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: 8929804
• 负责人: Robert Williams
• 金额: $ 164.67万
• 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/8929804
• 关键词: APTX gene; Active Sites; Architecture; Ataxia; Binding; Biochemical; Biological Models; C-terminal; Cell Cycle; Cell Proliferation; Cell Respiration; Cell physiology; Cells; Chromosomes; Chronic; Complex; DNA; DNA Adducts; DNA Binding; DNA Damage; DNA Double Strand Break; DNA Ligases; DNA Ligation; DNA Repair; DNA Single Strand Break; DNA Structure; DNA biosynthesis; DNA damage checkpoint; DNA glycosylase; DNA lesion; DNA replication fork; DNA strand break; DNA-Directed DNA Polymerase; DNA-protein crosslink; Defect; Diphosphates; Disease; Double Strand Break Repair; Enzymes; Eukaryota; Eukaryotic Cell; Event; Excision Repair; Exposure to; Failure; Family; Family Picornaviridae; Fission Yeast; Frequencies; Genetic Transcription; Genome; Genome Stability; Genomic Instability; Histidine; Homologous Gene; Human; Human poliovirus; Hydroxyl Radical; In Vitro; Inflammation; Ionizing radiation; Lead; Lesion; Ligase; Ligation; Limb structure; Link; Maintenance; Malignant Neoplasms; Mediating; Metabolism; Modeling; Molecular; Molecular Conformation; Mutation; N-terminal; Nerve Degeneration; Neurodegenerative Disorders; Nucleic Acids; Nucleoside Hydrolases; Pathway interactions; Pharmaceutical Preparations; Pharmacologic Substance; Phenotype; Phosphotyrosine; Poison; Polioviruses; Predisposition; Process; Production; Protein Conformation; Protein Dynamics; Proteins; RNA; RNA replication; Reaction; Research; Resolution; Rhinovirus; Ribonucleases; Ribonucleotides; Roentgen Rays; Role; S Phase; Saccharomycetales; Series; Side; Signal Transduction; Single-Stranded DNA; Structure; Surgical incisions; Syndrome; Topoisomerase; Topoisomerase II; Toxic Environmental Substances; Triad Acrylic Resin; Tyrosine; Urea; Vertebrates; Wood material; Work; Yeasts; adduct; adenylate; base; carcinogenesis; clastogen; coping; cytotoxic; dimer; emergency service responder; environmental stressor; homologous recombination; human DNA; improved; in vitro activity; in vivo; inhibitor/antagonist; inorganic phosphate; member; molecular recognition; nuclease; oculomotor; programs; repaired; response; seal; signal processing; structural biology; tyrosyl-DNA phosphodiesterase

I. Adenylated RNA-DNA repair by Aptx The frequency of ribonucleotide incorporation into DNA in cycling cells exceeds other known DNA damage combined. A major ribonucleotide excision repair (RER) pathway is initiated by RNase H2 incision on the 5 side of a DNA embedded ribonucleotide. We hypothesized that abundant incised ribonucleotide intermediates arising during RER might also be substrates for otherwise unanticipated reactions or side reactions during commonly occurring DNA transactions. One example of this is DNA ligation. Eukaryotic ATP-dependent DNA ligases catalyze nick sealing during DNA replication and repair with a three step mechanism involving active site adenylation of the ligase, adenylate transfer to the DNA 5′-phosphate, and DNA nick sealing with release of AMP. However, when DNA ligases engage nicked DNA substrates with preexisting DNA damage, for instance an RNA-DNA junction from RER, ligase can undergo abortive ligation where the enzyme dissociates prematurely from its substrate following DNA adenylation. In this case, rather than sealing a DNA nick to finalize DNA replication or repair, ligase catalyzes the addition of a bulky AMP adduct to the 5′ terminus of the nicked substrate. We established that RNA-DNA junctions arising from RER are indeed subject to abortive ligation by human DNA ligases 1 and 3 in vitro. RER intermediates trigger ligation failure and production of compounded DNA damage in the form of adenylated RNA-DNA lesions. Aprataxin (Aptx) is a member of the histidine triad (HIT) family of nucleoside hydrolases, and catalyzes direct reversal of DNA 5′-adenylation resulting from abortive ligation. Aptx therefore may be critical for genome stability in cells undergoing RER. Consistent with a roles for Aprataxins in metabolizing RNA derived damage, budding yeast cells with elevated ribonucleotides but lack the Aptx homolog deficient cells (hnt3Δ) display marked defects in cellular proliferation, activation of the S-phase DNA damage checkpoint, and are sensitive to the replication inhibitor hydroxyl urea (HU). These phenotypes are relieved in an rnh201Δ background, supporting a model where Aptx (Hnt3p) is required for efficient repair adenylated RNA-DNA junctions that arise from RNase H2 incision followed by abortive ligase metabolism. Human and yeast aprataxins all efficiently catalyze reversal of adenylated RNA-DNA in vitro, and a series of X-ray crystal structures of hAptx bound to adenylated RNA-DNA structures further establishes the molecular basis for adenylated RNA-DNA damage processing. The mechanism involves structure-specific A-form RNA-DNA recognition of the adenylated RNA 5′ terminus, and encirclement of the lesion pyrophosphate linkage to facilitate direct reversal of the adenylated RNA-DNA lesion. Notably high-resolution structural analysis of a disease causing Aptx mutation (K197Q) linked to Ataxia with Oculomotor Apraxia 1 (AOA1) distorts the Aptx RNA-DNA lesion binding pocket II. Repair of ribonucleotide linked Top2cc by Tdp2 Normally, the eukaryotic type II topoisomerases (Top2α and Top2β) regulate DNA topology by employing a dsDNA cleavage and religation cycle involving transient formation of Top2-DNA cleavage-complexes (Top2cc). Top2 catalytic intermediates are characterized by the topoisomerase covalently linked to the DNA 5′-terminus by an active site tyrosine residue (Fig. 1A). However, aberrant DNA structure or targeted chemotherapeutic disruption of the Top2 reaction can generate Top2cc, protein-DNA crosslinks that block transcription and/or collapse DNA replication forks. Interestingly, ribonucleotides stimulate the Top2α and Top2β DNA cleavage reactions, and therefore are potentially toxic to the Top2 reaction cycle by producing increased Top2RNA-DNA cleavage complex. We have been studying a major pathway for repair of Top2cc that is present vertebrates, but absent in lower eukaryotes including yeasts. This pathway involves direct reversal of Top2-DNA phosphortyrosyl linkages by tyrosyl DNA phosphodiesterase 2 (Tdp2). Tdp2 (aka Ttrap/Eap2/VPg unlinkase) also catalyzes reversal of protein-RNA covalent linkages during RNA replication of picornaviruses (e.g poliovirus and rhinovirus), suggestive of broader RNA repair functions for Tdp2. Recently we established Tdp2 also efficiently processes phosphotyrosine covalently adducted to 5′ ribonucleotides. To understand the molecular basis for RNA-protein processing by Tdp2, we solved a high-resolution structure of Tdp2 bound to a 5-ribonucleotide containing substrate. This work defines a mechanism through which RNA containing substrates are engaged by Tdp2 in a manner similar to that of DNA-only substrates. Overall these results are suggestive that genomic instability triggered by ribonucleotides in DNA might also be mediated by formation of Top2RNA-DNA cleavage complexes, and production of DNA single strand and double strand breaks. In an RNA-DNA repair role, emerging results suggest Tdp2 might also protect from RNA-triggered events in vivo. III. Mechanisms of DNA end processing by Ctp1Ctip/Sae2/Mre11/Rad50/Nbs1 DNA double strand breaks (DSBs) generated by clastogen exposures including ionizing radiation and topoisomerase poisons can sever entire chromosomes, thereby contributing to genomic instability and carcinogenesis. Error-free DSB repair of adducted DNA strand breaks by homologous recombination (HR) is initiated by the Mre11/Rad50/Nbs1 (MRN) complex. Ctp1CtIP/Sae2 collaborates with the Mre11-Rad50-Nbs1 (MRN) nuclease to modulate end processing, but the functional roles for Ctp1 remain unclear. We have established that Ctp1 harbors DNA-binding and bridging activities, but is not a nuclease. Our Ctp1 X-ray structures, small angle X-ray scattering (SAXS), and biophysical analysis define the salient features of Ctp1 architecture: an N-terminal interlocking tetrameric helical dimer of dimers domain (THDD), and an extended central intrinsically disordered region (IDR) linked to conserved C-terminal DNA-binding RHR motifs. The THDD, IDR and RHR regions are all required to support Ctp1 DNA bridging activity in vitro, and THDD or RHR disruption confers sensitivity of fission yeast to DNA damaging agents. Together, our results establish functional roles for tetrameric Ctp1 in the binding and coordination of DSB repair intermediates, and suggest that disruption of CtIP DNA binding activity by truncating mutations underlies CtIP-linked neurodegenerative Seckel and Jawad syndromes.

