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Regulation of DNA double-strand break repair pathway choice

DNA双链断裂修复途径选择的调控

基本信息

批准号：
10656805
负责人：
Wolf-Dietrich Heyer
金额：
$ 46.66万
依托单位：
UNIVERSITY OF CALIFORNIA AT DAVIS
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：
2023-07-01 至 2028-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10656805
关键词：
Abbreviations Affect BRCA2 gene Base Pairing Biochemical Biochemical Reaction C-terminal Camptothecin Cell Cycle Cells Chromosomal Rearrangement Clinical Complex Cyclin-Dependent Kinases DNA Binding DNA Binding Domain DNA Damage DNA Double Strand Break DNA Repair Gene DNA biosynthesis DNA damage checkpoint DNA lesion DNA replication fork DNA-Directed DNA Polymerase Data Defect Double Strand Break Repair Drug Targeting Energy Transfer Filament Fluorescence Fluorescence Resonance Energy Transfer Genes Genome Genome Stability Genomics Goals Human Image Immune checkpoint inhibitor In Vitro Individual Ionizing radiation Knowledge Length Mediating Mediator Mitosis Modality Modeling Mutagenesis Nonhomologous DNA End Joining Pathway interactions Patient Selection Phase Poly(ADP-ribose) Polymerase Inhibitor Poly(ADP-ribose) Polymerases Polymerase Predisposition Process Productivity Property Proteins Protocols documentation RAD52 gene Rad51 recombinase Radiation induced damage Reaction Reactive Oxygen Species Regimen Regulation Research Resistance Role S phase Side Source Testing Therapeutic Topoisomerase II Type I DNA Topoisomerases cancer cell cancer therapy chromosome fusion chromosome loss clinical application homologous recombination inhibitor innovation insight loss of function mutant novel personalized medicine protein purification reconstitution repaired response translational potential tumor

项目摘要

Project Summary The general goal of the proposed research is to define the mechanistic underpinning for the synthetic lethality network between a deficiency in homologous recombination (HR) and defects in single-strand annealing (RAD52) or POLq-mediated end-joining (TMEJ). Exciting preliminary data revealed that BRCA2 and RAD52 delay the repair of S-phase-associated DNA double-stranded breaks (DSB) by TMEJ until M-phase. This regulation avoids TMEJ-mediated chromosomal rearrangements of one-sided DSBs that are produced by replication fork breakage. Our approach combines innovative cell cycle phase resolved imaging of DNA repair proteins and DNA damage markers with mechanistic biochemical analysis using purified human proteins in reconstituted reactions. Our results will have potential translational implications for the clinical application of newly developed RAD52 and POLq inhibitors for the treatment of HR-deficient tumors with respect to application protocols, patient selection, and use of DNA damage response checkpoint inhibitors as well as the response to poly(ADP-ribose) polymerase inhibition. The Specific Aims are: 1. Define the mechanism of action of BRCA2 in DSB repair pathway control. We will test the model that the DNA binding properties of BRCA2 are critical for TMEJ inhibition. In Aim 1A, we conduct foundational studies to determine the fundamental DNA binding properties of full-length BRCA2. In Aim 1B, we will define which domains of BRCA2 are required for TMEJ inhibition in cells. This combination of cell-based and biochemical studies will define the functions and regions of BRCA2 that are required for TMEJ inhibition. 2. Define the mechanism of TMEJ inhibition by BRCA2 and RAD52. BRCA2 and RAD52 employ two different modes to inhibit the DNA polymerase activity of POLq which may affect additional reaction steps in the TMEJ process. We will reconstitute TMEJ in vitro with purified proteins to determine the mechanisms by which BRCA2 (Aim 2A) and RAD52 (Aim 2B) inhibit TMEJ. We will test inhibition of the overall TMEJ reaction and individual steps including 1) DNA binding, 2) end-alignment, and 3) DNA synthesis. Analysis of wild type and catalytic mutants of POLq will be conducted in vitro and in cells. 3. Define which HR defects are susceptible to RAD52 loss of function. It is an open question whether loss of RAD52 will lead to POLq-mediated chromosome fusions and lethality in all HR-deficient backgrounds (Aim 3A) or all BRCA2 mutants (Aim 3B). Our preliminary studies suggest a model that loading of BRCA2 is the critical step to limit TMEJ to M-phase and that HR defects past this step are not affected by RAD52 inhibition.

项目总结