Viral and Bacterial DNA Ligases

病毒和细菌 DNA 连接

• 批准号: 7728999
• 负责人: Stewart H Shuman
• 金额: $ 49.49万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-08-01 至 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7728999
• 关键词: 2-methyladenosine; Active Sites; Adenine; Adenosine; African Trypanosomiasis; Amino Acids; Anti-Bacterial Agents; Antibiotics; Architecture; Bacteria; Bacterial DNA; Binding; Biochemical Genetics; Biochemistry; Biological Assay; Biological Models; Case Study; Catalytic Domain; Cellular Stress Response; Chagas Disease; Chemicals; Chemistry; Chlorella; Chlorella virus DNA ligase; Complex; Crystallization; Cysteine; DNA; DNA Binding; DNA Damage; DNA Double Strand Break; DNA Ligases; DNA Repair; DNA Structure; DNA biosynthesis; Disabled Persons; Enzymes; Escherichia coli; Evolution; Excision; Family; Functional disorder; Funding; Genomics; Goals; Grant; Growth; Healed; Hereditary Disease; Human; Human Genetics; In Vitro; Leishmaniasis; Ligase; Ligation; Link; Lysine; Mammals; Maps; Measures; Methods; Modification; Molecular Genetics; Movement; Multi-Drug Resistance; Mutagenesis; Mycobacterium tuberculosis; Names; Nonhomologous DNA End Joining; Nucleic Acids; Nucleotides; Parasites; Pathway interactions; Plants; Poison; Polymerase; Polynucleotides; Process; Proteins; Pseudomonas aeruginosa; Public Health; RNA; RNA Editing; RNA Ligase (ATP); RNA Splicing; Reaction; Research; Rhizobium radiobacter; Ribonucleotides; Solutions; Structure; Substrate Specificity; Surface; Syndrome; System; Transfer RNA; Translations; Trypanosomiasis; Tuberculosis; Viral; Virus; Work; X-Ray Crystallography; adenylate; analog; antimicrobial; antimicrobial drug; base; design; drug development; drug discovery; healing; human DNA; infectious disease treatment; inhibitor/antagonist; inorganic phosphate; insight; interest; macrophage; mimetics; multidisciplinary; next generation; novel; paralogous gene; pathogen; phosphodiester; polypeptide; preference; public health relevance; repaired; research study; resistant strain; seal; single molecule; structural biology; tool

DESCRIPTION (provided by applicant): DNA ligases are ubiquitous enzymes that catalyze an essential final step in DNA replication and repair - the conversion of DNA nicks into phosphodiester bonds. RNA ligases participate in breakage-repair pathways that underlie tRNA splicing, post-transcriptional RNA editing, and cellular stress responses. The DNA and RNA ligases seal 5'-PO4 and 3'-OH polynucleotide ends via three chemical steps: (i) ligase reacts with ATP or NAD+ to form a covalent ligase-(lysyl-N6)-AMP intermediate; (ii) AMP is transferred from the ligase to the 5'-PO4 DNA or RNA strand to form a DNA/RNA-adenylate intermediate (AppDNA or AppRNA); (iii) ligase catalyzes attack by the 3'-OH on AppDNA/RNA to form a phosphodiester and release AMP. Our goals are to understand how ligase reaction chemistry is catalyzed, how ligases recognize "damaged" DNA or RNA ends, and how domain movements and active site remodeling are used to choreograph the end- joining pathway. We study these problems using three model systems: a eukaryal virus-encoded DNA ligase (Chlorella virus DNA ligase: ChVLig); a bacterial NAD+-dependent DNA ligase (E. coli LigA), and a viral ATP- dependent RNA ligase (T4 Rnl2). During the previous grant period, we determined the atomic structure of ChVLig-AMP bound at a 3'-OH/5'-PO4 nick and structures of LigA and T4 Rnl2 bound to their nicked polynucleotide-adenylate intermediates. These structures, and functional studies inspired by them, are revealing mechanistic principles shared by all DNA and RNA ligases, as well as the unique domain modules and substrate specificities that distinguish the various branches of the ligase superfamily. We have extended our interests in bacterial DNA ligases