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: 10307906
• 负责人: Kara A Bernstein
• 金额: $ 1.75万
• 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 2022-04-01
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10307906
• 关键词: Address; Alkylating Agents; Alkylation; Anemia; Antigen-Presenting Cells; Bypass; Cause of Death; Cells; Cessation of life; Complex; DNA; DNA Alkylation; DNA Damage; DNA Repair; DNA Repair Gene; DNA replication fork; DNA-(apurinic or apyrimidinic site) lyase; Development; Environment; Eukaryota; Exposure to; Fanconi' s Anemia; Genes; Genetic; Genomic Instability; Heritability; Human; Human Cell Line; Individual; Industrialization; Invaded; Lead; Learning; Malignant Neoplasms; Malignant neoplasm of ovary; Mediating; Mediator of activation protein; Mutate; Mutation; Pathway interactions; Process; Proteins; Rad51 recombinase; Regulation; Repair Complex; Risk; Role; Saccharomycetales; Somatic Mutation; Source; Structure; Testing; Toxic Environmental Substances; United States; Work; Yeasts; base; cancer predisposition; homologous recombination; insight; malignant breast neoplasm; paralogous gene; prevent; repaired; replication stress

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.

7.项目总结 DNA损伤修复不当是癌症的一个标志。我们发现，发芽酵母舒复合体是一种 保守的DNA修复调节因子，在RAD51调节中起中心作用。RAD51在高电压期间的功能 找到并侵入用于修复的同源模板的保真度同源重组途径 在复制分叉保护和重新启动期间。RAD51在细胞内受到辅助蛋白的严格调控， 统称为RAD51调解人，包括蜀国情结。在人类中，hRAD51或 其介体与癌症易感性(尤其是乳腺癌和卵巢癌)和Fanconi有关 贫血，也以贫血和癌症为特征。我们发现酵母舒复合体的破坏 导致细胞死亡，特别是当暴露在烷基化诱导的DNA损伤时。烷基化损伤是 由无数工业和消费者来源引起，在我们的环境中无处不在。脱氧核糖核酸 烷基化会导致复制应激和DNA损伤。如果DNA在复制过程中发生烷基化，那么 复制分叉可能停滞或崩溃，许多修复机制可用于容忍、绕过或修复 受损的DNA。细胞如何致力于特定的修复途径在很大程度上是已知的。在萌芽酵母中， 在被烷基化试剂损坏的复制叉子的加工过程中，Shu复合体是至关重要的。这个建筑群是 在真核生物中高度保守，含有结构相似的RAD51蛋白 与中心DNA修复蛋白RAD51结合，并在癌症中发生突变。在这项研究中，我们旨在阐明这一角色 酵母和人类Shu复合体在修复复制叉处DNA烷基化损伤中的作用。我们是 检验Shu复合体是复制时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介导的模板切换 并保护叉子免受AP内切酶诱导的双链断裂。利用我们在酵母中学到的东西 快速有效地确定关键底物、残基和蛋白质靶标，我们将把研究扩展到 我们将研究人类舒缩复合体在烷基化耐受性中的作用 损坏。此外，我们将识别携带这些重要基因突变的高危个体，这些突变可能 对DNA烷基化损伤更敏感，因此对癌症更敏感。总的来说，这些研究 将为Shu复合体在耐受DNA烷基化损伤中的作用提供关键的见解并阐明 这种复合体如何促进无错误的DNA修复，以防止遗传不稳定和癌症。