Metnase, PIKK, and RPA Roles in DNA Damage and Replication Stress Responses

描述（由申请人提供）：大多数癌症患者接受放射和/或化疗，会导致DNA损伤，从而阻止DNA复制。正常细胞和肿瘤细胞通过激活DNA修复、细胞周期阻滞（检查点）系统来应对DNA损伤和相关的复制压力，当损伤严重时，程序性死亡途径统称为DNA损伤反应（DDR）。DDR蛋白在肿瘤抑制和基因组稳定（癌症病因学）以及肿瘤对放疗和化疗的反应（癌症治疗）中起着至关重要的作用。DDR通路在DNA损伤的反应中决定细胞命运，包括细胞存活、基因组稳定性和细胞死亡/永久性生长停滞，包括细胞凋亡、自噬、坏死、衰老或有丝分裂灾难。细胞在S期特别容易受到DNA损伤，因为大多数DNA损伤会使复制分叉停滞，造成复制压力。本研究的重点是在DNA修复、检查点激活和复制应激恢复中发挥作用的几种蛋白质。Metnase和DNA- pk最初都是通过非同源末端连接（NHEJ）来修复DNA双链断裂（DSB）。最近的研究表明，Metnase和DNA-PK（以及复制蛋白A (RPA), ATM/ATR， Chk1等）也在检查点激活和复制应激恢复中发挥作用。复制检查点防止触发新的原点，并稳定已停止的复制分叉，以防止分叉崩溃，从而有时间进行修复和重新启动。持续的复制应激可导致分叉塌陷，产生以磷酸化为标志的单端dsb

DESCRIPTION (provided by applicant): Most cancer patients receive radio- and/or chemotherapy that causes DNA damage, which blocks DNA replication. Normal and tumor cells respond to DNA damage and associated replication stress by activating DNA repair, cell cycle arrest (checkpoint) systems, and when damage is severe, programmed death pathways, collectively termed the DNA damage response (DDR). DDR proteins play crucial roles in tumor suppression and genome stabilization (cancer etiology) as well as tumor response to radio- and chemotherapy (cancer treatment). DDR pathways determine cell fates in response to DNA damage, including cell survival, genome stability, and cell death/permanent growth arrest via apoptosis, autophagy, necrosis, senescence, or mitotic catastrophe. Cells are particularly vulnerable to DNA damage during S phase because most DNA lesions stall replication forks, causing replication stress. This proposal focuses on several proteins with roles in DNA repair, checkpoint activation, and recovery from replication stress. Metnase and DNA-PK were both initially characterized for their roles in DNA double-strand break (DSB) repair by non-homologous end joining (NHEJ). Recent studies demonstrate that Metnase and DNA-PK (along with replication protein A (RPA), ATM/ATR, Chk1, and others) also function in checkpoint activation and replication stress recovery. The replication checkpoint prevents new origin firing and stabilizes stalled replication forks to prevent fork collapse, allowing time for repair and for restart. Persistent replication stress can lead to fork collapse, producing one-ended DSBs marked by phosphorylated H2AX (γ-H2AX). RPA accumulates on single-stranded DNA at stalled forks and the RPA32 subunit is phosphorylated at multiple sites by phosphoinositide 3-kinase-related protein kinases (PIKKs) DNA-PK, ATM and ATR, leading to Chk1 activation and replication arrest. Metnase also regulates Chk1 activation and replication arrest. Our central hypothesis is that Metnase, DNA-PK, and RPA operate within the DDR to influence cell fate after genotoxic stress, including cell survival, genome stability, and death pathway activation. We will determine roles of Metnase (Aim 1) and PIKK phosphorylation of RPA (Aim 2) in replication stress responses including replication arrest, fork restart, genome stability, cell survival and cell death by apoptosis. In Aim 3 we will define epistatic relationships between Metnase and PIKK/RPA pathways, and test novel combinations of replication stress agents plus DDR inhibitors to enhance killing of breast, lung, pancreatic, colon, head and neck, and leukemic tumor cells. A better understanding of how DDR factors regulate cell fate decisions will drive the development of novel cancer therapies to improve local tumor control, and reduce the risk of therapy-induced tumor progression and secondary tumor induction.