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致癌作用。BRCA 1和BRCA 2蛋白是 对于同源重组（HR）介导的DSB修复至关重要。了解的机制 BRCA通路对人类健康具有广泛的意义。当复制叉遇到受损的DNA时， 它们停止，除非被适当处理，否则它们会崩溃，导致DNA断裂和基因组不稳定。到 为了避免它们的崩溃，停滞的分叉可以通过使两个新生链彼此退火来逆转， 由DNA移位酶如ZRANB 3催化的过程。BRCA蛋白将RAD 51装载在反向叉上， 以保护DNA末端不被核酸酶MRE 11降解。保护叉子免受 降解与DNA损伤敏感性相关。因此，复制叉保护对于DNA修复至关重要 和基因组稳定性。然而，如何保护停滞的复制叉免受溶核降解， 这是一个重大的知识差距。PARP家族至少有17个成员，其中包括各种和更少的成员。 比创始成员PARP 1更了解功能。PARP 14与多种细胞 过程，但机械细节一般稀疏。我们之前已经证明PARP 14的缺失会降低HR， 提高效率并使细胞对辐射敏感。最近，我们已经确定了PARP 14的一个新的作用， 复制叉降解、基因组不稳定性和DNA损伤敏感性，这是研究的重点 应用程序.对于这个应用程序，我们的目标是了解PARP 14如何促进分叉退化， BRCA缺陷细胞的DNA损伤敏感性。我们的总体假设是PARP 14干扰了 RAD 51-MRE 11控制DNA在反向复制叉处切除以触发新生链的机制 降解，从而增强BRCA缺陷细胞的DNA损伤敏感性。目标1是揭示 PARP 14对RAD 51介导的停滞复制叉的保护作用。我们假设PARP 14干扰 与BRCA独立稳定的反向叉上的RAD 51，以提高其降解。目标二是 揭示PARP 14如何与MRE 11接合以进行受损叉的溶核降解。我们假设 PARP 14与BRCA缺陷细胞中停滞的复制叉结合，并招募MRE 11启动溶核反应 在这些结构中新生DNA的降解。目的3是阐明KU在叉保护中的作用， 通过EXO 1和MRE 11的溶核切除。我们假设KU与反向分叉的结合保护了它们 针对EXO 1介导的降解，但能够通过MRE 11-PARP 14复合物进行新生链切除。 由于DNA损伤剂通过诱导新生链降解促进基因组不稳定性， 在其致癌作用的基础上，这些特定目标的成功实现将揭示一种新的 基因组稳定性和肿瘤抑制机制，以PARP 14为中心。它也可能揭示PARP 14是一种 在BRCA状态的背景下，肿瘤对放疗和遗传毒性化疗的反应的生物标志物。

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.