Structural Biology of Genome Maintenance and DNA repair

结合生化、突变和蛋白质结构分析(例如；x射线晶体学和小角x射线散射)，我们解剖功能关键的蛋白质构象，蛋白质-蛋白质和蛋白质-核酸界面。我们目前主要研究DNA末端加工因子Aprataxin （APTX）的结构/功能。APTX是一种保守的真核DNA修复酶，对保护细胞免受氧化性DNA损伤起重要作用，APTX突变可导致隐性遗传性神经退行性疾病共济失调伴动眼肌失用症1 （AOA1）。在DNA复制和修复过程的最后一步，DNA连接酶通过一种不完善的机制来封闭DNA缺口，当连接酶遇到含有氧化或DNA烷基化损伤产物的DNA末端时，这种机制可能会中断。这种“流产的结扎”产生了第二种形式的损伤，5'-腺苷化的dna末端，由APTX纠正以保护基因组的完整性。然而，由于缺乏蛋白质结构信息，APTX催化逆转5'腺苷化损伤的分子基础，以及APTX如何在神经退行性疾病共济失调伴眼动失用1 （AOA1）中失活，在很大程度上仍然未知。为了理解APTX的机制，我们开发了人类和酵母aprataxin同源物的强大过表达系统，我们的目标是定义APTX DNA修复的分子决定因素，以及APTX如何通过结合Xrcc1 （DNA单链断裂修复，SSBR）和Xrcc4 （DNA双链断裂修复，DSBR）与DNA断裂修复途径相互作用，整合到损伤修复途径中。我们具体测试的假设是：1) APTX组氨酸三联体（HIT）和锌指结构域（Znf）形成复合融合催化结构域，用于DNA结构特异性的镍结合、5'-AMP识别和DNA死基化加工；2)AOA1患者突变破坏APTX蛋白折叠和/或通过活性位点畸变直接损害APTX的催化活性。3) FHA结构域和FHA- hit连接子为CK2磷酸化的XRCC4和XRCC1 DNA修复支架提供了一个灵活的靶向APTX DNA死化活性的链带。来自外源性自然来源的电离辐射（IR）和非电离辐射，如宇宙射线，环境中的放射性元素，或来自包括诊断x射线在内的人工来源，不断攻击我们的基因组。在慢性炎症或暴露于环境因素时，线粒体呼吸的副产物活性氧产生的氧化DNA损伤对所有类型的细胞都构成威胁。因此，我们正在研究的DNA SSBR和DSBR修复机制的知识对环境健康具有重要意义。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，我们正在努力了解遗传性DNA修复缺陷是如何损害损伤监测并导致神经退行性变的。

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.