to two subfamilies of ATP-dependent strand joining enzymes (named LigD and LigC) that participate in a non-homologous end joining (NHEJ) pathway of bacterial DNA repair. LigC and LigD are unique among known ligases in that they require a 3'-OH monoribonucleotide in order to perform efficient nick sealing. LigD is doubly unique insofar as it is a multifunctional enzyme composed of three autonomous catalytic domains: a ligase (LIG); a polymerase (POL), and a phosphoesterase (PE). The POL and PE domains comprise a suite of DNA "end-healing" activities that remodel the 3' terminus of the DSB prior to sealing by the LIG component. We propose a multidisciplinary agenda (blending biochemistry, molecular genetics, and structural biology) to tackle a next generation of issues in the field. Our specific aims are: (i) to exploit the protein-DNA structures we've solved to guide a mutational analysis of amino acids at the ligase-DNA interface; (ii) to probe the mechanism of adenylate transfer to lysine, via structural methods and "chemical mutagenesis" - an approach that circumvents the limitations to the genetically programmable protein "tool kit"; (iii) to solve the structure of the LigD phosphoesterase domain, which exemplifies a new family of 3' end-modifying enzymes; and (iv) to illuminate the distinctive substrate preference of bacterial NHEJ ligases for a 3'-OH monoribonucleotide nick. We are confident that the experiments we propose will yield new insights to phosphoryl transfer reaction mechanisms, nucleic acid damage recognition, and the evolution of nucleic acid repair systems. . PUBLIC HEALTH RELEVANCE: Ligases are attractive targets for antimicrobial drug discovery. Inhibitors of bacterial NAD+-dependent DNA ligase (LigA) are promising candidates for broad-spectrum antibacterial therapy, given that: (i) NAD+- dependent ligases are present in all bacteria and are essential for bacterial growth in all cases studied, and (ii) LigA enzymes are structurally conserved among bacteria, but display unique substrate specificity and domain architecture compared to the ATP-dependent ligases of humans and other mammals. Our structure of E. coli LigA in complex with AppDNA inspires a strategy for inhibitor design. The LigA structure reveals a through-and-through "tunnel" - from the exterior surface of LigA to the adenosine- binding pocket - that completely exposes the edge of the adenine base. In particular the adenine C2 atom is pointed directly into the tunnel, which is formed by a cage of hydrophobic amino acids. This tunnel is present in all LigA enzymes. In contrast, there is no such tunnel emanating from the adenosine binding pockets of human DNA ligase or Chlorella virus ligase. This situation invites the structure-based design of C2-substituted derivatives of adenosine (or non-nucleotide mimics thereof) as unique and selective inhibitors of LigA. One such compound, 2-methyladenosine, has excellent antimicrobial activity against Mycobacterium tuberculosis, in culture and within human macrophages. There is now a pressing need for new antibiotics against human tuberculosis, as available treatment options degrade with the emergence of multi-drug- resistant strains. This is a serious public health problem. We expect our studies of LigA structure and mechanism will stimulate the discovery of new compounds that either interdict LigA binding to NAD+ or nicked DNA, or "poison" the ligation pathway by trapping a "toxic" nicked-adenylate intermediate. Similar considerations - a unique structural domain and distinctive nucleic acid substrate specificity - recommend Rnl2-type RNA ligases as targets for drug development for treatment of infectious diseases caused by protozoan parasites, specifically trypanosomiasis (African sleeping sickness and Chagas disease) and leishmaniasis. .

