Molecular analysis of mycobacterial NHEJ

描述（由申请人提供）：非同源末端连接（NHEJ）是细菌中双链断裂修复的新认识途径。NHEJ在细菌的一个子集中起作用，包括M。结核和M. SMEGALINE，其编码核心NHEJ组分：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，我们对M. smegalgae和M.结核病，并证明其依赖Ku和LigD，与ATP依赖性DNA连接酶C（LigC）的备份作用。我们已经表明，通过NHEJ修复5'突出端和平端双链断裂（DSB）通过LigD聚合酶结构域（LigD-POL）的活性是高度致突变的，LigD聚合酶结构域是一种新型细菌聚合酶，在NHEJ复合物中也起着关键的结构作用。我们已经开发了归巢核酸内切酶I-SceI，用于切割分枝杆菌染色体，并表明NHEJ是修复染色体DSB所必需的，这一过程也在修复的末端引入插入和缺失。在此基础上，我们现在提出了一个扩大的计划，生物化学和遗传学研究的分枝杆菌NHEJ途径，其关系到其他途径的DSB修复，其作用在M。结核病发病机制通过使用新开发的区分HR、NHEJ和单链退火（SSA）途径的染色体DSB修复试验，我们将确定途径使用的相对频率、分子结果以及DSB末端构型对野生型M中DSB修复的影响。耻垢病和NHEJ组分、HR组分或两者缺陷的突变体。根据我们的发现，UvrD 1是一个DNA依赖的ATP酶和一个Ku依赖的3 '-to-5' DNA解旋酶，我们将探讨UvrD 1在DNA修复中的作用。我们将通过对这些酶复合物进行详细的遗传和生化分析，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，我们将测试NHEJ和HR是否在M中扮演重叠的角色。结核病的持续性和潜伏期。这些研究将为原核NHEJ提供机制性见解，并确定NHEJ在发病机制中的作用，可能将此途径作为抗菌剂开发的靶点。 公共卫生相关性：本项目研究分枝杆菌中的非同源末端连接这一新的DNA修复途径，包括人类主要病原体分枝杆菌。结核病，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消除的理解，并可能导致分枝杆菌引起的感染的新药物策略。

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.