Replication fork dynamics and repair by Rad51 paralogues after DNA alkylation

DNA 烷基化后 Rad51 旁系同源物的复制叉动力学和修复

• 批准号: 9182822
• 负责人: Kara A Bernstein
• 金额: $ 44.72万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-01-01 至 2019-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9182822
• 关键词: Address; Alkylating Agents; Alkylation; Alpha Cell; Architecture; Binding; Biochemical; Biological Assay; Cause of Death; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Crystallization; DNA; DNA Alkylation; DNA Binding; DNA Damage; DNA Double Strand Break; DNA Repair; DNA Repair Gene; DNA Repair Pathway; DNA Structure; DNA lesion; DNA replication fork; DNA-Protein Interaction; Data; Development; Double Strand Break Repair; Environment; Eukaryota; Exposure to; Genes; Genetic; Genetic Polymorphism; Genetic Recombination; Genomic Instability; Heritability; Homologous Gene; Human; Human Cell Line; Hypersensitivity; Individual; Industrialization; Investigation; Lead; Lesion; Link; Malignant Neoplasms; Maps; Mediating; Methyl Methanesulfonate; Modeling; Mutation; Nucleotides; Outcome; Pathway interactions; Play; Point Mutation; Process; Proteins; Rad51 recombinase; Recruitment Activity; Regulation; Repair Complex; Research Personnel; Risk; Role; Saccharomycetales; Scheme; Somatic Mutation; Source; Stress; Structural Protein; Structure; Testing; Time; Toxic Environmental Substances; United States; Vitronectin; Work; Yeasts; base; cancer risk; chromatin immunoprecipitation; helicase; homologous recombination; human disease; insight; member; mutant; neoplastic cell; prevent; protein complex; protein protein interaction; public health relevance; repaired; transcription activator-like effector nucleases; tumor; tumorigenesis; yeast two hybrid system

 DESCRIPTION (provided by applicant): Misrepair of DNA damage is a hallmark of tumor cells. DNA damage can occur from many endogenous and exogenous sources including environmental toxicants. Alkylation damage caused by a myriad of industrial and consumer based sources is pervasive in the environment. DNA alkylation leads to replication stress and DNA damage. If DNA is alkylated during replication, then the replication fork can collapse and many repair mechanisms can be utilized to repair the damaged DNA. How a cell commits to a specific repair pathway is not completely understood. In budding yeast, the Shu complex is critical in the repair of DNA double-strand breaks (DSB) created by processing of replication forks damaged by DNA alkylating agents. This complex is highly conserved throughout eukaryotes and contains the Rad51 paralogues, proteins that are structurally similar to the central DNA repair protein Rad51. In this study, the investigators aim to elucidate the role of the yeast and human Shu complexes in repair of DNA alkylation damage at a replication fork. They are testing the hypothesis that the Shu complex is a critical key regulator of error-free DNA repair by shuttling DNA intermediates into homologous recombination pathway through its unique protein-protein interactions. Consistent with this hypothesis, mutation of the Shu complex proteins leads to repair by alternative error-prone repair mechanisms. The investigators will test the model that the Shu complex plays an essential role in the DNA repair pathway choice by 1) characterizing the association of the Shu complex with a damaged replication fork and the importance of its protein-protein interactions at the fork and 2) solving the structure of the Shu complex DNA binding subunits while bound to a replication fork-like substrate. These studies will elucidate how the Shu complex interactions with DNA and its protein-protein interactions direct these DNA intermediates into distinct repair mechanisms. Using what is learned in yeast to quickly and efficiently identify key substrates, residues, and protein targets, the investigators will expand their studies into human cell lines where they will investigate the role of the human Shu complex and RAD51 paralogues in repair of alkylation damage caused by environmental toxicants. The investigators will specifically characterize mutations and small nucleotide polymorphisms in the hRAD51 paralogues, which may make these individuals hypersensitive to DNA alkylators and could therefore increase cancer risk. Collectively, these studies will provide key insights into the role of the Shu complex in repair of DNA alkylation damage and elucidate how this complex promotes error-free DNA repair to prevent genetic instability. In addition, the investigators will identify at-risk individuals harboing mutations in these important genes that may be more sensitive to DNA alkylation damage and therefore susceptible to human disease.

 描述（由申请人提供）：DNA损伤的错误修复是肿瘤细胞的标志。DNA损伤可由许多内源性和外源性来源引起，包括环境毒物。由无数工业和消费者来源引起的烷基化损害在环境中普遍存在。DNA烷基化导致复制应激和DNA损伤。如果DNA在复制过程中被烷基化，那么复制叉可以崩溃，许多修复机制可以用来修复受损的DNA。细胞如何致力于特定的修复途径尚未完全了解。在芽殖酵母中，Shu复合物在修复DNA双链断裂（DSB）中是至关重要的，所述DNA双链断裂（DSB）是由DNA烷化剂损伤的复制叉加工产生的。该复合物在整个真核生物中高度保守，并含有Rad51旁系同源物，这些蛋白质在结构上与中心DNA修复蛋白Rad51相似。在这项研究中，研究人员旨在阐明 酵母和人Shu复合物修复复制叉处的DNA烷基化损伤。他们正在验证一个假设，即Shu复合物是无错误DNA修复的关键调节因子，通过其独特的蛋白质-蛋白质相互作用将DNA中间体穿梭到同源重组途径中。与这一假设相一致，Shu复合物蛋白的突变导致通过替代易错修复机制进行修复。研究人员将通过以下方式测试Shu复合物在DNA修复途径选择中起重要作用的模型：1）表征Shu复合物与受损复制叉的关联及其在叉处的蛋白质-蛋白质相互作用的重要性; 2）解决Shu复合物DNA结合亚基的结构，同时结合到复制叉样底物。这些研究将阐明舒复合物与DNA的相互作用及其蛋白质-蛋白质相互作用如何指导这些DNA中间体进入不同的修复机制。利用在酵母中学到的知识来快速有效地识别关键底物、残留物和蛋白质靶标，研究人员将把他们的研究扩展到人类细胞系中，在那里他们将研究人类Shu复合物和RAD 51旁系同源物在修复环境毒物引起的烷基化损伤中的作用。研究人员将具体描述hRAD 51旁系同源物中的突变和小核苷酸多态性，这可能使这些个体对DNA烷化剂过敏，因此可能增加癌症风险。总的来说，这些研究将提供关键的见解，舒复杂的修复作用， DNA烷基化损伤，并阐明这种复合物如何促进无错误的DNA修复，以防止遗传不稳定。此外，研究人员将确定在这些重要基因中携带突变的高危个体，这些突变可能对DNA烷基化损伤更敏感，因此易患人类疾病。