Shu complex and RAD52 function in DNA damage recognition and subsequent repair

Shu 复合物和 RAD52 在 DNA 损伤识别和后续修复中发挥作用

• 批准号: 10864084
• 负责人: Sarah R Hengel
• 金额: $ 24.9万
• 依托单位: TUFTS UNIVERSITY MEDFORD
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10864084
• 关键词: Affinity; Alkylating Agents; Alkylation; Area; Atomic Force Microscopy; BRCA2 gene; Binding; Biochemical; Biological Assay; Biophysics; Bypass; CRISPR/Cas technology; Cancer Center; Cells; Cellular biology; Complex; DNA; DNA Damage; DNA Double Strand Break; DNA lesion; DNA replication fork; DNA-Directed RNA Polymerase; Data; Development; ERCC6 gene; Electrophoretic Mobility Shift Assay; Environment; Equilibrium; Filament; Fluorescence Anisotropy; Fluorescence Microscopy; Fluorescence Resonance Energy Transfer; Genetic Transcription; Genomic Instability; Goals; Grant; Health; Human; Hybrids; In Vitro; Investigation; Knock-out; Label; Laboratories; Lead; Lesion; Malignant Neoplasms; Mediating; Mentors; Molecular; Pathway interactions; Pesticides; Phase; Play; Process; Proteins; Publications; RAD52 gene; RNA; Rad51 recombinase; Radon; Recombinants; Research; Resolution; Resources; Role; Series; Single-Stranded DNA; Site; Soil; Specificity; Structure; Technical Expertise; Techniques; Testing; Time; Tobacco; Training; Transcription-Coupled Repair; Universities; Water Purification; Work; Writing; Yeasts; analog; base; biophysical techniques; drinking; drinking water; experimental study; homologous recombination; human disease; insight; laser tweezer; member; mutant; novel; optic tweezer; programs; protein complex; prototype; repaired; replication stress; response; single molecule; skills; stoichiometry

PROJECT SUMMARY/ABSTRACT Alkylating agents in our environment from tobacco, pesticides, and produced during drinking water purification cause DNA lesions. These DNA lesions can cause replication fork stalling and lead to DNA double-strand breaks that are canonically repaired by the homologous recombination (HR) pathway. The RAD51 protein plays essential functions in the HR pathway and is regulated by proteins including the Shu complex (SWSAP1- SWS1-PDS5B-SPIDR), BRCA2, RAD52, and CSB. Misregulation of RAD51 regulators leads to genome instability and cancer. Recent studies from our lab and others identified novel additional roles of these proteins in non-canonical repair during DNA lesion recognition, response to replication stress, and transcription coupled repair of replication structures containing R-loops. Mechanistic insight from the yeast Shu complex determined a role during abasic lesion recognition and RAD51-mediated bypass mechanisms during replication. Our work shows that like the yeast Shu complex, the human Shu complex is sensitive to the prototype alkylating agent MMS and depletion of Shu complex components SWSAP1 and SWS1 cause reduced RAD51 foci. Whether the human Shu complex functions by a similar mechanism is unknown. Both RAD52 and Shu components SWSAP1 and SWS1 function during replication restart by an unknown mechanism. RAD52 may use its annealing functions during replication restart and R-loop resolution. The overall goal of this proposed research is to determine how the human Shu complex functions at stalled replication forks to recognize abasic lesions thus enabling either RAD51-dependent strand exchange or RAD52-dependent annealing repair activities. The experiments proposed in this research program will be conducted in two phases. During the mentored K99 phase, I will determine how the Shu complex functions to recognize alkylation-induced lesions like abasic lesions and modulate RAD51-dependent repair using training in cell biology, atomic force microscopy (AFM) and correlative optical tweezers-fluorescence microscopy (CTFM) techniques (Aim 1). During the mentored phase the candidate will take advantage of co-mentoring, resources available at the University of Pittsburgh and the UPMC Hillman Cancer Center for professional development to utilize these skills through research, mentoring, data presentation, and writing opportunities. During the independent R00 phase of the research program, technical skills obtained during the K99 phase will be applied to elucidate the role of Shu complex in RAD52-mediated replication fork restart (Aim 2). Also, during the R00 phase I will extend these approaches into a new area involving resolution of RNA-DNA hybrids by RAD52 protein complexes. These experiments will provide me with the data required for an early independent publication and preliminary data for R-series grants. Importantly, during the R00 phase the candidate will develop independence from their mentor and co-mentors by focusing on the dynamic interplay between the Shu complex and RAD52 in response to replication stress.

项目概要/摘要 我们环境中的烷基化剂来自烟草、农药以及饮用水净化过程中产生的 引起DNA损伤。这些 DNA 损伤会导致复制叉停滞并导致 DNA 双链 通过同源重组 (HR) 途径进行规范修复的断裂。 RAD51蛋白 在 HR 通路中发挥重要功能，并受 Shu 复合物 (SWSAP1- SWS1-PDS5B-SPIDR)、BRCA2、RAD52 和 CSB。 RAD51 调节剂的错误调节导致基因组 不稳定和癌症。我们实验室和其他人的最新研究发现了这些蛋白质的新的附加作用 DNA 损伤识别、复制应激反应和转录耦合过程中的非规范修复 修复含有 R 环的复制结构。从酵母 Shu 复合物中确定的机制见解 在复制过程中脱碱基损伤识别和 RAD51 介导的旁路机制中发挥作用。我们的工作 表明与酵母 Shu 复合物一样，人类 Shu 复合物对原型烷化剂敏感 MMS 和 Shu 复合体成分 SWSAP1 和 SWS1 的耗竭导致 RAD51 病灶减少。无论 人类舒复杂的功能通过类似的机制尚不清楚。 RAD52 和 Shu 组件 SWSAP1 和 SWS1 在复制重启期间通过未知机制发挥作用。 RAD52 可以使用其 复制重启和 R 环解析期间的退火功能。本研究的总体目标 目的是确定人类 Shu 复合体如何在停滞的复制叉上发挥作用来识别无碱基损伤 从而实现 RAD51 依赖性链交换或 RAD52 依赖性退火修复活动。这 本研究计划中提出的实验将分两个阶段进行。在指导K99期间 阶段，我将确定 Shu 复合体如何发挥作用来识别烷基化诱导的病变，例如脱碱基 利用细胞生物学、原子力显微镜 (AFM) 培训来调节 RAD51 依赖性修复 以及相关光镊荧光显微镜 (CTFM) 技术（目标 1）。辅导期间 阶段候选人将利用匹兹堡大学提供的共同指导和资源 和 UPMC Hillman 癌症中心进行专业发展，通过研究利用这些技能， 指导、数据演示和写作机会。研究的独立R00阶段 计划中，在 K99 阶段获得的技术技能将被应用于阐明 Shu 复合物在 RAD52 介导的复制叉重启（目标 2）。另外，在 R00 阶段我将扩展这些方法 进入一个涉及通过 RAD52 蛋白复合物解析 RNA-DNA 杂交体的新领域。这些实验将 向我提供早期独立出版物所需的数据以及 R 系列资助的初步数据。 重要的是，在 R00 阶段，候选人将独立于导师和共同导师 通过关注 Shu 复合体和 RAD52 之间响应复制压力的动态相互作用。