Regulation of DSB repair by 53BP1

描述(申请人提供)：DNA双链断裂(DSB)如果不修复或修复不当，是基因组中最致命的损伤之一。大多数癌症治疗，包括放射治疗，都会诱导多个DSB杀死癌细胞，但在非肿瘤细胞中同时诱导DSB会导致染色体重排，这可能是治疗相关肿瘤的一个来源。DSB修复通过两条主要途径进行，即同源定向修复(HDR)和非同源末端连接(NHEJ)，它们受到仔细的调控，以避免染色体异常的形成。DSB修复调控对人类健康非常重要，因为DSB修复途径的错误选择会引发基因重排，从而促进癌症的发生，而错误的DSB修复往往会产生异常的染色体结构，从而杀死细胞。53BP1是一种DNA损伤反应因子，影响HDR和NHEJ之间的选择，促进NHEJ和抑制HDR。53BP1最近引起了人们的注意，因为它参与了用PARP抑制剂(PARPI)治疗BRCA缺陷型乳腺癌和卵巢癌，PARPI在S/G2细胞中产生DSB。BRCA1缺陷细胞对PARPI处理敏感，因为它们不能通过同源重组(HR)修复PARPI诱导的DSB，并积累由NHEJ形成的异常连锁的致死染色体。然而，当53BP1缺失时，NHEJ不适当的DSB修复减少，HR似乎恢复，PARPI治疗不再有效。本研究旨在阐明53BP1调控NHEJ和HDR平衡的机制。我们建议使用我们实验室开发的基于端粒的检测系统的独特方面来确定53BP1的三个功能方面的机制和结果。在AIM 1中，我们将询问53BP1相互作用因子Rif1如何抑制DSB中CtIP依赖的切除，以及这一调控途径对DSB修复的影响，包括在PARPI处理的BRCA1缺陷细胞中。在AIM 2中，我们将使用第二个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在DNA损伤部位/附近增加染色质流动性的能力，这是我们在端粒功能障碍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，我们将解决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对DSB修复的调节的基本方面，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.