Mechanism and Architecture of EndoMS/NucS Mutation Avoidance in Mycobacteria

工程摘要 结核分枝杆菌等分枝杆菌病原体耐药性的主要驱动因素是遗传 突变，然而，这些基因中控制突变和避免突变的分子过程 生物体仍然知之甚少。在几乎所有其他生物体中，突变率都受到DNA的严格控制。 错配修复（MMR）途径，在复制后立即修复错配的核苷酸， 如果不纠正，就会变成永久性的基因突变，在衰老过程中，会抑制不适当的重组 事件大多数放线菌--包括分枝杆菌--尽管有相似的基础突变率， 似乎缺乏保守的MMR蛋白的任何同源物。相反，直到2017年， 鉴定出许多放线菌具有古细菌错配敏感性核酸内切酶的同源物 深海热球菌NucS和Kodakarensis EndoMS，并且天然MSMEG_4923基因 在耻垢分枝杆菌中的产物“EndoMS/NucS”（EN）蛋白赋予了类似的抗诱变和 典型定义典型MMR的抗重组表型。到目前为止，EN- 协调突变避免（ENMA）仍然是神秘的，人们对它的了解很少， EN蛋白在分子水平上促进突变避免的机制，甚至 还有哪些蛋白质参与了这个过程虽然ENMA可能代表了一个新的机会， 了解并潜在地对抗分支杆菌的耐药性和多药耐药性（MDR） 病原体，缺乏有关其机制和途径的基本知识将限制这些 机会因此，长期目标是定义机制和架构（组件和 相互作用），以便这些知识可用于理解和应对MDR的挑战 治疗分枝杆菌感染。为此，该R21的目的是应用一种能够 直接表征活大肠杆菌中的MMR样活性，作为解构 ENMA在活体M.恶臭新的测定法具有许多优点， 解构分枝杆菌中MMR样活性，这是研究MMR的传统方法所缺乏的， 配备了这种新的生物技术，我们将阐明ENMA的基本机制，以及它是如何 与典型的MMR反应相似或不同。结合下一代检测试剂盒执行本检测试剂盒 生物技术，如CRISPR，我们还将识别和表征疑似分枝杆菌的调节剂， ENMA或DNA修复相关毒性。这种独特的方法有望有效地阐明 ENMA的结构和机制。该项目将为雄心勃勃的R 01阶段奠定基础 分枝杆菌病原体突变和耐药机制的研究及其EN如何可能 被利用来引起分支杆菌细胞死亡。

Project Abstract The primary driver of drug resistance in mycobacterial pathogens like Mycobacterium tuberculosis is genetic mutation, however the molecular processes which govern mutation and mutation avoidance in these organisms remain poorly understood. In nearly all other organisms, mutation rate is tightly controlled by a DNA mismatch repair (MMR) pathway that, immediately after replication, repairs mismatched nucleotides that would become permanent genetic mutations if not corrected and, during senescence, inhibits improper recombination events. Most actinobacteria—which includes mycobacteria—despite having similar basal mutation rates, appear to lack any homologues of the conserved MMR proteins. Rather, it was not until 2017 when it was identified that many actinobacteria instead harbor homologues of archaeal mismatch-sensitive endonucleases Pyrococcus abyssi NucS and Thermococcus kodakarensis EndoMS, and that the native MSMEG_4923 gene product, the “EndoMS/NucS” (EN) protein, in Mycobacterium smegmatis conferred similar anti-mutagenic and anti-recombination phenotypes that typically define canonical MMR. To date, the mechanisms of EN- coordinated mutation avoidance (ENMA) remain cryptic and poorly understood, and little else is known about the mechanism by which the EN protein would promote mutational avoidance at the molecular level, or even what other proteins are involved in this process. While the ENMA might represent a new opportunity to understand and potentially counter drug resistance and multi-drug resistance (MDR) in mycobacterial pathogens, the absence of fundamental knowledge regarding its mechanism and pathway will limit those opportunities. The long-term goal is therefore to define the mechanism and architecture (components and interactions) of ENMA so that this knowledge can be used to understand and address the challenges of MDR in treating mycobacterial infections. To do so, the purpose of this R21 is to apply a novel assay that is capable of directly characterizing MMR-like activity in living Escherichia coli as an experimental basis for deconstructing the molecular mechanisms of ENMA in living M. smegmatis. The novel assay has many advantages to deconstructing MMR-like activities in mycobacteria that traditional approaches to studying MMR lack, and equipped with this novel biotechnology we will elucidate the foundational mechanisms of ENMA and how it is similar or differs from the canonical MMR reaction. Performing this assay in combination with next-generation biotechnologies like CRISPR, we will also identify and characterize suspected modulators of mycobacterial ENMA or DNA repair-associated toxicity. This unique approach holds the promise of efficiently elucidating the architecture and mechanism of ENMA. This project will then set the foundation for ambitious R01-stage investigation into mechanisms of mutation and drug resistance in mycobacterial pathogens and how it EN may be exploited to provoke mycobacterial cell death.