Structural Biology of Genome Maintenance and DNA repair

目前，我们专注于研究DNA末端加工因子，酪氨酰DNA磷酸二酯酶2（Tdp 2）（项目1）和Aprataxin（Aptx）（项目2）的结构/功能。 项目1（Tdp 2）概述和进展：拓扑异构酶II（topo II）DNA切割/连接反应可以被毒化（例如，在用化疗剂治疗之后）以产生DNA双链断裂（DSB），其中topo II共价结合到DNA。如果不进行处理，这种蛋白加合的DNA末端会损害DSB修复，从而导致致染色体断裂DSB的积累、基因组不稳定性、诱变和细胞死亡。酪氨酰-DNA磷酸二酯酶2（Tdp 2）通过逆转5-磷酸酪氨酰（5-Y）连接的topo II蛋白-DNA加合物来保护基因组完整性。Tdp 2在细胞拓扑异构酶II耐药性中起作用，也介导突变型p53获得功能表型。然而，Tdp 2拓扑II-DNA加合物修复活动的分子基础仍然不清楚的蛋白质结构信息的情况下，任何Tdp 2同系物。 为了了解Tdp 2的功能，我们确定了哺乳动物Tdp 2在三种DNA结合状态下的X射线晶体结构，并使用突变和功能研究来研究Tdp 2的活性，这些研究定义了Tdp 2 DNA-蛋白质共价加合物识别和逆转的决定因素。总的来说，这些结果支持可测试的基于结构的单一镁离子介导的催化机制，其中Tdp 2与DNA-蛋白质缀合物相互作用，使用两个结合口袋，一个用于DNA，第二个用于拓扑异构酶衍生的蛋白质加合物。Tdp 2 DNA结合沟由螺旋帽和新的β-2螺旋-β DNA损伤结合“抓握”组成，它们一起包裹暴露的5-加合的ssDNA末端。定制的DNA和5-加合物识别元件使Tdp 2与相关的DNA修复核酸内切酶如脱嘌呤核酸内切酶1（Ape 1）不同，并且Tdp 2从其活性位点排除完整的磷酸二酯骨架以确保选择性，并限制内切或外切核酸水解加工。总之，我们的研究结果提供了对Tdp 2相关癌症化疗耐药性机制的见解，并建立了开发Tdp 2抑制剂的框架，该抑制剂可用作常用拓扑异构酶II毒药（例如依托泊苷）的佐剂。 项目2（Aptx）概述和进展：（Aptx）是一种保守的真核生物DNA修复酶，对保护细胞免受氧化DNA损伤非常重要，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 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.