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.项目摘要 DNA损伤的错误修复是癌症的标志。我们发现芽殖酵母Shu复合体是一种 通过在Rad51调节中发挥中心作用的DNA修复的保守调节剂。Rad51功能在高 保真度同源重组途径，以找到并侵入用于修复的同源模板， 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， 统称为拉德51调解人，包括舒氏复合体。在人类中，hRAD 51或 它的介质与癌症易感性（特别是乳腺癌和卵巢癌）和范可尼有关。 贫血，其特征还在于贫血和癌症。我们发现酵母Shu复合物的破坏 特别是在暴露于烷基化诱导的DNA损伤时导致细胞死亡。烷基化损害是 由无数的工业和消费者为基础的来源，并在我们的环境中无处不在。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， 复制分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细胞如何致力于特定的修复途径在很大程度上是已知的。在芽殖酵母中， Shu复合物在被烷化剂破坏的复制叉的加工中是关键的。该复合物 在整个真核生物中高度保守，包含Rad51旁系同源物，即结构相似的蛋白质 中心DNA修复蛋白Rad51，并在癌症中发生突变。在这项研究中，我们的目的是阐明的作用， 酵母和人Shu复合物在复制叉处DNA烷基化损伤修复中的作用。我们 测试Shu复合物是复制时DNA损伤耐受性的关键关键调节剂的假设 通过特异性识别烷基化诱导的DNA损伤来促进Rad51介导的模板转换 并保护叉免受AP内切核酸酶的双链断裂诱导。利用我们在酵母中学到的东西 快速，有效地确定关键底物，残留物和蛋白质靶点，我们将扩大我们的研究， 人类细胞系，我们将研究人类Shu复合物在烷基化耐受性中的作用 损害此外，我们将确定在这些重要基因中携带突变的风险个体， 对DNA烷基化损伤更敏感，因此易患癌症。总的来说，这些研究 将提供关键的见解，舒复合物的作用，在耐受DNA烷基化损伤和阐明 这种复合物如何促进无错误的DNA修复，以防止遗传不稳定和癌症。