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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

基本信息

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
项目类别：
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

项目摘要

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/文摘