Regulation of DNA double-strand break repair pathway choice

项目摘要 拟议研究的总体目标是定义合成致命性的机制基础。 同源重组缺陷与单链退火法缺陷之间的网络 (RAD52)或POLQ介导的末端连接(TMEJ)。令人兴奋的初步数据显示，BRCA2和RAD52 延缓TMEJ对S期相关DNA双链断裂的修复至M期。这 调节避免TMEJ介导的单侧DSB的染色体重排，这些DSB是由 复制分叉断裂。我们的方法结合了DNA修复的创新细胞周期时相分辨成像 用纯化的人蛋白进行机械生化分析的蛋白质和DNA损伤标志物 重组反应。我们的结果将对临床应用具有潜在的翻译意义。 新开发的RAD52和POLQ抑制剂在HR缺陷肿瘤治疗中的应用 方案、患者选择、DNA损伤反应检查点抑制剂的使用以及对 聚(ADP-核糖)聚合酶抑制。 具体目标是： 1.明确BRCA2在DSB修复途径调控中的作用机制。我们将测试该模型 BRCA2的DNA结合特性是TMEJ抑制的关键。在目标1A中，我们进行基础性研究 以确定全长BRCA2的基本DNA结合特性。在目标1B中，我们将定义 BRCA2的结构域是细胞中抑制TMEJ所必需的。这种以细胞为基础的和生化的结合 研究将确定抑制TMEJ所需的BRCA2的功能和区域。 2.明确BRCA2和RAD52抑制TMEJ的机制。BRCA2和RAD52使用两个不同的 抑制POLQ DNA聚合酶活性的方式可能影响TMEJ中的额外反应步骤 进程。我们将在体外用纯化的蛋白重建TMEJ，以确定其机制 BRCA2(Aim 2A)和RAD52(Aim 2B)抑制TMEJ。我们将测试对整个TMEJ反应的抑制作用，并 各个步骤包括1)DNA结合，2)末端对齐，3)DNA合成。野生型和野生型分析 POLQ的催化突变体将在体外和细胞内进行。 3.确定哪些HR缺陷对RAD52功能丧失易感。损失是否会是一个悬而未决的问题 RAD52的缺失将导致POLQ介导的染色体融合和在所有HR缺陷背景中的致命性(目的 3A)或所有BRCA2突变体(目标3B)。我们的初步研究表明，BRCA2的负荷是 将TMEJ限制为M期关键步骤，且超过该步骤的HR缺陷不受RAD52抑制的影响。

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.