Structural Biology of Genome Maintenance and DNA repair

基因组维护和 DNA 修复的结构生物学

• 批准号: 8149120
• 负责人: Robert Williams
• 金额: $ 53.96万
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8149120
• 关键词: DNA Repair; Genome; Maintenance; structural biology

With combined biochemical, mutational, and protein structural analyses (eg. X-ray crystallography and Small angle X-ray scattering), we dissect functionally critical protein conformations, protein-protein and protein-nucleic acid interfaces. We are currently focused on examining structure/function of DNA end processing factor Aprataxin (APTX). APTX is a conserved eukaryotic DNA repair enzyme that is important for protection of cells from oxidative DNA damage, and APTX mutations cause the recessive hereditary neurodegenerative disorder Ataxia with Oculomotor Apraxia 1 (AOA1). In the ultimate step of DNA replication and repair processes, DNA ligases seal DNA nicks through an imperfect mechanism that can abort when the ligase encounters DNA termini harboring the products of oxidative or DNA-alkylation damage. Such "abortive ligation" generates a secondary form of damage, 5'-adenylated DNA-termini, which are corrected by APTX to protect genomic integrity. However, due to a lack of protein structural information, the molecular basis for APTX catalytic reversal of 5' adenylation damage, and how APTX is inactivated in the neurodegenerative disorder Ataxia with Oculomotor Apraxia 1 (AOA1) remain largely unknown. Towards understanding APTX mechanism we have developed robust overexpression systems for human and yeast aprataxin homologs and we aim to define molecular determinants of APTX DNA repair, and how APTX integrates into damage repair pathways through interactions with DNA break repair pathways through binding Xrcc1 (DNA single strand break repair, SSBR) and Xrcc4 (DNA double strand break repair, DSBR). We are specifically testing hypotheses that: 1) APTX Histidine triad (HIT) and Zinc finger (Znf) domains form a composite fused catalytic domain for DNA structure specific nick-binding, 5'-AMP recognition, and DNA-deadenylation processing, 2) AOA1 patient mutations disrupt APTX protein folding and/or directly impair APTX catalytic activities through active site distortion, and 3) The FHA domain and FHA-HIT linker provides a flexible leash targeting APTX DNA deadenylation activity to Caesin kinase 2 (CK2) phosphorylated XRCC4 and XRCC1 DNA repair scaffolds. Ionizing radiation (IR) and non-ionizing radiation from exogenous natural sources such as cosmic rays, radioactive elements in the environment, or from artificial sources including diagnostic X-rays mount a constant assault our genomes. Oxidative DNA damage from reactive oxygen species generated as by-products of mitochondrial respiration, during chronic inflammation, or upon exposure to environmental agents poses a threat to all cell types. Thus our knowledge of the DNA SSBR and DSBR repair mechanisms we are studying has critical implications for environmental health. Significantly, DNA repair defects underpin many human diseases associated with disorders of the nervous system and we are working to understand how heritable DNA repair defects impair damage surveillance and contribute to neurodegeneration.

通过合并的生化，突变和蛋白质结构分析（例如X射线晶体学和小角度X射线散射），我们在功能上进行了关键的蛋白质构象，蛋白质蛋白质和蛋白质核酸界面。目前，我们专注于检查DNA终端加工因子Aprataxin（APTX）的结构/功能。 APTX是一种保守的真核DNA修复酶，对于保护细胞免受氧化性DNA损伤很重要，APTX突变会导致隐性遗传性神经退行性疾病共济失调，其动眼作用1（AOA1）。在DNA复制和修复过程的最终步骤中，DNA连接酶通过一种不完善的机制密封DNA，当连接酶遇到具有氧化或DNA烷基化损伤产物的DNA末端时，该机制可能会流产。这种“流产的连接”产生了一种次要形式的损伤形式，即5'-腺苷酸化的DNA末端，通过APTX校正以保护基因组完整性。然而，由于缺乏蛋白质结构信息，APTX催化逆转5'腺苷酸化损伤的分子基础，以及在神经退行性疾病共济失调中使用眼动性失调1（AOA1）在神经退行性疾病中灭活的方法仍然很大。 Towards understanding APTX mechanism we have developed robust overexpression systems for human and yeast aprataxin homologs and we aim to define molecular determinants of APTX DNA repair, and how APTX integrates into damage repair pathways through interactions with DNA break repair pathways through binding Xrcc1 (DNA single strand break repair, SSBR) and Xrcc4 (DNA double strand break repair, DSBR). We are specifically testing hypotheses that: 1) APTX Histidine triad (HIT) and Zinc finger (Znf) domains form a composite fused catalytic domain for DNA structure specific nick-binding, 5'-AMP recognition, and DNA-deadenylation processing, 2) AOA1 patient mutations disrupt APTX protein folding and/or directly impair APTX catalytic activities through active site distortion, and 3) The FHA结构域和FHA-HIT接头提供了针对APTX DNA降苯式活性的柔性皮带，该链球菌2（CK2）磷酸化的XRCC4和XRCC1 DNA修复支架。 来自外源天然来源的电离辐射（IR）和非电离辐射，例如宇宙射线，环境中的放射性元件，或包括诊断X射线在内的人工来源，持续攻击我们的基因组。由线粒体呼吸，慢性炎症期间或暴露于环境剂时产生的活性氧造成的氧化DNA损伤对所有细胞类型构成威胁。因此，我们对正在研究的DNA SSBR和DSBR修复机制的了解对环境健康具有至关重要的意义。值得注意的是，DNA修复缺陷是许多与神经系统疾病相关的人类疾病的基础，我们正在努力了解可遗传的DNA修复缺陷如何损害损伤监测并导致神经变性。