I. APTX腺苷酸化的RNA-DNA修复 在循环细胞中，核糖核苷酸掺入DNA的频率超过了其他已知的DNA损伤。 RNase H2切口在DNA嵌入的核糖核苷酸的5侧通过RNase H2切口启动了主要的核糖核苷酸切除修复（RER）途径。我们假设在RER期间产生的大量切开的核糖核苷酸中间体也可能是底物的，对于通常发生的DNA交易期间，其他意外反应或副反应。一个例子是DNA连接。真核ATP依赖性的DNA连接酶在DNA复制过程中催化刻度密封并通过三个步骤的机理进行修复，涉及连接酶的活性位点腺苷酸化，腺苷酸化，腺苷酸转移至DNA 5'-磷酸盐，以及通过释放AMP释放的DNA Nick密封。但是，当DNA连接酶与已有DNA损伤（例如RER的RNA-DNA连接）接合成立的DNA底物时，连接酶会经过流产的连接，其中该酶在DNA腺苷酸化后过早地从其底物中分离出来。在这种情况下，连接酶没有密封DNA迹线以最终确定DNA复制或修复，而是催化在划痕底物的5'末端添加笨重的AMP加合物。 我们确定由RER引起的RNA-DNA连接确实受到人DNA连接酶1和3体外的堕胎结扎。 RER中间体以腺苷酸化的RNA-DNA病变的形式触发了连接失败和复合DNA损伤的产生。 Aprataxin（APTX）是核苷水解酶的组氨酸三合会（HIT）家族的成员，并且由于流产的连接而导致DNA 5'-腺苷酸化的催化。因此，APTX对于经历RER的细胞中的基因组稳定性可能至关重要。与Aprataxins在代谢RNA造成的损伤中的作用，核糖核苷酸升高但缺乏APTX同源性缺陷细胞（HNT3δ）在细胞增殖中显示出明显缺陷，S相DNA损伤检查点的激活，对复制抑制剂的复制敏感性显示出明显的缺陷。这些表型在RNH201Δ背景中得到缓解，支持一个模型，其中APTX（HNT3P）需要有效修复腺苷酸化的RNA-DNA连接，这是由RNase H2切口引起的，然后是流产的连接酶代谢。人和酵母菌素在体外有效地催化了腺苷酸化RNA-DNA的逆转，以及与腺苷酸化RNA-DNA结构结合的一系列X射线晶体结构进一步建立了腺苷酸化RNA DNA损伤处理的分子基础。 该机制涉及对腺苷酸化RNA 5'末端的结构特异性A形式RNA-DNA识别，以及焦磷酸病变链接的包围，以促进腺苷酸化RNA-DNA病变的直接反转。值得注意的是对aptx突变（K197Q）的疾病的高分辨率结构分析，该疾病与动脉taxia相关的动脉瘤1（AOA1）扭曲了APTX RNA-DNA病变结合口袋 ii。修复TDP2的核糖核苷酸链接TOP2CC 通常，真核II型拓扑异构酶（TOP2α和TOP2β）通过采用DSDNA裂解和宗教周期来调节DNA拓扑，涉及TOP2-DNA裂解 - 复合物（TOP2CC）的短暂形成。 TOP2催化中间体的特征是通过活性位点酪氨酸残基与DNA 5'-末端共价呈拓扑异构酶（图1A）。但是，TOP2反应的异常DNA结构或靶向化学治疗破坏会产生top2cc，蛋白-DNA交联可阻止转录和/或塌陷DNA复制叉。有趣的是，核糖核苷酸刺激TOP2α和TOP2βDNA裂解反应，因此通过产生增加的TOP2RNA-DNA裂解复合物，可能对TOP2反应周期有毒。 我们一直在研究脊椎动物的TOP2CC修复的主要途径，但在包括酵母在内的较低真核生物中不存在。该途径涉及通过酪酶DNA磷酸二酯酶2（TDP2）直接逆转TOP2-DNA磷酸酪糖基链接。 TDP2（又名TTRAP/EAP2/VPG脱链酶）还催化了picornaviruses（例如poliovirus and Rhinovirus）在RNA复制过程中蛋白RNA共价链接的逆转，提示TDP2的宽RNA修复功能。最近，我们建立了TDP2，还有效地处理了共价添加到5'核糖核苷酸的磷酸酪氨酸。为了理解TDP2 RNA蛋白质加工的分子基础，我们求解了与含有5-核核苷酸含有底物的TDP2的高分辨率结构。这项工作定义了一种机制，该机制通过TDP2与仅DNA底物相似的方式参与其中含有RNA的底物。总体而言，这些结果表明，由DNA中核糖核苷酸触发的基因组不稳定性也可能是由Top2RNA-DNA裂解复合物的形成以及DNA单链和双链断裂的产生来介导的。在RNA-DNA修复作用中，新出现的结果表明TDP2也可能可以保护体内RNA触发的事件。 iii。 CTP1CTIP/SAE2/MRE​​11/RAD50/NBS1的DNA终端处理机理 DNA双链断裂（DSB）是由包括电离辐射（电离辐射和拓扑异构酶毒物）在内的碎屑原暴露产生的，可以切断整个染色体，从而有助于基因组不稳定性和癌变。通过同源重组（HR）对加合的DNA链的无误DSB修复是由MRE11/RAD50/NBS1（MRN）复合物启动的。 CTP1CTIP/SAE2与MRE11-RAD50-NBS1（MRN）核酸酶合作以调节终端处理，但CTP1的功能作用尚不清楚。我们已经确定CTP1具有DNA结合和桥接活性，但不是核酸酶。 Our Ctp1 X-ray structures, small angle X-ray scattering (SAXS), and biophysical analysis define the salient features of Ctp1 architecture: an N-terminal interlocking tetrameric helical dimer of dimers domain (THDD), and an extended central intrinsically disordered region (IDR) linked to conserved C-terminal DNA-binding RHR motifs.在体外支持CTP1 DNA桥接活性，THDD，IDR和RHR区域都是必需的，THDD或RHR破坏赋予了裂变酵母对DNA损害剂的敏感性。总之，我们的结果在DSB修复中间体的结合和协调中确立了四聚体CTP1的功能作用，并表明通过截断突变通过CTIP链接神经降解型SECKEL和JAWAD综合症来破坏CTIP DNA结合活性。