DNA Ligases in Mycobacterial DNA repair & Pathogenesis

描述（由申请人提供）：DNA连接酶通过恢复DNA磷酸二酯骨架的完整性催化DNA修复和复制的最后共同步骤。所有已知的细菌基因组编码NAD依赖性DNA连接酶（LigA），其对于生存力是必需的。一些细菌基因组编码另外的功能未知的ATP依赖性连接酶。分枝杆菌，包括主要的人类病原体结核分枝杆菌（Mtu）和遗传上易处理的M。smeglycin（MSm），除了LigA外，还编码3种推测ATP依赖性DNA连接酶（LigB、LigC、LigD）。这些ATP依赖性连接酶在分枝杆菌DNA修复系统和分枝杆菌发病机制中的功能尚不清楚。我们的初步数据表明，这3种连接酶在体外是ATP依赖性连接酶，但具有不同的催化性质。MSm和Mtu中每种连接酶的突变体表明，这些连接酶单独和组合对于生长是非必需的，但三种替代的ATP依赖性连接酶不能挽救M中LigA的必需性。恶臭此外，ligC和ligD都参与了原核非同源末端连接（NHEJ）的新途径，该途径是有效的，但保真度低。NHEJ连接的序列分析表明，聚合酶和核酸酶的活动参与平端分枝杆菌NHEJ。本实验旨在阐明M. smegalgae和M.结核我们提出了一个多学科的研究计划，包括遗传学，生物化学和微生物的发病机制，将解决的假设，分枝杆菌的DNA连接酶是一种新的DNA修复系统，防御分枝杆菌染色体对双链断裂在体内生长和持久性。

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.