Molecular analysis of mycobacterial NHEJ

描述（申请人提供）：非同源末端连接（Nonhomologous end-joining， NHEJ）是细菌双链断裂修复的新途径。NHEJ在一些细菌中起作用，包括结核分枝杆菌和耻垢分枝杆菌，它们编码NHEJ的核心成分：DNA末端结合蛋白Ku和ATP依赖性DNA连接酶D （LigD）。在该奖项支持的研究中，我们表征了耻垢分枝杆菌和结核分枝杆菌的NHEJ途径，并证明了其对Ku和LigD的依赖性，以及atp依赖性DNA连接酶C （LigC）的辅助作用。我们已经证明，NHEJ对5'悬垂和钝端双链断裂（DSBs）的修复是高度诱变的，通过LigD聚合酶域（LigD- pol）的活性，一种新型的细菌聚合酶，在NHEJ复合体中也起着关键的结构作用。我们已经开发了用于分枝杆菌染色体切割的归巢内切酶I-SceI，并表明NHEJ是染色体dsb修复所必需的，这一过程也在修复端引入插入和缺失。在此基础上，我们现在提出了一个扩展的分枝杆菌NHEJ途径的生化和遗传研究计划，它与DSB修复的其他途径的关系，以及它在结核分枝杆菌发病机制中的作用。通过一项新开发的染色体DSB修复测定，区分HR、NHEJ和单链退火（SSA）途径，我们将确定野生型臭毛分枝杆菌和缺乏NHEJ成分、HR成分或两者都缺乏的突变体中，途径使用的相对频率、分子结果以及DSB末端构型对DSB修复的影响。根据我们的发现，UvrD1是DNA依赖性atp酶和ku依赖性3‘到5’ DNA解旋酶，我们将探讨UvrD1在DNA修复中的作用。我们将通过对这些酶复合物的详细遗传和生化分析来确定RecBCD和新型分枝杆菌解旋酶/核酸酶AdnAB对DNA修复和NHEJ缺失形成的贡献。最后，我们将在小鼠模型中测试NHEJ和HR是否在结核分枝杆菌的持续和潜伏期中发挥重叠作用。这些研究将提供对原核生物NHEJ机制的深入了解，并确定NHEJ在发病机制中的作用，有可能推进这一途径作为抗菌药物开发的靶点。

DESCRIPTION (provided by applicant): Nonhomologous end-joining (NHEJ) is a newly appreciated pathway of double strand break repair in bacteria. NHEJ operates in a subset of bacteria, including M. tuberculosis and M. smegmatis, that encode the core NHEJ components: the DNA end-binding protein Ku and ATP- dependent DNA ligase D (LigD). In studies supported by this award, we have characterized the NHEJ pathway in M. smegmatis and M. tuberculosis and demonstrated its dependence on Ku and LigD, with a backup role for ATP-dependent DNA ligase C (LigC). We have shown that repair of 5' overhang and blunt-end double strand breaks (DSBs) by NHEJ is highly mutagenic through the activity of the LigD polymerase domain (LigD-POL), a novel bacterial polymerase that also plays a key structural role in the NHEJ complex. We have developed the homing endonuclease I-SceI for cleavage of the mycobacterial chromosome and shown that NHEJ is required for repair of chromosomal DSBs, a process which also introduces insertions and deletions at repaired ends. Building on this foundation, we now propose an expanded program of biochemical and genetic investigation of the mycobacterial NHEJ pathway, its relationship to other pathways of DSB repair, and its role in M. tuberculosis pathogenesis. By using a newly developed assay of chromosomal DSB repair that discriminates HR, NHEJ and single-strand annealing (SSA) pathways, we will determine the relative frequency of pathway use, molecular outcomes, and effects of DSB end-configuration on DSB repair in wild-type M. smegmatis and mutants deficient in NHEJ components, HR components, or both. Prompted by our findings that UvrD1 is a DNA-dependent ATPase and a Ku-dependent 3'-to-5' DNA helicase, we will probe the role of UvrD1 in DNA repair. We will determine the contribution of RecBCD and the novel mycobacterial helicase/nuclease AdnAB to DNA repair and NHEJ deletion formation through a detailed genetic and biochemical analysis of these enzyme complexes. Finally, we will test whether NHEJ and HR play overlapping roles in M. tuberculosis persistence and latency in the murine model. These studies will provide mechanistic insight into prokaryotic NHEJ and determine the role of NHEJ in pathogenesis, potentially advancing this pathway as a target for antimicrobial development.