The Role of Mono-ADP-Ribosylation by PARP14 in Radioresistance

项目摘要 DNA双链断裂(DSB)干扰细胞活力，但也启动染色体 易位导致基因组不稳定并促进癌症的发生。BRCA1和BRCA2蛋白是 对于同源重组(HR)介导的双链断裂修复是必不可少的。了解这些问题的机制 BRCA途径对人类健康具有广泛的意义。当复制叉遇到损坏的DNA时， 除非得到适当的处理，否则它们会被阻止并崩溃，导致DNA断裂和基因组不稳定。至 避免它们的崩溃，停滞不前的叉子可以通过将两条新生的叉子彼此加热来逆转，在 由ZRANB3等DNA易位酶催化的过程。BRCA蛋白在倒叉上装载RAD51 以保护DNA末端免受核酸酶Mre11的降解。保护叉子免受攻击的能力 降解与DNA损伤敏感性相关。因此，复制分叉保护对于DNA修复是必不可少的 和基因组的稳定性。然而，如何保护停滞的复制叉子免受核溶解降解 已取得的成就代表着一项重大的知识差距。PARP家族至少有17名成员，有各种或更少的成员 比创始成员PARP1更了解职能。PARP14与多个细胞 过程，但机械性细节通常是稀疏的。我们之前已经证明，PARP14的损失会降低HR 提高效率，并使细胞对辐射敏感。最近，我们发现了PARP14在促进 复制分叉降解、基因组不稳定性和DNA损伤敏感性，这是这方面的重点 申请。对于本应用程序，我们的目标是了解PARP14如何促进分叉降级，从而导致 BRCA缺陷细胞的DNA损伤敏感性。我们的总体假设是PARP14干扰了 RAD51-Mre11控制反向复制叉处DNA切除以触发新生链的机制 降解，从而增强BRCA缺陷细胞对DNA损伤的敏感性。目标1是揭示 RAD51介导的复制分叉停滞保护的PARP14。我们假设PARP14干扰 通过BRCA非依赖的RAD51在倒叉上的稳定，以增强其降解性。目标2是 揭示PARP14如何与Mre11结合，以促进受损叉子的核溶解降解。我们假设 PARP14与BRCA缺陷细胞中停滞的复制分叉结合并招募Mre11来启动核溶解 新生的DNA在这些结构上的降解。目的3是阐明KU在叉子保护中的作用。 EXO1和Mre11的核溶解切除。我们假设KU与反向叉子的结合保护了它们 抗EXO1介导的降解，但允许Mre11-PARP14复合体切除新生的链。 由于DNA损伤剂通过诱导新生链的降解来促进基因组的不稳定性，潜在地 在它们致癌的基础上，这些特定目的的成功实现将揭示一种新的 基因组稳定和肿瘤抑制的机制，以PARP14为中心。它还可能揭示PARP14作为一种 在BRCA状态下，肿瘤对放射和基因毒性化疗反应的生物标记物。

Project Summary DNA double stranded breaks (DSBs) interfere with cellular viability, but also initiate chromosomal translocations resulting in genomic instability and promoting carcinogenesis. BRCA1 and BRCA2 proteins are essential for homologous recombination (HR)-mediated repair of DSBs. Understanding the mechanisms of the BRCA pathway has broad implications for human health. When replication forks encounter damaged DNA, they arrest and unless properly processed, they collapse leading to DNA breaks and genomic instability. To avoid their collapse, stalled forks can be reversed by annealing the two nascent strands to each other, in a process catalyzed by DNA translocases such as ZRANB3. The BRCA proteins load RAD51 on reversed forks to protect the DNA ends against degradation by the nuclease MRE11. The ability to protect forks against degradation corelates with DNA damage sensitivity. Thus, replication fork protection is essential for DNA repair and genomic stability. However, how protection of stalled replication forks against nucleolytic degradation is achieved represents a major knowledge gap. The PARP family at least 17 members, with various and lesser understood functions than the founding member PARP1. PARP14 has been associated with multiple cellular processes, but mechanistic details are generally sparse. We previously showed that PARP14 loss reduces HR efficiency and sensitizes cells to radiation. Recently, we have identified a novel role of PARP14 in promoting replication fork degradation, genomic instability and DNA damage sensitivity, which is the focus on this application. For this application, our goal is to understand how PARP14 promotes fork degradation, resulting in DNA damage sensitivity of BRCA-deficient cells. Our overall hypothesis is that PARP14 interferes with the RAD51-MRE11 mechanism of control of DNA resection at reversed replication forks to trigger nascent strand degradation, thus enhancing DNA damage sensitivity in BRCA-deficient cells. Aim 1 is to reveal the impact of PARP14 on RAD51-mediated protection of stalled replication forks. We hypothesize that PARP14 interferes with BRCA-independent stabilization of RAD51 on reversed forks, to enhance their degradation. Aim 2 is to uncover how PARP14 engages MRE11 for nucleolytic degradation of damaged forks. We hypothesize that PARP14 binds to stalled replication forks in BRCA-deficient cells and recruits MRE11 to initiate nucleolytic degradation of nascent DNA at these structures. Aim 3 is to elucidate the role of KU in fork protection against nucleolytic resection by EXO1 and MRE11. We hypothesize that KU binding to reversed forks protects them against EXO1-mediated degradation, but enables nascent strand resection by the MRE11-PARP14 complex. Since DNA damaging agents promote genomic instability by inducing nascent strand degradation, potentially underlying their carcinogenesis, successful accomplishment of these Specific Aims would reveal a new mechanism of genome stability and tumor suppression, centered on PARP14. It may also reveal PARP14 as a biomarker for the tumor response to radiation and genotoxic chemotherapy, in the context of the BRCA status.