Chromatin Modulation Associated with DNA Breakage and Repair in Human Cells

与人类细胞 DNA 断裂和修复相关的染色质调节

基本信息

批准号：
8657907
负责人：
Michael B Kastan
金额：
$ 31.6万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-07-01 至 2016-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8657907
关键词：
Acetylation Address Affect Ataxia-Telangiectasia-Mutated protein kinase BRCA1 Protein BRCA1 gene Binding Biochemical Cancer-Predisposing Gene Cell Line Cell Survival Cells Cellular biology Chromatin Complex DNA DNA Damage DNA Double Strand Break DNA Ligation DNA Repair Development Disease Double Strand Break Repair Endonuclease I Enzymes Eukaryota Event Functional disorder Generations Genetic Transcription Homing Human Human Genome Kinetics Laboratories Lead Ligation Malignant Neoplasms Mammalian Cell Medicine Methods Modeling Modification Molecular Molecular Probes Movement Mutagenesis Nature Nucleosomes Outcome Phosphorylation Predisposition Process Protein Binding Proteins Radiation therapy Regulation Reporting Role Signal Pathway Signal Transduction Pathway Site Stress System Techniques Ubiquitination base cancer therapy chemotherapy chromatin protein endonuclease human disease improved insight interest malignant breast neoplasm novel novel strategies nuclease prevent repaired research study response restoration tumor yeast genome

项目摘要

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.

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。 DNA 损伤反应尤其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关键方面，近年来在阐明此类信号转导途径中的生化步骤方面取得了重大进展。澄清这些步骤可以调节这些反应，从而可以加强研究，并可以产生预防和治疗这些疾病的新药物。该申请描述了 Kastan 实验室开发的一项新技术的增强，其中在人类基因组的指定位点引入 DNA 双链断裂 (DSB)。引入定点 DNA 损伤可以研究 DSB 处及其周围发生的分子事件，并直接评估 DNA 断裂的修复（重新连接）。该技术的增强允许对 DSB 的引入进行更大的时间控制，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提出的实验中，以阐明 DSB 处及其周围发生的蛋白质运动/修饰的动态变化，并探索可能决定 DNA 断裂修复效率的因素。一个主要焦点是探索 DSB 位点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会发生大量修饰；然而，许多这些修饰的功能作用，尤其是与 DNA 修复相关的修饰，仍有待阐明。建议进行实验，使用这个新系统来探索 DSB 周围的这些染色质变化对细胞修复（重新连接）DNA 断裂的能力的潜在影响。此外，还将研究乳腺癌易感基因产物Brca1对此过程的机制和影响。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。