Mechanism of APE1 in DNA damage response

项目总结/摘要 DNA单链断裂（SSB）可由氧化应激或各种氧化应激的中间产物引起。 DNA代谢包括DNA复制和损伤修复。未修复的氧化性DNA损伤和 SSB可能导致复制叉崩溃或转录机制故障。氧化性DNA损伤 和SSB是基因组稳定性的关键挑战，当它们不稳定时， 迅速或适当地修复。检查点的分子机制研究进展 对氧化性DNA损伤和SSB的信号传导和调节机制是有限的， 由于缺乏可行的实验系统，而APE 1（AP内切核酸酶1）是 已知其在碱基切除修复和转录调控中的关键功能，目前它是 目前还不清楚APE 1是否在DNA损伤反应（DDR）通路中起重要作用。我们的出版 工作和大量的初步数据表明，APE 1是激活ATR依赖性 DDR途径在氧化应激中的作用，即一个独特的ATR-Chk 1检查点反应被一个定义的 基于质粒的SSB结构，并且APE 1与ATRIP和TopBP 1相关联。我们的主要假设是 APE 1在响应氧化应激和SSB的检查点信号传导中起着至关重要的作用。测试 我们的具体目标包括：（1）确定APE 1是否在 通过其对SSB信号传导的核酸外切酶活性，在3 '-5方向上启动SSB末端切除;（2） 确定APE 1在DDR通路中如何与ATRIP相互作用，以及（3）确定TopBP 1在DDR通路中如何与ATRIP相互作用。 调节以激活ATR-Chk 1检查点信号，以及APE 1在DDR中的作用是否 在胰腺癌细胞中是保守的我们建立了两种互补的方法来研究 检查点信号通路：（1）过氧化氢诱导的多个SSB随机分布在 染色质在复制非洲爪蟾LSS系统，和（2）质粒为基础的位点特异性SSB结构在复制非洲爪蟾LSS系统。 非复制型Xenopus HSS系统。使用创新的生化和结构功能分析， 非洲爪蟾卵提取物，我们将展示如何氧化DNA损伤和SSBs的认识， 由APE 1与ATRIP和TopBP 1协调处理以调节检查点信令。我们将 也验证了我们从非洲爪蟾卵提取物系统在哺乳动物细胞，包括胰腺细胞中的发现。 癌细胞这项研究项目的预期成果将帮助我们更好地了解如何 基因组稳定性在对氧化性DNA损伤和SSB的细胞应答中得以维持。总之，这 研究项目将在概念上推进我们关于癌症如何发展的科学知识， 新的治疗策略，特别是胰腺癌。

Project Summary/Abstract DNA single-strand breaks (SSBs) can be caused by oxidative stress, or intermediate products of various DNA metabolisms including DNA replication and damage repair. Unrepaired oxidative DNA damage and SSBs may result in replication fork collapse or transcription machinery failure. Oxidative DNA damage and SSBs are critical challenges to genomic stability and can lead to tumorigenesis when they are not repaired quickly or properly. Current understanding of molecular mechanisms underlying checkpoint signaling and regulatory mechanisms in response to oxidative DNA damage and SSBs is limited or indirect because of the lack of feasible experimental systems. Whereas APE1 (AP endonuclease 1) is known for its critical functions in base excision repair and transcriptional regulation, it is currently unknown whether APE1 plays an essential role in DNA damage response (DDR) pathway. Our published work and substantial preliminary data suggest that APE1 is essential for activating the ATR-dependent DDR pathway in oxidative stress, that a distinct ATR-Chk1 checkpoint response is activated by a defined plasmid-based SSB structure, and that APE1 associates with ATRIP and TopBP1. Our major hypothesis is that APE1 plays an vital role in checkpoint signaling in response to oxidative stress and SSBs. To test this directly, our specific aims include: (1) to determine whether APE1 plays an important role in the initiation of SSB end resection in the 3'-5 direction via its exonuclease activity for the SSB signaling; (2) to determine how APE1 interacts with ATRIP in DDR pathway, and (3) to determine how TopBP1 is regulated to activate the ATR-Chk1 checkpoint signaling and whether the role of APE1 in DDR is conserved in pancreatic cancer cells. We have established two complementary approaches to study checkpoint signaling pathway: (1) hydrogen peroxide-induced multiple SSBs randomly distributed on chromatin in a replicating Xenopus LSS system, and (2) plasmid-based site-specific SSB structures in a nonreplicating Xenopus HSS system. Using innovative biochemical and structure-function analysis in Xenopus egg extracts, we will demonstrate how oxidative DNA damage and SSBs are recognized and processed by APE1 in coordination with ATRIP and TopBP1 to regulate checkpoint signaling. We will also validate our findings from Xenopus egg extract system in mammalian cells including pancreatic cancer cells. The anticipated outcomes of this research project will help us better understand how genome stability is maintained in cellular response to oxidative DNA damage and SSBs. All together, this research project will advance our scientific knowledge conceptually on how cancers develop, and open avenues to new therapeutic strategies, especially for pancreatic cancer.