Molecular analysis of mycobacterial NHEJ

描述(申请人提供)：非同源末端连接(NHEJ)是一种新发现的细菌双链断裂修复途径。NHEJ在包括结核分枝杆菌和耻垢分枝杆菌在内的细菌亚群中发挥作用，这些细菌编码NHEJ的核心成分：DNA末端结合蛋白Ku和ATP依赖的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，并证明了它对Ku和LigD的依赖，以及对ATP依赖的DNA连接酶C(LigC)的支持作用。我们已经证明，NHEJ通过LigD聚合酶结构域(LigD-POL)的活性修复5‘端悬垂和钝端双链断裂(DSB)是高度突变的，LigD-POL是一种新的细菌聚合酶，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切割分枝杆菌染色体的归巢内切酶I-SCEI，并表明NHEJ是修复染色体DSB所必需的，这一过程也引入了修复端的插入和缺失。在此基础上，我们现在提出了分枝杆菌NHEJ途径的生化和遗传学研究的扩展计划，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。通过使用一种新开发的区分HR、NHEJ和单链退火(SSA)途径的染色体DSB修复分析，我们将确定途径使用的相对频率、分子结果，以及DSB末端配置对野生型耻垢分枝杆菌和缺乏NHEJ成分、HR成分或两者都缺乏的突变株DSB修复的影响。根据我们的发现，UvrD1是一种DNA依赖的ATPase和Ku依赖的3‘-5’DNA解旋酶，我们将探讨UvrD1在DNA修复中的作用。我们将通过详细的遗传和生化分析来确定RecBCD和新的分枝杆菌解旋酶/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久性和潜伏期中扮演重叠的角色。这些研究将提供对原核生物NHEJ的机制洞察，并确定NHEJ在发病机制中的作用，潜在地推动这一途径成为抗菌药物开发的靶点。 公共卫生相关性：该项目调查分枝杆菌中非同源末端连接的新的DNA修复途径，包括人类的主要病原体结核分枝杆菌，结核病的病因。这些研究将促进我们对分枝杆菌如何抵抗宿主清除的理解，并可能导致治疗分枝杆菌感染的新药物策略。

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. PUBLIC HEALTH RELEVANCE: This project investigates the novel DNA repair pathway of Nonhomologous end-joining in mycobacteria, including the major human pathogen M. tuberculosis, cause of the disease Tuberculosis. These studies will advance our understanding of how mycobacteria resist elimination by the host and may lead to novel drug strategies for infections caused by mycobacteria.