Regulation of DNA double-strand break repair pathway choice

项目摘要 拟议研究的总体目标是确定合成杀伤力的机制基础 同源重组（HR）缺陷和单链退火缺陷之间的网络 （RAD 52）或POLq介导的末端连接（TMEJ）。令人兴奋的初步数据显示，BRCA 2和RAD 52 延迟TMEJ对S期相关DNA双链断裂（DSB）的修复，直至M期。这 调节避免了TMEJ介导的单侧DSB的染色体重排， 复制叉断裂。我们的方法结合了创新的细胞周期相分辨成像的DNA修复 蛋白质和DNA损伤标记物，并使用纯化的人蛋白质进行机械生化分析， 重组反应。我们的研究结果将有潜在的翻译意义的临床应用， 新开发的用于治疗HR缺陷型肿瘤的RAD 52和POLq抑制剂 方案、患者选择和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BRCA 2在DSB修复途径控制中的作用机制。我们将测试模型， BRCA 2的DNA结合特性对于TMEJ抑制至关重要。在目标1A中，我们进行基础研究 以确定全长BRCA 2的基本DNA结合特性。在目标1B中，我们将定义 BRCA 2的结构域是细胞中TMEJ抑制所必需的。这种基于细胞和生物化学的结合 研究将确定TMEJ抑制所需的BRCA 2的功能和区域。 2.明确BRCA 2和RAD 52抑制TMEJ的机制。BRCA 2和RAD 52采用两种不同的 抑制POLq的DNA聚合酶活性的模式，这可能影响TMEJ中的其他反应步骤 过程我们将在体外用纯化的蛋白质重建TMEJ，以确定其机制， BRCA 2（Aim 2A）和RAD 52（Aim 2B）抑制TMEJ。我们将测试总体TMEJ反应的抑制， 单个步骤包括1）DNA结合，2）末端比对和3）DNA合成。分析野生型和 将在体外和细胞中进行POLq的催化突变。 3.定义哪些HR缺陷易受RAD 52功能丧失的影响。这是一个悬而未决的问题， RAD 52的表达将导致POLq介导的染色体融合和所有HR缺陷背景中的致死性（目的 3A）或所有BRCA 2突变体（Aim 3B）。我们的初步研究表明，BRCA 2的负载是一个模型， 这是将TMEJ限制在M期的关键步骤，并且经过该步骤的HR缺陷不受RAD 52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.