Viral DNA and RNA Ligases

病毒 DNA 和 RNA 连接

• 批准号: 6966522
• 负责人: Stewart H Shuman
• 金额: $ 33.48万
• 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 2009-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6966522
• 关键词: DNA damage; DNA repair; adenosine triphosphate; catalyst; enzyme activity; enzyme mechanism; enzyme structure; enzyme substrate; phosphoester ligase; protein structure function; site directed mutagenesis; structural biology

DESCRIPTION (provided by applicant): Breaks in the phosphodiester backbone of DNA and essential RNA molecules can lead to cell death if not repaired. This project aims to illuminate the mechanisms and structures of the DNA ligase and RNA ligase enzymes that rectify such breaks. Polynucleotide ligases catalyze the joining of a 5'-PO4 strand to a 3'-OH end via 3 chemical steps: (i) ligase reacts with ATP or NAD+ to form a covalent ligase-adenylate intermediate and release pyrophosphate or NMN; (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 of the nick on AppDNA or AppRNA to form a phosphodiester and release AMP. Our long-range goals are to understand how ligase reaction chemistry is catalyzed and how ligases recognize "damaged" DNA or RNA ends. We are approaching these problems using a eukaryotic virus-encoded DNA ligase (Chlorella virus DNA ligase), a bacterial NAD+-dependent DNA ligase (E. coli LigA), and a bacteriophage ATP-dependent RNA ligase (T4 Rnl2) as models. Rnl2 was discovered by this laboratory during the previous funding period and quickly developed into a model-of-choice for RNA repair enzymology. Rnl2 exemplifies a new and growing family of RNA ligases found in all phylogenetic domains. Our studies, which integrate biochemistry, molecular genetics, and structural biology, have revealed mechanistic principles shared by all DNA and RNA ligases, as well as the unique structural features and substrate specificities that distinguish the various branches of the polynucleotide ligase superfamily. In particular, our work indicates that progression through the sequential steps of the ligation pathway is coupled to large protein domain movements and serial remodeling of the active site. Specific aims of this proposal are: (1) To identify the functional groups of Chlorella virus DNA ligase, E. coli LigA, and T4 Rnl2 that contribute to DNA/RNA recognition and nucleotidyl transfer; (2) To biochemically define the interface between ligase-adenylate and nicked DNA/RNA substrates; and (3) To determine atomic structures of ligase-adenylate bound at a DNA/RNA nick and of ligases bound to the 5'-adenylated polynucleotide intermediate.

描述（由申请人提供）：如果不修复，DNA和必需RNA分子的磷酸二酯主链中断裂可能会导致细胞死亡。该项目旨在阐明纠正此类断裂的DNA连接酶和RNA连接酶的机理和结构。多核苷酸连接酶通过3个化学步骤催化5'-PO4链的连接到3'-OH端：（i）连接酶与ATP或NAD+反应形成共价连接酶辅助酶中间体并释放焦磷酸或NMN； （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）连接酶在AppDNA或Applna上催化nick的3'-OH攻击形成磷酸酯并释放AMP。我们的远距离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。我们正在使用真核病毒编码的DNA连接酶（Chlorella病毒DNA连接酶），细菌NAD+依赖性的DNA连接酶（大肠杆菌）和ATP依赖性的RNA连接酶（T4 RNL2）来解决这些问题。该实验室在上一个资金期间发现了RNL2，并迅速发展为用于RNA修复酶学的选择模型。 RNL2例证了在所有系统发育结构域中发现的一个新的且不断增长的RNA连接酶家族。我们的研究整合了生物化学，分子遗传学和结构生物学，已经揭示了所有DNA和RNA连接酶共有的机械原理，以及区分多核苷酸连接酶超级家族的各个分支的独特结构特征和底物特异性。特别是，我们的工作表明，通过连接途径的顺序步骤进行进程与大型蛋白质结构域运动和活动位点的串行重塑。 该提案的具体目的是：（1）确定有助于DNA/RNA识别和核苷酸转移的小球藻病毒DNA连接酶，大肠杆菌和T4 RNL2的官能团； （2）在生化上定义了连接酶 - 腺苷和成形的DNA/RNA底物之间的界面； （3）确定在DNA/RNA迹线和连接酶结合的连接酶 - 腺酸的原子结构，结合了5'-腺苷酸化的多核苷酸中间体。