Mechanism of APE1 in DNA damage response

项目摘要/摘要 DNA单链断裂(SSB)可由氧化应激或各种 DNA代谢包括DNA复制和损伤修复。未修复的氧化DNA损伤和 SSB可能会导致复制分叉崩溃或转录机器故障。DNA氧化损伤 SSB是对基因组稳定性的关键挑战，当它们不是时，可能会导致肿瘤发生 迅速地或适当地修复的。当前对检查点分子机制的理解 响应DNA氧化损伤和SSB的信号和调控机制是有限的或 间接因为缺乏可行的实验体系。而APE1(AP核酸内切酶1)是 以其在碱基切除修复和转录调控中的关键功能而闻名，目前 目前尚不清楚APE1是否在DNA损伤反应(DDR)通路中发挥重要作用。我们出版的 工作和大量的初步数据表明，APE1是激活ATR依赖的必不可少的 氧化应激中的DDR途径，即不同的ATR-Chk1检查点反应由定义的 以质粒为基础的SSB结构，APE1与Trip和TopBP1结合。我们的主要假设 APE1在应对氧化应激和SSB的检查点信号中起着至关重要的作用。为了测试 直接来说，我们的具体目标包括：(1)确定APE1是否在 通过SSB信号的核酸外切酶活性启动SSB末端切除；(2) 确定APE1如何与DDR途径中的ATIP相互作用，以及(3)确定TopBP1如何 调节激活ATR-Chk1检查点信号，以及APE1在DDR中的作用是否 在胰腺癌细胞中保守。我们已经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随机分布的SSB 复制的非洲爪哇LSS系统中的染色质，以及(2)基于质粒的位置特异的SSB结构 非复制型非洲爪哇HSS系统。利用创新的生化和结构-功能分析 ，我们将演示氧化DNA损伤和SSB是如何识别和 由APE1与TRIP和TopBP1协调处理，以调节检查点信令。我们会 也在包括胰腺在内的哺乳动物细胞中验证了我们从非洲爪哇卵提取系统中的发现 癌细胞。这一研究项目的预期结果将帮助我们更好地了解 基因组的稳定性在细胞对DNA氧化损伤和SSB的反应中得以维持。总而言之，这 研究项目将从概念上推进我们对癌症如何发展的科学知识，并开放 新的治疗策略的途径，特别是对胰腺癌。

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.