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（由应用提供）：DNA损伤的虚假措施是肿瘤细胞的标志。 DNA损伤可能来自包括环境有毒物质在内的许多内源和外源性来源。在环境中，由无数工业和消费者来源造成的烷基化损害在环境中普遍存在。 DNA酗酒会导致复制应力和DNA损伤。如果在复制过程中烷基化的DNA，则复制叉可能会塌陷，并且可以利用许多维修机制来修复受损的DNA。细胞如何遵守特定的修复途径尚不完全了解。在发射酵母中，SHU复合物在修复DNA双链断裂（DSB）中至关重要。该复合物在整个真核生物中都高度保守，并包含Rad51旁系同源物，这些蛋白质在结构上与中央DNA修复蛋白RAD51相似。在这项研究中，研究人员旨在阐明 在复制叉上修复DNA烷基化损伤的酵母菌和人类SHU复合物。他们正在测试以下假设：SHU复合物是通过将DNA中间体通过其独特的蛋白质 - 蛋白质相互作用将DNA中间体将无错误DNA修复的关键调节剂。与这一假设一致，SHU复合蛋白的突变导致通过替代错误的修复机制进行修复。研究人员将测试该模型，即SHU复合物在DNA修复途径选择中起着至关重要的作用，通过1）表征SHU复合物与损坏的复制叉的关联以及其在叉子上的蛋白质 - 蛋白质相互作用的重要性和2）求解Shu复合物DNA结合的结构，同时与复制的fork fork类似于类似于复制。这些研究将阐明SHU复合物与DNA及其蛋白质蛋白相互作用如何将这些DNA中间体引导到不同的修复机制。利用酵母中学到的知识来快速有效地识别关键底物，恢复和蛋白质靶标，研究人员将将其研究扩展到人类细胞系中，其中他们将研究人类SHU复合物和RAD51旁系同源物在修复环境有毒物质引起的酒精损害中的作用。研究人员将特别表征HRAD51副群中突变和小核苷酸多态性，这可能使这些人对DNA烷基烷高度敏感，因此可能会增加癌症的风险。总的来说，这些研究将提供有关SHU综合体在修复的作用的关键见解 DNA酒精损害并阐明该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具有突变的高风险个体，这些个体可能对DNA酗酒损害更敏感，因此容易受到人类疾病的影响。