Structural Biology of Genome Maintenance and DNA repair

目前，我们主要研究DNA末端加工因子Tyrosyl DNA磷酸二酯酶2(Tdp2)(项目1)和Aprataxin(AptX)(项目2)的结构/功能。 项目1(Tdp2)综述和进展：拓扑异构酶II(Topo II)DNA切割/连接反应可中毒(如化疗后)，产生DNA双链断裂(DSB)，Topo II与DNA共价结合。如果不进行处理，这种蛋白质加成的DNA末端会损害DSB的修复，从而导致断裂性DSB的积累、基因组的不稳定、突变和细胞死亡。酪氨酰DNA磷酸二酯酶2(Tdp2)通过逆转5&8242；-磷酸酪氨酰(5&8242；-Y)连接的Topo II蛋白-DNA加合物来保护基因组的完整性。Tdp2在细胞Topo II耐药中发挥作用，也介导突变型P53功能表型的获得。然而，在缺乏任何Tdp2同源物的蛋白质结构信息的情况下，Tdp2拓扑II-DNA加合物修复活性的分子基础仍然不清楚。为了了解Tdp2的功能，我们测定了哺乳动物Tdp2在三种DNA结合状态下的X射线晶体结构，并通过突变和功能研究来研究Tdp2的活性，这些研究定义了Tdp2 DNA-蛋白质共价加合物识别和逆转的决定因素。总体而言，这些结果支持基于可测试结构的单一镁离子介导的催化机制，其中Tdp2通过两个结合口袋与DNA-蛋白质结合，一个结合DNA，另一个结合拓扑异构酶衍生的蛋白质加合物。Tdp2 DNA结合槽由螺旋帽和新型的β-2螺旋-βDNA损伤结合“抓握”组成，它们共同包裹着一个暴露的单链DNA末端。量身定制的DNA和加合物识别元件使Tdp2有别于相关的DNA修复内切酶，如脱氧核糖核酸内切酶1(APE1)，Tdp2将完整的磷酸二酯骨架从其活性部位排除在外，以确保选择性，并限制内或外核溶解过程。总之，我们的结果为Tdp2相关癌症化疗耐药的机制提供了洞察力，并为Tdp2抑制剂的开发建立了一个框架，这些抑制剂可以用作常用拓扑异构酶II毒物(如依托泊苷)的佐剂。 项目2(AptX)综述及进展：aptX是一种保守的真核DNA修复酶，对保护细胞免受DNA氧化损伤具有重要作用，aptX突变导致遗传性神经退行性疾病共济失调伴动眼失用症1(AOA1)。在DNA复制和修复过程的最终步骤中，DNA连接酶通过一种机制封闭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，该机制可以终止。这种“流产的连接”会产生第二种形式的损伤，即5‘-腺化DNA末端，aptX会纠正这种末端，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‘-腺基化损伤的分子基础仍然很大程度上不清楚。此外，aptX在疾病中是如何失活的还不清楚。为了了解aptX的功能，我们确定了裂殖酵母aptX-DNA-AMP-锌复合体的结构，揭示了HIT(组氨酸三联体)核苷酸水解酶与前所未有的DNA小槽结合C2HE锌指(ZIF)形成的DNA相互作用裂隙。这项工作突出了aptXα螺旋楔形体如何询问DNA碱基堆栈以检测DNA末端/缺口。结构和突变数据支持楔形枢轴切割HIT-ZIF催化机制识别和移除5&AMP加合物，并提示突变影响蛋白折叠、活性部位口袋和枢轴导致神经退行性疾病共济失调动眼失用1(AOA1)的aptX功能障碍。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DNA单链断裂修复(DNA Single strand Break Repair，SSBR)和DNA双链断裂修复(DNA Double Strand Break Repair，DSBR)与DNA断裂修复途径相互作用，整合到损伤修复途径中。我们正在测试以下假设：1)aptX组氨酸三联体(HIT)和锌指(Znf)结构域形成一个复合融合催化结构域，用于DNA结构特异性缺口结合、5‘-AMP识别和DNA死烯基化处理；2)AOA1患者突变通过活性位点扭曲破坏aptX蛋白质折叠和/或直接削弱aptX催化活性；3)FHA结构域和FHA-Hit连接区提供灵活的靶向靶向aptX DNA去烯化活性的磷酸化XRCC4和XRCC1 DNA修复支架。

Currently, we are focused on examining structure/function of the DNA end processing factors, Tyrosyl DNA phosphodiesterase 2 (Tdp2) (project 1) and Aprataxin (Aptx) (project2). Project1 (Tdp2) summary and progress: Topoisomerase II (topo II) DNA incision/ligation reactions can be poisoned (e.g following treatment with chemotherapeutics) to generate DNA double strand breaks (DSBs) with topo II covalently bound to DNA. Left un-processed, such protein-adducted DNA ends can impair DSB repair, thereby contributing to accumulation of clastogenic DSBs, genomic instability, mutagenesis, and cell death. Tyrosyl-DNA phosphodiesterase 2 (Tdp2) protects genomic integrity by reversing 5′-phosphotyrosyl (5′-Y) linked topo II protein-DNA adducts. Tdp2 functions in cellular topo II drug resistance, and also mediates mutant p53 gain of function phenotypes. However, the molecular basis underlying Tdp2 topo II-DNA adduct repair activities remains unclear in the absence of protein structural information for any Tdp2 homolog. To understand Tdp2 functions we determined X-ray crystal structures of mammalian Tdp2 in three DNA bound states, and studied Tdp2 activities using mutational and functional studies that define determinants of Tdp2 DNA-protein covalent adduct recognition and reversal. Overall, these results support a testable structure-based single magnesium ion mediated catalytic mechanism whereby Tdp2 interacts with a DNA-protein conjugate using two binding pockets, one for the DNA, and a second for the Topoisomerase derived protein adduct. The Tdp2 DNA binding groove is composed of a helical cap and novel beta-2 helix-beta DNA damage binding "grasp" that together envelop an exposed 5′-adducted ssDNA terminus. Tailored DNA and 5′-adduct recognition elements make Tdp2 distinct from the related DNA repair endonucleases such as Apurinic endonuclease 1 (Ape1), and Tdp2 excludes an intact phosphodiester backbone from its active site to ensure selectivity, and restrict endo- or exonucleolytic processing. Together, our results provide insights to the mechanism of Tdp2-linked cancer chemotherapeutic resistance, and establish a framework for the development of Tdp2 inhibitors that could be employed as adjuvants for commonly employed topoisomerase II poisons (e.g Etoposide). Project 2 (Aptx) summary and progress: (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.