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.

项目摘要/摘要 我们的环境中的烷基化剂，烟草，农药和饮用水净化期间生产的烷基化剂 引起DNA病变。这些DNA病变会导致复制叉的失速并导致DNA双链 通过同源重组（HR）途径来修复的断裂。 RAD51蛋白 在HR途径中发挥重要功能，并受到包括SHU复合物在内的蛋白质的调节（SWSAP1-- SWS1-PDS5B-SPIDR），BRCA2，RAD52和CSB。 RAD51调节剂的不调节导致基因组 不稳定性和癌症。我们实验室和其他人的最新研究确定了这些蛋白质的新作用 在DNA病变识别期间的非经典修复中，对复制应力的反应和转录耦合 修复包含R环的复制结构。确定的酵母菌复合物的机械洞察力 在Abasic病变识别和RAD51介导的旁路机制中的作用。我们的工作 表明像酵母菌复合物一样，人类SHU复合物对原型烷基化剂敏感 MMS和SHU复合物成分SWSAP1和SWS1的耗竭会导致RAD51焦点减少。无论 人类SHU复合物通过类似机制的功能尚不清楚。 RAD52和SHU组件 复制过程中的SWSAP1和SWS1通过未知机制重新启动。 Rad52可能会使用它的 复制期间的退火功能重新启动和R环分辨率。这项拟议研究的总体目标 是确定人类SHU复合物如何在停滞的复制叉处发挥作用以识别无骨病变 从而实现Rad51依赖性链交换或Rad52依赖性退火修复活动。这 该研究计划中提出的实验将分为两个阶段。在指导的K99期间 阶段，我将确定SHU复合物如何识别烷基化诱导的病变（如abasic） 使用细胞生物学，原子力显微镜（AFM）中的训练来调节RAD51依赖性修复 和相关光学镊子荧光显微镜（CTFM）技术（AIM 1）。在指导期间 阶段候选人将利用匹兹堡大学获得的资源来利用冠军 以及UPMC Hillman癌症专业发展中心通过研究利用这些技能， 指导，数据演示和写作机会。在研究的独立R00阶段 计划，在K99阶段获得的技术技能将应用于阐明Shu Complex在 RAD52介导的复制叉重新启动（AIM 2）。另外，在R00阶段，我将扩展这些方法 进入一个新区域，涉及RAD52蛋白复合物分辨出RNA-DNA杂交的区域。这些实验会 为我提供早期独立出版物和R系列赠款的初步数据所需的数据。 重要的是，在R00期间 通过重点关注SHU复合物与Rad52之间的动态相互作用，以响应复制应力。