Structural Biology of Genome Maintenance and DNA repair

目前，我们主要研究DNA末端加工因子Aprataxin（Aptx）的结构和功能。APTX是一种保守的真核DNA修复酶，对保护细胞免受氧化DNA损伤很重要，APTX突变导致遗传性神经退行性疾病共济失调伴眼用失用1（AOA 1）。在DNA复制和修复过程的最终步骤中，DNA连接酶通过一种机制密封DNA切口，当连接酶遇到含有氧化或DNA烷基化损伤产物的DNA末端时，该机制可以中止。这种“失败的连接”产生第二种形式的损伤，5 '-腺苷酸化的DNA末端，其被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在很大程度上仍然未知。此外，Aptx在疾病中如何失活尚不清楚。为了了解APTX的功能，我们确定了裂殖酵母Aptx-DNA-AMP-Zn复合物的结构，揭示了通过融合HIT（组氨酸三联体）核苷酸水解酶与前所未有的DNA小沟结合C2 HE锌指（Znf）形成的活性位点和DNA相互作用裂缝。这项工作突出了Aptx α-螺旋楔如何询问DNA末端/切口传感的DNA碱基堆栈。结构和突变的数据支持一个楔形支点切割的HIT-Znf催化机制5-AMP加合物的识别和去除，并建议突变影响蛋白质折叠，活性位点口袋，和枢轴Aptx功能障碍的神经退行性疾病共济失调眼失用症1（AOA 1）。我们的目标是进一步确定APTX DNA修复的分子决定因素，以及APTX如何通过结合Xrcc 1（DNA单链断裂修复，SSBR）和Xrcc 4（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。我们正在检验以下假设：1）APTX组氨酸三联体（HIT）和锌指（Znf）结构域形成复合融合催化结构域，用于DNA结构特异性切口结合、5 ′-AMP识别和DNA-去腺苷化加工，2）AOA 1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催化活性，和3）FHA结构域和FHA-HIT接头提供了将APTX DNA去腺苷化活性靶向磷酸化XRCC 4和XRCC 1 DNA修复支架的柔性链。

Currently, we are 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 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 with a 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 is 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 remains largely unknown. Furthermore, how Aptx is inactivated in disease is unknown. To understand APTX function, we determined the structure of a Schizosaccharomyces pombe Aptx-DNA-AMP-Zn complex revealing active site and DNA interaction clefts formed by fusing a HIT (histidine triad) nucleotide hydrolase with an unprecedented DNA minor groove binding C2HE Zn-finger (Znf). This work highlights how an Aptx alpha-helical wedge interrogates the DNA base stack for DNA end/nick sensing. Structural and mutational data support a wedge-pivot-cut HIT-Znf catalytic mechanism for 5′-AMP adduct recognition and removal, and suggest mutations impacting protein folding, the active site pocket, and the pivot underlie Aptx dysfunction in the neurodegenerative disorder Ataxia Oculomotor Apraxia 1 (AOA1). We aim to further 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 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 phosphorylated XRCC4 and XRCC1 DNA repair scaffolds.