Viral DNA and RNA Ligases

描述（由申请人提供）：DNA和基本RNA分子的磷酸二酯主干断裂如果不修复，可能导致细胞死亡。本项目旨在阐明修复这种断裂的DNA连接酶和RNA连接酶的机制和结构。多核苷酸连接酶通过3个化学步骤催化5'-PO4链与3'-OH末端的连接：(i)连接酶与ATP或NAD+反应形成共价连接酶-腺苷酸中间体并释放焦磷酸或NMN；（ii） AMP从连接酶转移到5'-PO4 DNA或RNA链上，形成DNA/RNA腺苷酸中间体（AppDNA或AppRNA）；（iii）连接酶催化AppDNA或AppRNA上缺口的3'-OH攻击，形成磷酸二酯并释放AMP。我们的长期目标是了解连接酶反应化学是如何催化的，以及连接酶是如何识别“受损”的DNA或RNA末端的。我们正在使用真核病毒编码的DNA连接酶（小球藻病毒DNA连接酶）、细菌依赖于NAD+的DNA连接酶（大肠杆菌LigA）和噬菌体依赖于atp的RNA连接酶（T4 Rnl2）作为模型来解决这些问题。Rnl2是该实验室在之前的资助期内发现的，并迅速发展成为RNA修复酶学的首选模型。Rnl2是在所有系统发育域中发现的一个新的、不断增长的RNA连接酶家族的例证。我们的研究结合了生物化学、分子遗传学和结构生物学，揭示了所有DNA和RNA连接酶共有的机制原理，以及区分多核苷酸连接酶超家族不同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，通过连接途径的连续步骤的进展与大的蛋白质结构域运动和活性位点的连续重塑相结合。

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.