Viral and Bacterial DNA Ligases

描述(申请人提供)：DNA连接酶是普遍存在的酶，催化DNA复制和修复的关键最后一步-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5‘-PO4和3’-OH多核苷酸末端通过三个化学步骤结束：(I)连接酶与ATP或NAD+反应形成共价连接酶-(赖氨酰-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催化的，连接酶如何识别“受损”的DNA或RNA末端，以及结构域移动和活性部位重塑如何被用来编排末端连接途径。我们使用三个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：ChVLig)、细菌依赖NAD+的DNA连接酶(E.coliLIGA)和病毒依赖的RNA连接酶(T4Rnl2)。在上一个授权期内，我们测定了ChVLig-AMP在3‘-OH/5’-PO4缺口上的原子结构，以及LIGA和T4Rnl2与其镍多核苷酸-腺苷中间体结合的结构。这些结构以及受其启发的功能研究揭示了所有DNA和RNA连接酶所共有的机制原理，以及区别连接酶超家族不同分支的独特结构模块和底物特异性。我们已经将我们对细菌DNA连接酶的兴趣扩展到参与细菌DNA修复的非同源末端连接(NHEJ)途径的两个亚家族的ATP依赖的链连接酶(称为LigD和LigC)。LigC和LigD在已知的连接酶中是独一无二的，因为它们需要3‘-OH单核苷酸来执行有效的缺口封闭。LigD具有双重独特性，因为它是由三个自主催化域组成的多功能酶：连接酶(LIG)、聚合酶(POL)和磷酸酯酶(PE)。POL和PE结构域包括一套DNA“末端修复”活动，这些活动在被LIG组分封闭之前重塑DSB的3‘末端。我们提出了一个多学科的议程(融合生物化学、分子遗传学和结构生物学)来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界面的氨基酸的突变分析；(Ii)通过结构方法和“化学突变”-一种绕过遗传可编程蛋白质“工具包”限制的方法-来探索腺苷酸转移到赖氨酸的机制；(Iii)解决LigD磷酸酯酶结构域的结构，这是一种新的3‘末端修饰酶家族的例证；以及(Iv)阐明细菌NHEJ连接酶对3’-OH单核苷酸缺口的明显底物偏好。我们相信，我们提出的实验将对磷酸转移反应机制、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.