Mechanisms of Healing and Sealing DNA and RNA Ends

DNA 和 RNA 末端的愈合和密封机制

• 批准号: 8728467
• 负责人: Stewart H Shuman
• 金额: $ 26.27万
• 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 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8728467
• 关键词: Active Sites; Address; African Trypanosomiasis; Antibiotics; Archaea; Bacteria; Bacterial DNA; Biochemistry; Biological; Biological Models; Catalytic Domain; Cellular Stress Response; Chagas Disease; Chemicals; Chemistry; Chlorella virus DNA ligase; Communicable Diseases; Complex; DNA; DNA Damage; DNA Fingerprinting; DNA Ligases; DNA Primers; DNA Repair; DNA biosynthesis; Development; Developmental Delay Disorders; Disease; Drug Targeting; Enzymes; Escherichia coli; Evolution; Excision; Family; Functional disorder; Funding; Genomics; Goals; Healed; Health; Hereditary Disease; Human Genetics; Immunologic Deficiency Syndromes; Kinetics; Leishmaniasis; Lesion; Ligase; Link; Lysine; Microcephaly; Molecular Genetics; Mutagenesis; Mutation; Names; Nature; Nonhomologous DNA End Joining; Nucleic Acids; Nucleosides; Parasites; Pathway interactions; Phosphoric Monoester Hydrolases; Phosphotransferases; Polymerase; Polynucleotide 5' -Hydroxyl-Kinase; Polynucleotides; Property; Proteins; RNA; RNA Editing; RNA Ligase (ATP); RNA Splicing; Radiation Tolerance; Reaction; Research; Ribonucleosides; Seizures; Specificity; Structure; Substrate Specificity; Syndrome; System; Transfer RNA; Viral; Virus; Work; adenylate; antimicrobial; antimicrobial drug; base; cofactor; drug discovery; early onset; enzyme substrate; healing; human DNA; inorganic phosphate; insight; multidisciplinary; nervous system disorder; novel; phosphodiester; phosphoric diester hydrolase; polypeptide; repair enzyme; repaired; research study; seal; stereochemistry; 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. 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-N-zeta)-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 directs an attack by the 3'-OH on AppDNA/RNA to form a phosphodiester bond and release AMP. Our aims are to understand how ligase reaction chemistry is catalyzed and how ligases recognize "damaged" DNA or RNA ends. 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). Many physiologically important types of DNA and RNA damage result in strand breaks with 3'-PO4 or 2',3'-cyclic-PO4 (>p) ends, which cannot be sealed by classic DNA/RNA ligases. Such broken ends must be "healed" - converted to a 3'-OH by a phosphoesterase - before they can be sealed. Nature has devised a remarkably diverse enzymatic tool-kit to deal with the end-healing problem. We are focused in this project on two types of end-healing systems: T4 polynucleotide kinase-phosphatase (Pnkp) and LigD phosphoesterase (LigD PE). T4 Pnkp exemplifies a large family of DNA and RNA repair proteins that convert 3'-PO4/5'-OH (or 2',3'>p/5'-OH) ends into ligatable 3'-OH/5'-PO4 ends. The mechanism of RNA cyclic-phosphate removal by T4 Pnkp is unique and entails four chemical steps and two covalent enzyme- substrate intermediates. LigD PE catalyzes two types of end-healing reactions on a DNA primer-template containing either a 3'-diribonucleotide or a 3'-PO4. The 3'-terminal nucleoside of a 3'-diribonucleotide is removed by LigD PE's phosphodiesterase activity to yield a primer strand with a ribonucleoside 3'-PO4. The 3'-PO4 is hydrolyzed by a LigD PE phosphomonoesterase activity to a 3'-OH. The atomic structure of the LigD PE domain and its active site are novel. Indeed, LigD PE defines a new superfamily of repair enzymes distributed widely in bacteria, archaea, and eukarya. We aim to understand how T4 Pnkp and LigD PE enzymes recognize their substrates and cofactors and perform their distinctive phosphoryl transfer chemistries. We propose a multidisciplinary agenda, blending biochemistry, molecular genetics, and structural biology. Our experiments will yield new insights to phosphoryl transfer reaction mechanisms and the evolution of nucleic acid repair systems. .

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）韧带与ATP或NAD+反应形成共价性连接酶 - （莱赛-N-Zeta）-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）连接酶指示3'-OH对AppDNA/RNA的攻击形成磷酸二酯键并释放AMP。我们的目标是了解连接酶反应化学是如何催化的，以及连接酶如何识别“受损”的DNA或RNA末端。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DNA连接酶（大肠杆菌）和病毒ATP依赖性RNA连接酶（T4 RNL2）。许多生理上重要的DNA和RNA损伤会导致3'-PO4或2'，3'-循环-PO4（> p）末端断裂，这不能被经典的DNA/RNA连接酶密封。必须将这种破碎的末端“治愈” - 通过磷酸酯酶转换为3' -OH - 才能密封。大自然已经设计了一种非常多样化的酶促工具，以解决最终治疗问题。我们专注于这两种类型的最终治疗系统：T4多核苷酸激酶 - 磷酸酶（PNKP）和LIGD磷酸酯酶（LIGD PE）。 T4 PNKP例证了一大批DNA和RNA修复蛋白，这些蛋白会转换3'-PO4/5'-OH（或2'，3'，3'，3'> p/5'-OH）末端，以可操的3'-OH/5'-PO4末端。 T4 PNKP通过T4 PNKP去除RNA环磷酸的机制是独特的，需要四个化学步骤和两个共价酶 - 底物中间体。 LIGD PE催化了包含3'-二甲核苷酸或3'-PO4的DNA引物 - 板上的两种类型的终止治疗反应。通过LIGD PE的磷酸二酯酶活性去除3'-二硝基核苷酸的3'-末端核苷，从而产生带有核糖核苷3'PO4的引物链。 3'-PO4通过LIGD PE PE磷酸酯酶活性水解至3'-OH。 LIGD PE域及其活性位点的原子结构是新颖的。实际上，LIGD PE定义了一个新的修复酶的超家族，该酶广泛分布在细菌，古细菌和eukarya中。我们旨在了解T4 PNKP和LIGD PE酶如何识别其底物和辅因子并执行其独特的磷酸化化学化学。我们提出了多学科议程，混合生物化学，分子遗传学和结构生物学。我们的实验将为磷酸转移反应机制和核酸修复系统的演变提供新的见解。 。