Mechanism of APE1 in DNA damage response

APE1在DNA损伤反应中的机制

基本信息

批准号：
9492890
负责人：
Shan Yan
金额：
$ 34.18万
依托单位：
UNIVERSITY OF NORTH CAROLINA CHARLOTTE
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-02-13 至 2023-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9492890
关键词：
APEXL2 Gene B-Lymphocytes Base Excision Repairs Binding Biochemical Cancer cell line Cell Cycle Cell Line Cells Chromatin DNA DNA Damage DNA Repair DNA Repair Pathway DNA Replication Damage DNA Single Strand Break DNA replication fork DNA-(apurinic or apyrimidinic site) lyase Data Disease Excision Exonuclease Failure Genetic Transcription Genome Stability Genomic Instability Goals Human Hydrogen Peroxide In Vitro Knowledge Lead Learning Malignant Neoplasms Malignant neoplasm of pancreas Mammalian Cell Mediating Metabolism Molecular Mus Neurodegenerative Disorders Outcomes Research Oxidation-Reduction Oxidative Stress Pathway interactions Phase Phosphodiesterase I Plasmids Play Process Publishing Rana Reactive Oxygen Species Recombinant Proteins Research Project Grants Role Signal Pathway Signal Transduction Site Speed Structure System TREX1 gene Testing Time Transcriptional Regulation Work Xenopus base cancer cell cancer therapy chemotherapeutic agent egg environmental agent inhibitor/antagonist innovation insight novel novel therapeutic intervention oxidative DNA damage pancreatic cancer cells prevent recruit repaired response tumorigenesis

项目摘要

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.

项目概要/摘要 DNA 单链断裂 (SSB) 可能是由氧化应激或各种中间产物引起的 DNA 代谢包括 DNA 复制和损伤修复。未修复的氧化DNA损伤和 SSB 可能会导致复制叉崩溃或转录机器故障。 DNA氧化损伤和 SSB 是基因组稳定性的关键挑战，当它们不存在时可能导致肿瘤发生快速或正确地修复。目前对检查点分子机制的理解响应氧化 DNA 损伤和 SSB 的信号传导和调节机制有限或间接原因是缺乏可行的实验系统。而 APE1（AP 核酸内切酶 1）是它以其在碱基切除修复和转录调控中的关键功能而闻名，目前尚不清楚 APE1 是否在 DNA 损伤反应 (DDR) 途径中发挥重要作用。我们发表的工作和大量初步数据表明 APE1 对于激活 ATR 依赖性氧化应激中的 DDR 通路，独特的 ATR-Chk1 检查点反应由定义的激活基于质粒的 SSB 结构，并且 APE1 与 ATRIP 和 TopBP1 相关。我们的主要假设 APE1 在响应氧化应激和 SSB 的检查点信号传导中发挥着至关重要的作用。测试直接地说，我们的具体目标包括：（1）确定APE1是否在通过其对 SSB 信号传导的核酸外切酶活性，启动 3'-5 方向的 SSB 末端切除； (2) 确定 APE1 如何与 DDR 通路中的 ATRIP 相互作用，以及 (3) 确定 TopBP1 如何作用调节激活 ATR-Chk1 检查点信号传导以及 APE1 在 DDR 中的作用是否在胰腺癌细胞中保守。我们建立了两种互补的研究方法检查点信号通路：（1）过氧化氢诱导的多个SSB随机分布在复制爪蟾 LSS 系统中的染色质，以及 (2) 复制中基于质粒的位点特异性 SSB 结构非复制型 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，我们将演示如何识别和识别氧化 DNA 损伤和 SSB 由 APE1 与 ATRIP 和 TopBP1 协调处理以调节检查点信号传导。我们将还验证了我们在哺乳动物细胞（包括胰腺细胞）中从非洲爪蟾卵提取物系统中获得的发现癌细胞。该研究项目的预期成果将帮助我们更好地了解如何细胞对氧化 DNA 损伤和 SSB 的反应维持了基因组稳定性。总而言之，这研究项目将从概念上推进我们关于癌症如何发展的科学知识，并开放新的治疗策略的途径，特别是对于胰腺癌。