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: 8553800
• 负责人: Robert Williams
• 金额: $ 134.15万
• 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/8553800
• 关键词: APTX gene; Active Sites; Adjuvant; Ataxia; Binding; Biochemical; Catalytic Domain; Cell Death; Cell Respiration; Cell physiology; Chronic; Cleaved cell; Complex; Cytoprotection; DNA; DNA Adducts; DNA Alkylation; DNA Binding; DNA Damage; DNA Double Strand Break; DNA Groove Binding; DNA Ligases; DNA Ligation; DNA Minor Groove Binding; DNA Repair; DNA Repair Endonuclease; DNA Repair Enzymes; DNA Single Strand Break; DNA Structure; DNA biosynthesis; DNA glycosylase; DNA strand break; DNA-(apurinic or apyrimidinic site) lyase; DNA-Directed DNA Polymerase; Data; Development; Disease; Double Strand Break Repair; Drug resistance; Elements; Ensure; Etoposide; Eukaryotic Cell; Excision; Exposure to; FHA Domain; Fingers; Fission Yeast; Functional disorder; Genome; Genomic Instability; Genomics; Histidine; Homologous Gene; Human; Hydrolase; Hydroxyl Radical; In Vitro; Inflammation; Inherited; Ionizing radiation; Lead; Left; Ligase; Ligation; Limb structure; Link; Maintenance; Malignant Neoplasms; Mediating; Metabolism; Molecular; Molecular Conformation; Mutagenesis; Mutation; Neurodegenerative Disorders; Nucleic Acids; Nucleotides; Pathway interactions; Patients; Pharmaceutical Preparations; Pharmacologic Substance; Phenotype; Poison; Poisoning; Predisposition; Process; Production; Protein Conformation; Protein Dynamics; Proteins; Reaction; Research; Resistance; Roentgen Rays; Signal Transduction; Single Strand Break Repair; Structural Protein; Structure; Surgical incisions; Testing; Topoisomerase; Topoisomerase II; Toxic Environmental Substances; Triad Acrylic Resin; Vertebral column; Wood material; Work; XRCC1 gene; XRCC4 gene; Yeast Model System; Zinc Fingers; adduct; base; coping; cytotoxic; emergency service responder; environmental stressor; flexibility; functional hypothalamic amenorrhea; gain of function; grasp; improved; in vivo; inhibitor/antagonist; inorganic phosphate; insight; magnesium ion; molecular recognition; mutant; novel; oculomotor; oxidative DNA damage; phosphodiester; programs; protein folding; repaired; response; scaffold; seal; structural biology; tyrosyl-DNA phosphodiesterase

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.

目前，我们专注于检查DNA末端加工因子的结构/功能，酪酶DNA磷酸二酯酶2（TDP2）（项目1）和Aprataxin（Aptx）（APTX）（Project2）。 Project1（TDP2）摘要和进度：拓扑异构酶II（TOPO II）DNA切口/连接反应可能被毒化（例如，在化学疗法治疗后处理后），以产生与TOPO II共同结合与DNA的DNA双链断裂（DSB）。剩下的未加工，这种蛋白质的DNA末端可能会损害DSB的修复，从而有助于层生成DSB的积累，基因组不稳定性，诱变和细胞死亡。酪酶-DNA磷酸二酯酶2（TDP2）通过逆转5'-磷酸酪糖基（5'-Y）链接的TOPO II蛋白DNA加合物来保护基因组完整性。 TDP2在细胞Topo II耐药性中起作用，还介导了突变体p53功能表型的增益。然而，在没有任何TDP2同源物的蛋白质结构信息的情况下，尚不清楚分子基础TDP2 TOPO II-DNA修复活动尚不清楚。 为了了解TDP2函数，我们确定了在三个DNA结合态下哺乳动物TDP2的X射线晶体结构，并使用突变和功能研究研究了TDP2活性，这些突变和功能研究定义了TDP2 DNA蛋白共价添加识别和逆转的决定因素。总体而言，这些结果支持可测试的基于结构的单镁介导的催化机制，从而使用两个结合口袋与DNA-蛋白质结合物相互作用，一个用于DNA，第二个用于拓扑酶衍生的蛋白质加合物。 TDP2 DNA结合凹槽由螺旋盖和新型β-2螺旋 - β-beta DNA损伤结合“ grasp”组成，共同包含暴露的5'-添加ssDNA末端。量身定制的DNA和5'-ADDUCT识别元件使TDP2与相关的DNA修复核酸内切酶（如肾上腺素核酸内切酶1（APE1））不同，而TDP2则将完整的磷酸二酯主链排除在其活性部位中，以确保选择性，并限制内核或外核酸或外生核酸化处理。总之，我们的结果为TDP2连接的癌症化学治疗性的机制提供了见解，并为开发TDP2抑制剂的框架建立了一个框架，这些抑制剂可作为常用的拓扑异构酶II毒药作为佐剂（例如，依托托固醇）。 项目2（APTX）摘要和进度：（APTX）是一种保守的真核DNA修复酶，对于保护细胞免受氧化DNA损伤至关重要，APTX突变引起遗传神经退行性疾病共济失调，其眼球瘤1（AOA1）（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密封DNA划痕，该机制可以在连接酶遇到具有氧化或DNA烷基化损伤产物的DNA末端时流产。这种“流产的连接”产生了一种次要损伤形式，即5'-腺苷酸化的DNA末端，通过APTX纠正以保护基因组完整性。但是，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仍然很大未知。此外，APTX如何在疾病中灭活是未知的。为了了解APTX函数，我们确定了通过融合了命中（组氨酸三合会）核苷酸水解酶与前所未有的DNA次要DNA次要DNA次要DNA次要的DNA次要DNA次要型grove C2HE Zn Zn Zn Zn Finger（Znf）形成的棘糖疗法POMBE POMBE APTX-DNA-AMP-ZN复合物的结构。这项工作强调了APTXα-螺旋楔如何询问DNA基堆堆的DNA端/nick传感。结构和突变数据支持用于5'-AMP加合物识别和去除的楔形 - 螺旋切割式HIT-ZNF催化机制，并提出了影响蛋白质折叠的突变，主动位点口袋和枢轴基础APTLIE APTX aptLie APTX功能障碍在神经性疾病障碍性障碍性障碍性障碍性障碍性障碍性障碍性障碍性无动性痛胺1（AOAA 1（AOAA）中）。我们旨在进一步定义APTX DNA修复的分子决定因素，以及APTX如何通过与DNA断裂修复途径的相互作用通过结合XRCC1（DNA单链断裂修复，SSBR）和XRCC4（DNA DNA双重链断裂修复，DSBR，DSBR）整合到损伤修复途径中。 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域和FHA撞击接头提供了靶向APTX DNA降苯基活性的柔性皮带，可针对磷酸化的XRCC4和XRCC1 DNA修复支架。