Regulation of DSB repair by 53BP1

描述（由申请人提供）：DNA双链断裂（DSBs）是基因组中最致命的病变之一，如果它们没有被修复或如果它们的修复被错误地执行。大多数癌症治疗，包括放射治疗，诱导多个dsb杀死癌细胞，但在非肿瘤细胞中同时诱导dsb可导致染色体重排，这可能是治疗患者治疗相关肿瘤的一个来源。DSB修复主要通过两种途径进行，即同源定向修复（HDR）和非同源末端连接（NHEJ），这两种途径受到精心调节以避免染色体畸变的形成。DSB修复调控对人类健康非常重要，因为DSB修复途径的选择错误会引发基因重排，从而促进癌症的发生，而DSB修复错误往往会产生异常的染色体结构，从而杀死细胞。53BP1是一种影响HDR和NHEJ选择、促进NHEJ和抑制HDR的DNA损伤应答因子，本研究关注53BP1对DSB修复的调控。53BP1最近引起了人们的关注，因为它参与使用PARP抑制剂（PARPi）治疗brca缺乏的乳腺癌和卵巢癌，PARP抑制剂（PARPi）在S/G2中产生dsb。brca1缺陷细胞对PARPi治疗敏感，因为它们不能通过同源重组（HR）修复PARPi诱导的DSBs，并积累由NHEJ形成的异常连接的致死染色体。然而，当53BP1缺失时，NHEJ对DSB的不适当修复减少，HR似乎得到恢复，PARPi治疗不再有效。本研究旨在阐明53BP1控制NHEJ和HDR之间平衡的机制。我们建议使用我们实验室开发的基于端粒的分析系统的独特方面来确定53BP1的三个功能方面的机制和后果。在AIM 1中，我们将探讨53bp1相互作用因子Rif1如何抑制DSB中ctip依赖性切除，以及这种调节途径对DSB修复的后果，包括在parpi处理的brca1缺陷细胞中。在AIM 2中，我们将使用第二种新开发的基于端粒的系统来确定53BP1如何抑制独立于CtIP的5'切除途径。最后，在AIM 3中，我们将重点关注53BP1增加DNA损伤位点/附近染色质流动性的能力，这是我们在端粒功能障碍的背景下发现的一个属性。53BP1改变染色质运动的机制将被确定，我们将研究这一途径在多大程度上影响全基因组dsb的修复。这些实验旨在深入了解53BP1调控DSB修复的基本方面，最终目的是为使用PARP抑制剂治疗乳腺癌和卵巢癌以及其他癌症治疗提供有价值的信息，包括放射治疗，其中DSB修复是治疗结果的核心。

DESCRIPTION (provided by applicant): Double-strand breaks in DNA (DSBs) are among the most lethal lesions in the genome if they are not repaired or if their repair is executed incorrectly. Most cancer therapies, including radiation therapy, induces multiple DSBs to kill cancer cells but concurrent induction of DSBs in non-tumor cells can result in chromosome rearrangements that might be a source of therapy related tumors in treated patients. DSB repair takes place through two main pathways, homology-directed repair (HDR) and non-homologous end-joining (NHEJ) that are carefully regulated to avoid the formation of chromosomal aberrations. DSB repair regulation is of great importance to human health since errors in the choice of DSB repair pathway can incite gene rearrangements that promote cancer and faulty DSB repair often generates aberrant chromosomal structures that kill cells. This proposal is focused on the regulation of DSB repair by 53BP1, a DNA damage response factor that affects the choice between HDR and NHEJ, promoting NHEJ and inhibiting HDR. 53BP1 has recently attracted attention because of its involvement in the treatment of BRCA-deficient breast and ovarian cancers with PARP inhibitors (PARPi) that generate DSBs in S/G2. Brca1-deficient cells are sensitive to PARPi treatment because they fail to repair PARPi-induced DSBs by homologous recombination (HR) and accumulate aberrantly linked lethal chromosomes formed by NHEJ. However, when 53BP1 is absent, inappropriate DSB repair by NHEJ is diminished, HR appears to be restored, and the PARPi treatment is no longer effective. This proposal aims to elucidate the mechanisms by which 53BP1 controls the balance between NHEJ and HDR. We propose to use unique aspects of a telomere-based assay system developed in our laboratory to determine the mechanism and consequences of three functional aspects of 53BP1. In AIM 1, we will ask how the 53BP1-interacting factor Rif1 inhibits CtIP-dependent resection at DSBs and what the consequences are of this regulatory pathway for DSB repair, including in PARPi-treated Brca1-deficient cells. In AIM 2, we will use a second, newly-developed telomere-based system to determine how 53BP1 inhibits a 5' resection pathway that is independent of CtIP. Finally, in AIM 3, we will focus on the ability of 53BP1 to increase the mobility of chromatin at/near sites of DNA damage, an attribute we discovered in the context of telomere dysfunction. The mechanism by which 53BP1 acts to change chromatin movement will be determined and we will address to what extent this pathway affects the repair of genome-wide DSBs. The experiments are designed to gain insights into fundamental aspects of the regulation of DSB repair by 53BP1 with the ultimate objective to provide information valuable to the use of PARP inhibitors for the treatment of breast and ovarian cancer and other cancer therapies, including radiation therapy in which DSB repair is central to the treatment outcome.