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 我们环境中的烷化剂来自烟草、杀虫剂和饮用水净化过程中产生的 造成DNA损伤。这些DNA损伤可导致复制叉停滞并导致DNA双链 由同源重组(HR)途径典型修复的断裂。RAD51蛋白 在HR途径中发挥重要作用，并受包括Shu复合体(SWSAP1- SWS1-PDS5B-SPIDR)、BRCA2、RAD52和CSB。RAD51调节器的错误调控导致基因组 不稳定和癌症。我们实验室和其他实验室最近的研究确定了这些蛋白质的新的额外作用 在DNA损伤识别、复制应激反应和转录耦合过程中的非典型修复 修复含有R-环的复制结构。酵母菌复合体的机械学洞察 在基本损伤识别和复制过程中RAD51介导的旁路机制中的作用。我们的工作 结果表明，与酵母菌Shu复合体一样，人类Shu复合体对原型烷基化试剂敏感 MMS和Shu复合成分SWSAP1和SWS1的耗竭导致RAD51病灶减少。是否 人类舒复合体通过类似的机制发挥作用尚不清楚。RAD52和舒适性组件 SWSAP1和SWS1在复制重启期间以未知机制运行。RAD52可以使用其 在复制重启和R-循环解析期间的退火功能。这项拟议研究的总体目标是 是为了确定人类Shu复合体如何在停滞的复制叉处发挥作用来识别基本病变 从而实现依赖于RAD51的链交换或依赖于RAD52的退火修复活动。这个 本研究计划中提出的实验将分两个阶段进行。在被指导的K99期间 阶段，我将确定舒复合体如何识别烷基化引起的损害，如abasic 利用细胞生物学、原子力显微镜(AFM)的训练，损伤和调节RAD51依赖的修复 和相关的光钳-荧光显微镜(CTFM)技术(目标1)。在接受指导的过程中 阶段候选人将利用匹兹堡大学提供的共同指导资源 和UPMC希尔曼癌症专业发展中心通过研究利用这些技能， 辅导、数据演示和写作机会。在研究的独立R00阶段 计划中，将应用在K99阶段中获得的技术技能来阐明舒情结在 RAD52介导的复制分叉重新启动(目标2)。此外，在R00阶段，我将扩展这些方法 进入了一个涉及RAD52蛋白质复合体拆分RNA-DNA杂交物的新领域。这些实验将 为我提供早期独立出版所需的数据和R系列赠款的初步数据。 重要的是，在R00阶段，候选人将独立于他们的导师和共同导师 通过关注Shu复合体和RAD52之间的动态相互作用来响应复制压力。