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.

项目总结/摘要 烷基化剂在我们的环境从烟草，农药，和饮用水净化过程中产生 导致DNA损伤。这些DNA损伤可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途径中发挥重要作用，并受包括Shu复合物（SWSAP1- SWS1-PDS5B-SPIDR）、BRCA 2、RAD 52和CSB。RAD51调节器的误调节导致基因组 不稳定和癌症。我们实验室和其他人最近的研究发现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偶联过程中的非典型修复中， 修复含有R环的复制结构。从机理上洞察酵母舒复合物的确定 在无碱基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 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复合体组分SWSAP1和SWS1的耗尽导致减少的RAD 51灶。是否 人类Shu复合体的功能也是未知的。RAD52和Shu组件 SWSAP1和SWS1在复制重新启动期间通过未知机制运行。RAD52可以使用其 在复制重新启动和R环解析期间退火功能。这项研究的总体目标是 是为了确定人类Shu复合体如何在停滞的复制叉中发挥作用， 因此能够实现依赖于RAD 51的链交换或依赖于RAD 52的退火修复活性。的 这项研究计划的实验将分两个阶段进行。在K99的指导下 阶段，我将确定Shu复合物如何发挥功能，以识别烷基化诱导的病变，如脱碱基 损伤和调节RAD51依赖的修复使用细胞生物学，原子力显微镜（AFM）的培训 和相关光镊-荧光显微镜（CTFM）技术（目的1）。在指导期间， 在这个阶段，候选人将利用匹兹堡大学提供的共同指导和资源 和UPMC希尔曼癌症中心的专业发展，以利用这些技能，通过研究， 指导、数据展示和写作机会。在研究的独立R00阶段 计划，在K99阶段获得的技术技能将被应用于阐明舒复杂的作用， RAD52介导的复制叉重启（Aim 2）。此外，在R00阶段，I将扩展这些方法 进入了一个新领域，涉及通过RAD 52蛋白复合物解析RNA-DNA杂交体。这些实验将 为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，候选人将独立于他们的导师和共同导师 通过关注Shu复合物和RAD 52之间响应复制应激的动态相互作用。