DNA Ligases in Mycobacterial DNA repair & Pathogenesis

描述(申请人提供)：DNA连接酶通过恢复DNA磷酸二酯骨架的完整性来催化DNA修复和复制的最后一个常见步骤。所有已知的细菌基因组都编码一种依赖NAD的DNA连接酶(LIGA)，这是生存所必需的。一些细菌基因组编码一种额外的功能未知的ATP依赖的连接酶。分枝杆菌，包括人类主要病原体结核分枝杆菌(MTU)和遗传易感性污垢分枝杆菌(MSM)，除LIGA外，还编码3种可能的ATP依赖DNA连接酶(LigB、LigC、LigD)。这些三磷酸腺苷依赖的连接酶在分枝杆菌DNA修复系统和分枝杆菌发病机制中的作用尚不清楚。我们的初步数据表明，这三种连接酶在体外都是ATP依赖的连接酶，但具有不同的催化性质。MSM和MTU中每个连接酶的零突变表明，这些连接酶对单独和组合生长都不是必需的，但三种替代的ATP依赖连接酶不能挽救耻垢分枝杆菌中LIGA的必要性。此外，LigC和LigD都参与了原核生物非同源末端连接(NHEJ)的一种新途径，该途径是有效的，但保真度较低。NHEJ连接的序列分析表明，钝端分枝杆菌NHEJ参与了聚合酶和核酸酶的活性。本实验旨在阐明耻垢分枝杆菌和结核分枝杆菌中由ATP依赖的DNA连接酶介导的非同源末端连接(NHEJ)的分子机制、生理作用和致病意义。我们提出了一个涵盖遗传学、生物化学和微生物发病机制的多学科研究计划，该计划将解决分枝杆菌DNA连接酶是一种新的DNA修复系统的假设，该系统在体内生长和持续期间保护分枝杆菌染色体免受双链断裂的影响。

DESCRIPTION (provided by applicant): DNA ligases catalyze the final common step of DNA repair and replication by restoring the integrity of the DNA phosphodiester backbone. All known bacterial genomes encode an NAD dependent DNA ligase (LigA) which is essential for viability. Some bacterial genomes encode an additional ATP dependent ligase of unknown function. Mycobacteria, including the major human pathogen Mycobacterium tuberculosis (Mtu) and the genetically tractable M. smegmatis (MSm), encode 3 putative ATP dependent DNA ligases (LigB, LigC, LigD) in addition to LigA. The function of these ATP dependent ligases in mycobacterial DNA repair systems and mycobacterial pathogenesis is unknown. Our preliminary data shows that these 3 ligases are ATP dependent ligases in vitro, but with distinct catalytic properties. Null mutants of each ligase in MSm and Mtu indicate that these ligases are nonessential for growth individually and in combination but that three alternative ATP dependent ligases does not rescue the essentiality of LigA in M. smegmatis. In addition both ligC and ligD participate in a novel pathway of prokaryotic Non homologous end joining (NHEJ) which is efficient but low fidelity. Sequence analysis of NHEJ junctions indicates involvement of polymerase and nuclease activities in blunt end mycobacterial NHEJ. The purpose of the experiments proposed herein is to elucidate the molecular mechanisms, physiologic role, and pathogenetic importance of Non homologous end joining (NHEJ) mediated by ATP dependent DNA ligases in M. smegmatis and M. tuberculosis. We propose a multidisciplinary research program encompassing genetics, biochemistry and microbial pathogenesis that will address the hypothesis that the DNA ligases of mycobacteria are a novel DNA repair system that defends the mycobacterial chromosome against double strand breaks during in vivo growth and persistence.