描述（由申请人提供）：DNA连接酶是无处不在的酶，它催化了DNA复制和修复的必要最后一步 - DNA镍转化为磷酸二酯键。 RNA连接酶参与了Breakage-REPAIR途径，该途径是tRNA剪接，转录后RNA编辑和细胞应激反应的基础。 DNA和RNA连接酶密封5'-PO4和3'-OH多核苷酸通过三个化学步骤末端：（i）连接酶与ATP或NAD+反应形成共价连接酶 - （Lysyl-N6） - Amp中间体； （ii）将AMP从连接酶转移到5'-PO4 DNA或RNA链中，形成DNA/RNA-腺酸中间体（appDNA或ASSEDNA）； （iii）连接酶催化3'-OH对AppDNA/RNA的攻击形成磷酸酯并释放AMP。我们的目标是了解连接酶反应化学是如何催化的，连接酶如何识别“受损”的DNA或RNA末端，以及域的运动和主动位点重塑如何用于编舞末端连接途径。我们使用三个模型系统研究了这些问题：真核病病毒编码的DNA连接酶（Chlorella病毒DNA连接酶：CHVLIG）；细菌NAD+依赖性的DNA连接酶（大肠杆菌）和病毒ATP依赖性RNA连接酶（T4 RNL2）。在上一个赠款期间，我们确定了在3'-OH/5'-PO4划痕的Chvlig-Amp结合的原子结构以及LIGA和T4 RNL2的结构与它们的昵称的多核苷酸 - 辅助中间体结合。这些结构以及受其启发的功能研究揭示了所有DNA和RNA连接酶共有的机械原理，以及区分连接酶超家族各个分支的独特域模块和底物特异性。我们已经将对细菌DNA连接酶的兴趣扩展到了参与细菌DNA修复的非同源末端连接（NHEJ）途径的两个依赖ATP依赖性链的亚家族。 LIGC和LIGD在已知的连接酶中是独一无二的，因为它们需要3'-OH单核核苷酸才能执行有效的划痕密封。 LIGD是双重独特的，因为它是由三个自主催化结构域组成的多功能酶：连接酶（LIG）；聚合酶（POL）和磷酸酯酶（PE）。 POL和PE结构域包含一套DNA“端盖”活动，该活动在通过LIG组件密封之前重塑了DSB的3'末端。我们提出了一个多学科议程（混合生物化学，分子遗传学和结构生物学），以解决该领域的下一代问题。我们的具体目的是：（i）利用我们已解决的蛋白质-DNA结构来指导连接酶-DNA界面上氨基酸的突变分析； （ii）通过结构方法和“化学诱变”探测腺苷酸转移到赖氨酸的机制，一种方法可以规避遗传可编程蛋白“工具包”的局限性； （iii）解决了LIGD磷酸酯酶结构域的结构，该结构域说明了一个新的3'末端改装酶的家族； （iv）阐明细菌NHEJ连接酶的独特底物偏好对3'-OH单核苷核苷酸刻痕。我们相信，我们提出的实验将为磷酸转移反应机制，核酸损伤识别以及核酸修复系统的演变提供新的见解。 。 公共卫生相关性：连接酶是抗菌药物发现的有吸引力的靶标。鉴于：（i）NAD+ - 依赖性连接酶在所有细菌中都存在，在所有细菌中都至关重要，在所有细菌中至关重要的是，在所有细菌中至关重要的是，在所有情况下，在所有细菌中，在所有细菌中，liga Enzymes均具有独特的liga enzymes，但（II）与构造的rigainia is在所有细胞中，liga enzymes在所有细胞中都是必不可少的，但（II）与结构上的rigainia均与结构性的典范相结合，依赖于：（i）nAD+ - 依赖性连接均至关重要，并且（II）与结构上的独特症相比人类和其他哺乳动物的ATP依赖性连接酶。我们与AppDNA复合体的大肠杆菌结构激发了抑制剂设计的策略。 LIGA结构揭示了从LIGA的外表面到腺苷结合袋的通行“隧道” - 完全暴露了腺嘌呤碱基的边缘。特别是将腺嘌呤C2原子直接指向隧道，该原子是由疏水氨基酸的笼子形成的。该隧道都存在于所有西甲酶中。相比之下，没有人DNA连接酶或小球藻病毒连接酶的腺苷结合袋中发出的这种隧道。这种情况将基于结构的腺苷（或非核苷酸模拟物模拟物）的C2取代衍生物作为独特的和选择性抑制剂的衍生物。一种这样的化合物，2-甲基腺苷，在培养和人类巨噬细胞中对结核分枝杆菌的抗菌活性出色。现在，对人类结核病的新抗生素有紧迫的需求，因为随着多药抗性菌株的出现，可用的治疗选择降低了。这是一个严重的公共卫生问题。我们期望我们对LIGA结构和机制的研究将刺激发现新化合物，这些化合物要么通过捕获“有毒的”成甲基化的辅助甲基化中间体来固定与NAD+或切成立的DNA结合或“毒害”结扎途径。类似的考虑 - 独特的结构结构域和独特的核酸底物特异性 - 建议RNL2型RNA连接酶作为药物开发的靶标，用于治疗由原生动物寄生虫引起的传染病，特别是锥虫病（特别是非洲睡病和木偶疾病）和Leishmaniasis。 。