EEPD1 Repair of Stressed Replication Forks

EEPD1 修复压力复制叉

• 批准号: 10585067
• 负责人: Robert A Hromas
• 金额: $ 38.75万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 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 2027-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10585067
• 关键词: Address; Affect; Aging; Amino Acids; BRCA1 gene; Base Excision Repairs; Brain; Brain Injuries; Catalytic Domain; Cell Aging; Cell Death; Cell Line; Cell Survival; Cells; DNA; DNA Binding; DNA Damage; DNA Repair Pathway; DNA lesion; DNA replication fork; Dangerousness; Development; Dimerization; Divalent Cations; EXO1 gene; Electrostatics; Environment; Enzymes; Eukaryota; Excision; Excision Repair; Exposure to; Face; Genome; Genome Stability; Genomic Instability; Glioblastoma; Glioma; Human; Hypoxia; Leisures; Length; Lesion; Malignant Neoplasms; Malignant neoplasm of brain; Mediating; Minority; Mutation; Neoplastic Cell Transformation; Nucleotides; Organ; Organism; Oxidative Stress; Oxygen; Pathway interactions; Physiological; Play; Proteins; Reporting; Resected; Role; Site; Stress; Structure; Structure-Activity Relationship; Testing; X-Ray Crystallography; base; chelation; differential expression; dimer; endonuclease; homologous recombination; in vitro Assay; in vivo; inhibitor; insight; monomer; new therapeutic target; nuclease; oxidation; oxidative DNA damage; oxidative damage; public health relevance; rational design; recombinational repair; recruit; repaired; replication stress; stressor; tumor

PROJECT SUMMARY Since DNA bases are continuously damaged by oxidation, cells have evolved a robust pathway to repair this type of DNA base damage, termed base excision repair (BER). Most oxidative damage can be repaired at the cell’s leisure except at a replication fork, where oxidative damage can cause replication fork collapse. Collapsed forks are a far greater danger to the cell than oxidative damage elsewhere in the genome, but the mechanism of BER at oxidatively damaged replication forks is less well understood compared to BER elsewhere. The 5’ abasic endonuclease APE1 plays a key role in BER repair at oxidatively stressed replication forks. However, there is significant evidence for an alternative pathway; some cancers lack APE1 yet replicate without difficulty, and several aging organs lose expression of APE1 without deleterious effects. We previously found that the 5’ endonuclease EEPD1 can initiate homologous recombination (HR) repair of stressed replication forks by cleaving the lagging parental strand of a stalled fork and loading EXO1 for 5’ end resection in a BRCA1-indepednent manner. In further characterization of EEPD1, we found that it has 5’ abasic endonuclease activity similar but not identical to APE1. EEPD1 can replace APE1 in BER assays in vitro and in vivo. EEPD1 depletion also harmed the repair and restart of oxidatively damaged replication forks. EEPD1 depletion or deletion also resulted in significantly decreased cell survival in the presence of oxidative or alkylative stressors, which cause DNA lesions repaired by BER. EEPD1 has a high differential expression in glioblastoma (GBM) compared to adjacent normal brain or other cancers. GBM exist in a hypoxic environment and are sensitive to oxidative injury, and EEPD1 is required for the survival in every GBM cell line tested. Our SEC-MALS studies found that EEPD1 exists as a dimer in physiologic solution. We resolved the X-ray crystallographic structure of the EEPD1 nuclease domain to 3.0 Å. The tertiary structure of the EEPD1 monomer is similar to the AlphFold2-predicted EEPD1 nuclease domain structure. The EEPD1 crystal structure also has similarities to and distinctions from the APE1 structure. Thus, EEPD1 represents a unique opportunity to gain insight into the structural basis for abasic endonuclease activity and how this activity promotes repair of oxidatively-stressed replication forks. Understanding the structure-function relationship of EEPD1 will lead to regions to target for development of rationally designed inhibitors, for which we have candidate compounds. This is especially important in GBM, for which new therapeutic targets are sorely needed. This renewal application will assess how the structure of EEPD1 functions to repair of replication forks stressed by oxidative DNA damage in GBM cells by addressing three questions: 1) Is EEPD1 dimerization essential for its activity? 2) What EEPD1 domains mediate its 5’ abasic endonuclease activity? 3) What are the distinct roles for EEPD1 versus APE1?

项目总结 由于DNA碱基不断被氧化破坏，细胞已经进化出一条强大的途径来修复这一点 DNA碱基损伤的类型，称为碱基切除修复(BER)。大多数氧化损伤可以在 细胞的闲置状态，但在复制叉处除外，在复制叉处氧化损伤会导致复制叉处坍塌。 与基因组其他地方的氧化损伤相比，倒塌的叉子对细胞的危险要大得多，但 与BER相比，复制叉处氧化损伤的BER机制还不是很清楚 其他地方。5‘碱性内切酶APE1在氧化应激复制的误码率修复中起关键作用 叉子。然而，有重要的证据表明有另一种途径；一些癌症缺乏APE1但仍可复制 没有困难，几个老化的器官失去了APE1的表达，没有任何不良影响。我们之前 发现5‘内切酶EEPD1可启动应激状态下的同源重组(HR)修复 通过切割停滞的叉子滞后的亲本链并装载EXO1进行5‘端切除来复制叉子 以独立于BRCA1的方式。在对EEPD1的进一步表征中，我们发现它具有5‘碱基 核酸内切酶活性与APE1相似，但不完全相同。EEPD1可替代APE1用于体外和体外误码率测定 活着。EEPD1的耗尽也损害了氧化损伤的复制叉子的修复和重新启动。EEPD1 耗尽或缺失也会导致细胞存活率在氧化或缺失的情况下显著降低 烷化应激源，导致DNA损伤通过BER修复。EEPD1具有较高的差异表达 胶质母细胞瘤(GBM)与邻近的正常脑或其他癌症相比。GBM存在于低氧环境中 并且对氧化损伤敏感，EEPD1是每个被测试的GBM细胞系存活所必需的。我们的 Sec-mals研究发现EEPD1在生理溶液中以二聚体的形式存在。我们解决了X光 EEPD1核酸酶结构域的晶体结构为3.0？EEPD1的三级结构 单体类似于AlphFold2预测的EEPD1核酸酶结构域结构。EEPD1晶体 结构与APE1结构既有相似之处也有不同之处。因此，EEPD1代表唯一的 有机会深入了解基本核酸内切酶活性的结构基础，以及这种活性是如何 促进氧化应激复制叉的修复。理解结构与功能的关系 EEPD1将导致以开发合理设计的抑制剂为目标的区域，我们已经 候选化合物。这在GBM中尤其重要，对于GBM来说，新的治疗靶点非常重要 需要的。此更新应用程序将评估EEPD1的结构如何作用于复制分叉的修复 通过解决三个问题来应对GBM细胞的DNA氧化损伤：1)EEPD1二聚化 对其活性至关重要？2)什么结构域调节其5‘基本内切酶活性？3)什么 EEPD1与APE1的角色是否截然不同？