Repair of oxidative DNA damage in mammalian cells

修复哺乳动物细胞中的氧化DNA损伤

• 批准号: 7248355
• 负责人: Tadahide Izumi
• 金额: $ 1.9万
• 依托单位: LSU HEALTH SCIENCES CENTER
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-03-10 至 2008-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7248355
• 关键词: DNA damage; DNA repair; acetylation; cell line; cells; endonuclease; fibroblasts; free radical oxygen; gel electrophoresis; gel mobility shift assay; necrosis; nucleosomes; oxidation; pentosyltransferase; surface plasmon resonance; tissue /cell culture; transfection

DESCRIPTION (provided by applicant): DNA single-strand breaks (SSBs), generated by reactive oxygen species (ROS) directly or indirectly, activate poly (ADP-ribose) polymerase (PARP) whose role in the DNA base excision repair (BER) is implicated. However, if overactivated, PARP can cause necrosis and inflammation in many pathophysiological conditions such as ischemia/reperfusion. Our studies indicate that the mammalian apurinic/apyrimidinic endonuclease (APE1), an essential BER protein, forms a stable complex with SSBs, and thus protects the lesions from being recognized by PARP. We have also found that APE1 can be acetylated by the histone acetyltransferase p300, which decreases APE1's affinity for the cleaved DNA. The central hypothesis of this project is that APE1's ability to bind to SSBs is crucial for cellular recovery from ROS-generated DNA damage, and for protection of cells from necrosis caused by PARP overactivation. Our hypothesis emphasizes the role of non-enzymatic APE1 functions, and will be tested by investigating (1) how APE1 competes with PARP for SSB binding; (2) how the acetylation modulates APE1 activity; and (3) how APE1 suppresses PARP overactivation in vitro and in vivo. We will determine the binding patterns and association constants of APE1 and PARP for various types of SSBs by electrophoretic mobility and DNA footprinting assays, and by fluorescence anisotropy measurement. Various site-specific APE1 mutants, including polymorphic alleles, will be examined to identify and elucidate the role of amino acid side chains critical for SSB binding, which our preliminary results imply is modulated independently of catalysis. The DNA binding domain of PARP will also be separately characterized for its binding mechanism. We will examine how nucleosomal structure affects the accessibility of these proteins to SSBs. We will perform cell biological studies to understand the effects of the APE1 level on PARP activation and the cytotoxicity induced by ROS and other damage-inducing reagents. These studies will help us understand the cellular defense mechanism against ROS more precisely, and will advance our long-term goal: to correlate the cellular BER capacity to cancer risk and age-related pathophysiology, and to seek better ways for their prevention.

描述（由申请人提供）：DNA单链断裂（SSB），由反应性氧（ROS）直接或间接产生，激活涉及DNA碱基切除修复（BER）的作用的Poly（ADP-核糖）聚合酶（PARP）。但是，如果过度活化，PARP会在许多病理生理状况（例如缺血/再灌注）中引起坏死和炎症。我们的研究表明，哺乳动物的肾上腺素/肾上腺素核酸内切酶（APE1）是一种必需的BER蛋白，与SSB形成稳定的复合物，从而保护病变免于被PARP识别。我们还发现，APE1可以通过组蛋白乙酰转移酶P300进行乙酰基，从而降低了APE1对切割DNA的亲和力。该项目的中心假设是，APE1与SSB结合的能力对于从ROS生成的DNA损伤中恢复细胞至关重要，并且对于保护细胞免受PARP过度激活引起的坏死的能力至关重要。我们的假设强调了非酶APE1功能的作用，并将通过研究（1）APE1如何与PARP竞争SSB结合来测试； （2）乙酰化如何调节APE1活性； （3）APE1如何在体外和体内抑制PARP过度活化。我们将通过电泳迁移率和DNA足迹测定法以及通过荧光各向异性测量来确定各种SSB的APE1和PARP的结合模式和关联常数。将检查各种位点特异性的APE1突变体，包括多态性等位基因，以识别和阐明对SSB结合至关重要的氨基酸侧链的作用，我们的初步结果暗示与催化无关。 PARP的DNA结合结构域也将其结合机理分别表征。我们将研究核小体结构如何影响这些蛋白质对SSB的可及性。我们将进行细胞生物学研究，以了解APE1水平对PARP激活的影响以及ROS和其他诱导损伤试剂引起的细胞毒性。这些研究将帮助我们更精确地了解针对ROS的细胞防御机制，并将促进我们的长期目标：将细胞BER的能力与癌症风险和与年龄有关的病理生理学相关联，并寻求更好的预防方法。