EEPD1 Repair of Stressed Replication Forks

EEPD1 修复压力复制叉

• 批准号: 9082924
• 负责人: Robert A Hromas
• 金额: $ 34.31万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-01 至 2021-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9082924
• 关键词: Address; BRCA1 gene; Biology; CDC2 Protein Kinase; CHEK1 gene; Camptothecin; Cell Cycle; Cells; Cessation of life; Chromosome abnormality; Cisplatin; Cleaved cell; Colon Carcinoma; Colon Lymphoma; Complex; Cytogenetics; DNA; DNA Damage; DNA Double Strand Break; DNA biosynthesis; DNA replication fork; DNA-PKcs; Data; Deoxyribonuclease I; Developmental Delay Disorders; Double Strand Break Repair; Embryo; Embryonic Development; Escherichia coli; Excision; Exonuclease; G22P1 gene; Genetic Recombination; Genome Stability; Genomic Instability; Genomics; Homologous Gene; Malignant Neoplasms; Mammals; Mediating; Modeling; Neoplasms; Nonhomologous DNA End Joining; Nuclear; Organism; Pathway interactions; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphorylation Site; Play; Process; Proliferating; Proteins; Radiation; Recombinants; Recruitment Activity; Resistance; Role; Sequence Homologs; Source; Stress; Structure; Surgical Flaps; Ultraviolet Rays; Zebrafish; base; cancer cell; cancer therapy; chemotherapy; chromosome fusion; endonuclease; falls; homologous recombination; hydroxyurea; inhibitor/antagonist; insight; neoplastic; novel; nuclease; p53-binding protein 1; prevent; public health relevance; recombinational repair; repaired; response; stressor

 DESCRIPTION (provided by applicant): Replication fork stalling and collapse is a major source of genomic instability and subsequent neoplasia. Such stressed forks can be conservatively repaired and restarted using homologous recombination (HR) repair, initiated by generating an endogenous nick at the fork junction. While there are candidate nucleases for generating this DSB, most mammalian stressed forks can be restarted without these nucleases, and the origin of this nick remains undefined. This nick permits the 5' end resection that initiate HR and prohibits non- homologous end-joining (NHEJ), the pathway leading to genomic instability during replication stress. We found that the previously uncharacterized nuclease EEPD1 is an essential initiating step in HR repair of stalled forks. EEPD1 has two amino terminal Helix-hairpin-Helix domains that resemble prokaryotic fork repair component RuvA and a carboxy terminal DNase I-like endonuclease. After replication stress, EEPD1 is recruited to stalled forks and increases nicking. EEPD1 enhances 5' DNA end resection and restart of stalled forks. It is required for proper ATR and CHK1 phosphorylation, and γ-H2Ax, Rad51 and RPA32 foci formation. Consistent with this, purified recombinant EEPD1 protein has unique 5' DNA endonuclease activity which enhances Exo1 nuclease activity at fork structures. Both Exo1 and EEPD1 (T134) are phosphorylated in S/G2 by CDK1. EEPD1 depletion generates nuclear and cytogenetic anomalies, made worse by replication stress. Inhibiting 53BP1 partially rescues the nuclear and cytogenetic abnormalities seen with EEPD1 depletion. Examining the role of EEPD1 in a rapidly proliferating organism, we found that Zebrafish embryos depleted of EEPD1 demonstrate significant nuclear abnormalities, and increased developmental delay and death. These data demonstrate that genomic stability during replication stress is maintained by EEPD1, yet the mechanism by which EEPD1 performs this is not defined. This project will assess how EEPD1 is regulated in 4 aims: 1) What structures of EEPD1 are important for stressed replication fork repair? 2) How does CDK1 phosphorylation of EEPD1 regulate stressed fork repair? 3) What role does EEPD1 play in pathway choice for stressed fork repair? 4) Does EEPD1 mediate response to replication stressing cancer therapy?

 描述（由申请人提供）：复制叉停滞和塌陷是基因组不稳定性和后续瘤形成的主要来源。这种应激叉可以保守地修复，并使用同源重组（HR）修复重新启动，通过在叉连接处产生内源性切口来启动。虽然存在用于产生这种DSB的候选核酸酶，但大多数哺乳动物应激叉可以在没有这些核酸酶的情况下重新启动，并且这种切口的起源仍然不确定。该切口允许启动HR的5'末端切除并阻止非同源末端连接（NHEJ），非同源末端连接是在复制应激期间导致基因组不稳定性的途径。我们发现，以前未表征的核酸酶EEPD 1是一个重要的启动步骤，在HR修复停滞的叉。EEPD 1具有两个类似于原核生物叉修复组分RuvA的氨基末端螺旋发夹Hispins结构域和一个羧基末端DNase I样内切核酸酶。在复制应激后，EEPD 1被招募到停滞的分叉并增加切口。EEPD 1增强5' DNA末端切除和停滞叉的重新启动。它是正确的ATR和CHK 1磷酸化以及γ-H2 Ax、Rad 51和RPA 32灶形成所必需的。与此一致，纯化的重组EEPD 1蛋白具有独特的5' DNA内切核酸酶活性，其增强了叉结构处的Exo 1核酸酶活性。Exo 1和EEPD 1（T134）在S/G2中均被CDK 1磷酸化。EEPD 1缺失产生核和细胞遗传学异常，复制应激使其变得更糟。抑制53 BP 1部分挽救了EEPD 1缺失所见的核和细胞遗传学异常。研究EEPD 1在快速增殖的生物体中的作用，我们发现EEPD 1耗尽的斑马鱼胚胎表现出显着的核异常，并增加发育延迟和死亡。这些数据表明，在复制应激过程中的基因组稳定性是由EEPD 1维持的，但EEPD 1执行这一功能的机制尚未确定。本项目将评估EEPD 1是如何调节的4个目标：1）EEPD 1的哪些结构对应激复制叉修复很重要？2)EEPD 1的CDK 1磷酸化如何调节应激叉修复？3)EEPD 1在应激叉修复的途径选择中起什么作用？4)EEPD 1介导对复制应激癌症治疗的反应吗？