Viral and Bacterial DNA Ligases

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- n6）- 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）连接酶催化3'- oh对AppDNA/RNA的攻击，形成磷酸二酯并释放AMP。我们的目标是了解连接酶反应化学是如何被催化的，连接酶是如何识别“受损”的DNA或RNA末端的，以及如何利用结构域运动和活性位点重塑来设计末端连接途径。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）。在之前的资助期间，我们确定了chvligi - amp与3'-OH/5'-PO4缺口结合的原子结构，以及LigA和T4 Rnl2与其缺口多核苷酸-腺苷酸中间体结合的结构。这些结构以及受其启发的功能研究揭示了所有DNA和RNA连接酶共有的机制原理，以及区分连接酶超家族不同分支的独特结构域模块和底物特异性。我们已经将我们对细菌DNA连接酶的兴趣扩展到两个atp依赖性链连接酶亚家族（命名为LigD和LigC），它们参与细菌DNA修复的非同源末端连接（NHEJ）途径。在已知的连接酶中，LigC和LigD是独特的，因为它们需要一个3'-OH单核糖核苷酸才能进行有效的缺口密封。LigD是双重独特的，因为它是一种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“末端愈合”活性，在被LIG组分密封之前重塑DSB的3'端。我们提出了一个多学科的议程（混合生物化学，分子遗传学和结构生物学），以解决该领域的下一代问题。我们的具体目标是：(i)利用我们已经解决的蛋白质- 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”探索腺苷酸转移到赖氨酸的机制，这是一种绕过基因可编程蛋白质“工具箱”限制的方法；（iii）解决了LigD磷酸酯酶结构域的结构，这是一个新的3'端修饰酶家族的例子；（iv）阐明细菌NHEJ连接酶对3'-OH单核糖核苷酸缺口的独特底物偏好。我们相信，我们提出的实验将对磷酸化转移反应机制、核酸损伤识别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. 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. .