EEPD1 Repair of Stressed Replication Forks

项目摘要 由于DNA碱基不断被氧化损伤，细胞已经进化出一种强大的途径来修复这种损伤。 一种DNA碱基损伤，称为碱基切除修复（BER）。大多数氧化损伤可以在 细胞的休闲除了在复制叉，氧化损伤可导致复制叉崩溃。 折叠的叉子对细胞的危险远大于基因组其他部位的氧化损伤， 与BER相比，氧化损伤复制叉处的BER机制不太清楚 其他地方5'脱碱基核酸内切酶APE 1在氧化应激复制的BER修复中起关键作用 叉子然而，有重要的证据表明存在替代途径;一些癌症缺乏APE 1，但可以复制 并且几个衰老器官失去APE 1的表达而没有有害作用。我们之前 发现5'内切核酸酶EEPD 1可以启动应激的同源重组（HR）修复， 通过切割停滞叉的落后亲本链并加载EXO 1进行5'端切除来复制叉 与BRCA 1无关。在对EEPD 1的进一步表征中，我们发现它具有5'脱碱基， 核酸内切酶活性与APE 1相似但不相同。EEPD 1可以在体外BER测定中取代APE 1， vivo. EEPD 1缺失也损害了氧化损伤的复制叉的修复和重新启动。EEPD1 消耗或缺失也导致在氧化或还原存在下细胞存活率显著降低。 烷基化应激源，其导致BER修复的DNA损伤。EEPD 1在人乳腺癌组织中有高差异表达， 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，并且EEPD 1是每种测试的GBM细胞系中存活所需的。我们 SEC-MALS研究发现EEPD 1在生理溶液中以二聚体形式存在。我们通过X光 将EEPD 1核酸酶结构域的晶体结构调整至3.0 μ m。EEPD 1的三级结构 单体类似于AlphFold 2预测的EEPD 1核酸酶结构域结构。EEPD 1晶体 结构与APE 1结构既有相似之处，又有区别。因此，EEPD 1表示唯一的 有机会深入了解无碱基核酸内切酶活性的结构基础以及这种活性如何 促进氧化应激复制叉的修复。了解结构-功能关系 EEPD 1将导致区域的目标，为发展合理设计的抑制剂，我们已经 候选化合物。这在GBM中尤其重要，对于GBM，新的治疗靶点非常重要。 needed.此更新应用程序将评估EEPD 1的结构如何修复复制叉 通过解决以下三个问题来强调GBM细胞中的氧化DNA损伤：1）EEPD 1二聚化 对于其活动至关重要？2)EEPD 1的哪些结构域介导其5'脱碱基核酸内切酶活性？3)什么 EEPD 1与APE 1的作用不同吗？

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?