Replication fork dynamics and repair by Rad51 paralogs 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: 10162586
• 负责人: Kara A Bernstein
• 金额: $ 37.26万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10162586
• 关键词: Address; Alkylating Agents; Alkylation; Anemia; Antigen-Presenting Cells; BRCA2 gene; Binding; Biochemical; Biological Assay; Bypass; Cause of Death; Cell Death; Cells; Cellular biology; Cessation of life; Clustered Regularly Interspaced Short Palindromic Repeats; Comb animal structure; Complex; DNA; DNA Alkylation; DNA Damage; DNA Repair; DNA Repair Gene; DNA Sequence; DNA Structure; DNA replication fork; DNA sequencing; DNA-(apurinic or apyrimidinic site) lyase; DNA-Protein Interaction; Data; Development; Environment; Eukaryota; Exhibits; Exposure to; Fanconi' s Anemia; Filament; Genes; Genetic; Genetic Recombination; Genome Stability; Genomic Instability; Heritability; Human; Human Cell Line; Hypersensitivity; In Vitro; Individual; Induced Mutation; Industrialization; Invaded; Knock-out; Lead; Learning; Lesion; Malignant Neoplasms; Malignant neoplasm of ovary; Mediating; Mediator of activation protein; Methyl Methanesulfonate; Mutate; Mutation; Nature; Nucleoproteins; Outcome; Pathway interactions; Positioning Attribute; Process; Proteins; Protocols documentation; RAD51C gene; Rad51 recombinase; Regulation; Repair Complex; Replication Origin; Risk; Role; Saccharomyces cerevisiae; Saccharomycetales; Sequence Analysis; Somatic Mutation; Source; Specificity; Structure; Testing; Toxic Environmental Substances; United States; Work; XRCC2 gene; XRCC3 gene; Yeasts; autonomously replicating sequence; base; cancer predisposition; genotoxicity; homologous recombination; in vivo; innovation; insight; malignant breast neoplasm; member; mutant; novel; paralogous gene; prevent; recruit; repaired; replication stress; tumorigenesis

7. Project Summary Misrepair of DNA damage is a hallmark of cancer. We discovered that the budding yeast Shu complex is a conserved regulator of DNA repair through a central role in Rad51 regulation. Rad51 functions during the high fidelity homologous recombination pathway to find and invade a homologous template for repair and also during replication fork protection and restart. Rad51 is tightly regulated in cells by accessory proteins, collectively called the Rad51 mediators, including the Shu complex. In humans, misregulation of hRAD51 or its mediators is associated with cancer predisposition (particularly breast and ovarian cancers) and Fanconi anemia, which is also characterized by anemia and cancer. We found that disruption of the yeast Shu complex leads to cellular death specifically upon exposure to alkylation induced DNA damage. Alkylation damage is caused by a myriad of industrial and consumer-based sources and is pervasive in our environment. DNA alkylation leads to replication stress and DNA damage. If DNA is alkylated during replication, then the replication fork can stall or collapse, and many repair mechanisms can be utilized to tolerate, bypass, or repair the damaged DNA. How a cell commits to a specific repair pathway is largely known. In budding yeast, the Shu complex is critical in the processing of replication forks damaged by alkylating agents. This complex is highly conserved throughout eukaryotes and contains the Rad51 paralogs, proteins that are structurally similar to the central DNA repair protein Rad51 and are mutated in cancer. In this study, we aim to elucidate the role of the yeast and human Shu complexes in repair of DNA alkylation damage at a replication fork. We are testing the hypothesis that the Shu complex is a critical key regulator of DNA damage tolerance at a replication fork by specifically recognizing alkylation induced DNA damage to promote Rad51-mediated template switch and protect forks from double-strand break induction by AP endonucleases. Using what we learn in yeast to quickly and efficiently identify key substrates, residues, and protein targets, we will expand our studies into human cell lines where we will investigate the role of the human Shu complex in tolerance of alkylation damage. In addition, we will identify at risk individuals harboring mutations in these important genes that may be more sensitive to DNA alkylation damage and therefore susceptible to cancer. Collectively, these studies will provide key insights into the role of the Shu complex in tolerance of DNA alkylation damage and elucidate how this complex promotes error-free DNA repair to prevent genetic instability and cancer. !

七、项目概要 DNA 损伤修复不当是癌症的一个标志。我们发现出芽酵母 Shu 复合体是 通过在 Rad51 调节中发挥核心作用，DNA 修复的保守调节因子。 Rad51 在高电平期间起作用 保真同源重组途径寻找并侵入同源模板进行修复 在复制分叉保护和重新启动期间。 Rad51 在细胞中受到辅助蛋白的严格调控， 统称为 Rad51 介体，包括 Shu 复合体。在人类中，hRAD51 的错误调节或 其介质与癌症易感性（特别是乳腺癌和卵巢癌）和 Fanconi 相关 贫血，其特征还在于贫血和癌症。我们发现酵母 Shu 复合体的破坏 导致细胞死亡，特别是在暴露于烷基化诱导的 DNA 损伤时。烷基化损伤是 由无数工业和消费者来源引起，并且在我们的环境中普遍存在。脱氧核糖核酸 烷基化会导致复制应激和 DNA 损伤。如果DNA在复制过程中被烷基化，那么 复制叉可能会停止或崩溃，并且可以利用许多修复机制来容忍、绕过或修复 受损的DNA。细胞如何参与特定的修复途径已广为人知。在芽殖酵母中， Shu 复合物在烷化剂损坏的复制叉的加工过程中至关重要。这个综合体是 在真核生物中高度保守，包含 Rad51 旁系同源蛋白，结构相似的蛋白质 核心 DNA 修复蛋白 Rad51 并在癌症中发生突变。在这项研究中，我们的目的是阐明 酵母和人类 Shu 复合物修复复制叉处的 DNA 烷基化损伤。我们是 检验 Shu 复合体是复制过程中 DNA 损伤耐受性的关键调节因子的假设 通过特异性识别烷基化诱导的 DNA 损伤促进 Rad51 介导的模板转换 并保护叉免受 AP 核酸内切酶诱导的双链断裂。利用我们在酵母中学到的知识 为了快速有效地识别关键底物、残基和蛋白质靶点，我们将把我们的研究扩展到 我们将研究人类 Shu 复合物在烷基化耐受性中的作用的人类细胞系 损害。此外，我们将识别出这些重要基因中携带突变的高危个体，这些突变可能会导致 对 DNA 烷基化损伤更敏感，因此容易患癌症。总的来说，这些研究 将为 Shu 复合物在耐受 DNA 烷基化损伤中的作用提供重要见解并阐明 这种复合物如何促进无错误的 DNA 修复以预防遗传不稳定和癌症。 ！