated. The proposed studies will provide novel insights into molecular mechanisms associated with the DNA DSB repair process in human cells.