Mechanisms of Healing and Sealing DNA and RNA Ends

描述（由申请人提供）：DNA连接酶是一种普遍存在的酶，它催化DNA复制和修复中必不可少的最后一步-将DNA缺口转化为磷酸二酯键。RNA连接酶参与tRNA剪接、转录后RNA编辑和细胞应激反应的断裂修复途径。DNA和RNA连接酶通过三个化学步骤封闭5'- po4和3'- oh多核苷酸末端：(i)连接酶与ATP或NAD+反应形成共价连接酶-（赖氨酸- n - ζ）- amp中间体；（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）连接酶引导3'-OH攻击AppDNA/RNA，形成磷酸二酯键并释放AMP。我们的目的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。我们使用三种模型系统来研究这些问题：真核病毒编码的DNA连接酶（小球藻病毒DNA连接酶：chvligg）；细菌依赖NAD+的DNA连接酶（大肠杆菌LigA）和病毒依赖atp的RNA连接酶（T4 Rnl2）。许多生理上重要的DNA和RNA损伤类型导致3'-PO4或2',3'-cyclic-PO4 （>p）末端的链断裂，这是经典的DNA/RNA连接酶无法密封的。这些断裂的末端必须“愈合”——通过磷酸酯酶转化为3'- oh——然后才能被密封。大自然设计了一个非常多样化的酶工具箱来处理最终愈合的问题。我们在这个项目中专注于两种类型的末端愈合系统：T4多核苷酸激酶-磷酸酶（Pnkp）和LigD磷酸酯酶（LigD PE）。T4 Pnkp是将3'-PO4/5'-OH（或2',3'>p/5'-OH）末端转化为可连接的3'-OH/5'-PO4末端的一大DNA和RNA修复蛋白家族的例证。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。在含有3'-二核糖核苷酸或3'-PO4的DNA引物模板上，LigD PE催化两种类型的末端愈合反应。3‘-二核糖核苷酸的3’端核苷被LigD PE的磷酸二酯酶活性去除，产生具有3'-PO4核糖核苷的引物链。3'-PO4被LigD - PE磷酸单酯酶水解成3'-OH。LigD - PE结构域的原子结构及其活性位点是新颖的。事实上，LigD PE定义了一个新的修复酶超家族，广泛分布于细菌、古细菌和真核生物中。我们的目标是了解T4 Pnkp和LigD PE酶如何识别它们的底物和辅因子，并执行它们独特的磷酸化转移化学。我们提出一个多学科的议程，混合生物化学，分子遗传学，和结构生物学。我们的实验将对磷酸化转移反应机制和核酸修复系统的进化产生新的见解。

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. .