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: 7389431
• 负责人: Tadahide Izumi
• 金额: $ 11.62万
• 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/7389431
• 关键词: Abbreviations; Acetylation; Adenosine Diphosphate Ribose; Affect; Affinity; Alleles; Amino Acids; Base Excision Repairs; Binding; Biological; Biological Assay; Brain; Catalysis; Cells; Chromatin Structure; Cleaved cell; Complex; Condition; Cytoprotection; DNA; DNA Binding; DNA Binding Domain; DNA Damage; DNA Footprint; DNA Single Strand Break; Defense Mechanisms; Disease; EP300 gene; Fluorescence Anisotropy; Functional disorder; Funding; Generations; Goals; In Vitro; Inflammation; Inflammatory Response; Ischemia; Lesion; Link; Lysine; Mammalian Cell; Mammals; Measurement; Mediating; Modification; Mutation; N-terminal; Necrosis; Nuclear; Nuclear Protein; Nuclear Proteins; Nucleosomes; Organism; Pathway interactions; Pattern; Physiological; Physiological reperfusion; Play; Poly(ADP-ribose) Polymerases; Predisposition; Prevention; Process; Proteins; Reactive Oxygen Species; Reagent; Recovery; Reperfusion Therapy; Reporting; Respiration; Role; Side; Single Strand Break Repair; Site; Structure; Testing; Toxic effect; age related; cancer risk; cytotoxicity; endonuclease; histone acetyltransferase; human APEX1 protein; improved; in vivo; inorganic phosphate; mutant; novel; oxidative DNA damage; phosphoglycolate; prevent; repaired; response

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 thc 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 APE I's affinity for the cleaved DNA. The central hypothesis of this project is that APEI'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 overactlvation in vitro and in vivo. We will determine the binding patterns and association constants of APE1 and PARP for various types of SSBs by electorophoretic 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.

DNA单链断裂（SSB），由反应性氧（ROS）直接或间接产生，激活 涉及在DNA碱基切除修复（BER）中的作用的聚（ADP-核糖）聚合酶（PARP）。 但是，如果过度活化，PARP可能会导致许多病理生理的坏死和炎症 缺血/再灌注等疾病。我们的研究表明THC哺乳动物肾上腺素/肾上腺素素 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 受PARP认可的病变。我们还发现，APE1可以被组蛋白乙酰化 乙酰基转移酶P300，降低了猿I对切割DNA的亲和力。中心假设 项目是APEI与SSB结合的能力对于从ROS生成的DNA中恢复细胞至关重要 损害，以保护细胞免受PARP过度活化引起的坏死。我们的假设 强调非酶APE1函数的作用，并将通过研究（1）APE1如何进行测试 与PARP竞争SSB结合； （2）乙酰化如何调节APE1活性； （3）APE1如何 在体外和体内抑制PARP过度分泌。我们将确定结合模式和关联 APE1和PARP的常数用于各种类型的SSB，通过选举流动性和DNA足迹 测定和荧光各向异性测量。各种特定地点的APE1突变体，包括 将检查多态性等位基因，以识别和阐明氨基酸侧链的作用至关重要 SSB结合，我们的初步结果暗示这是独立于催化的调节。 DNA- PARP的结合结构域也将分别以其结合机理为特征。我们将检查 核小体结构如何影响这些蛋白质对SSB的可及性。我们将执行细胞生物学 了解APE1水平对PARP激活和ROS诱导的细胞毒性的影响的研究 和其他造成损伤的试剂。这些研究将有助于我们了解细胞防御机制 更精确地反对ROS，并将推进我们的长期目标：将蜂窝ber的能力与 癌症风险和与年龄有关的病理生理学，并寻求更好的预防方法。