Mechanism and Architecture of EndoMS/NucS Mutation Avoidance in Mycobacteria

分枝杆菌 EndoMS/NucS 突变避免的机制和架构

• 批准号: 9809008
• 负责人: Eric Alan Josephs
• 金额: $ 14.55万
• 依托单位: UNIVERSITY OF NORTH CAROLINA GREENSBORO
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-06-11 至 2021-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9809008
• 关键词: Actinobacteria class; Address; Affect; Animal Model; Antibiotics; Architecture; Bacteria; Biochemical; Biological Assay; Biotechnology; Cell Death; Closure by clamp; Clustered Regularly Interspaced Short Palindromic Repeats; Coupled; DNA; DNA Damage; DNA Repair; DNA Sequence Alteration; DNA biosynthesis; DNA-Directed DNA Polymerase; Drug resistance; Escherichia coli; Event; Foundations; Genes; Genetic Recombination; Genetic Transcription; Genome; Genomics; Genus Mycobacterium; Goals; Health; Homologous Gene; Human; Investigation; Knock-out; Knowledge; Laboratories; Lesion; Mismatch Repair; Modeling; Molecular; Multi-Drug Resistance; Mutation; Mycobacterium Infections; Mycobacterium smegmatis; Mycobacterium tuberculosis; Nucleotides; Oligonucleotides; Organism; Pathway interactions; Phenotype; Point Mutation; Process; Property; Proteins; Pyrococcus abyssi; Reaction; Reporting; Research Personnel; Resistance; Stretching; Techniques; Testing; Thermococcus; Toxic effect; Tuberculosis; Variant; Work; cancer drug resistance; combat; endonuclease; gene product; genetic manipulation; insight; knock-down; mycobacterial; next generation; novel; pathogen; repaired; senescence

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.

项目摘要 分枝杆菌病原体（如结核分枝杆菌）中耐药性的主要驱动力是遗传 但是，突变，但是这些分子过程控制着突变和避免突变的突变 有机体仍然了解不足。在几乎所有其他生物体中，突变率都由DNA紧密控制 不匹配维修（MMR）途径，复制后立即修复了不匹配的核动脉底座 如果不纠正，将成为永久的遗传突变，并且在感应期间抑制重组不当 事件。大多数肌细菌（包括分枝杆菌），尽管具有相似的基本突变率，但 似乎缺乏保守的MMR蛋白的任何同源物。相反，直到2017年才 确定许多静脉细菌具有不过 Abyssi nuc和kodakarensis thermococcus abyssi nuc和天然MSMEG_4923基因 产物，“内膜/NUC”（EN）蛋白，在smegmatis的分枝杆菌中授予类似的抗糊状剂和 通常定义规范MMR的抗重组表型。迄今为止，en-机制 避免协调突变（ENMA）仍然是密码学且理解不足，对 EN蛋白会在分子水平促进突变的机制，甚至 其他蛋白质参与了这一过程。虽然ENMA可能代表了一个新的机会 理解并潜在地对抗分枝杆菌中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 机会。因此，长期目标是定义机制和体系结构（组件和 ENMA的互动），以便可以使用这些知识来理解和应对MDR的挑战 在治疗分枝杆菌感染中。为此，此R21的目的是应用一个有能力的新颖测定法 直接表征活体大肠杆菌中MMR样活性作为解构的实验基础 ENMA的分子机制在生存的史氏菌中。小说分析有许多优势 解构类似MMR的活动中的分枝杆菌研究MMR缺乏的传统方法， 配备了这种新型生物技术，我们将阐明ENMA的基础机制及其如何 与规范的MMR反应相似或不同。与下一代结合进行此评估 CRISPR等生物技术，我们还将确定并表征分枝杆菌的可疑调节剂 ENMA或DNA修复相关的毒性。这种独特的方法具有有效阐明 Enma的建筑和机制。然后，该项目将为雄心勃勃的R01阶段奠定基础 研究分枝杆菌病原体中突变和耐药性机制及其如何可能 被探索以促进分枝杆菌细胞死亡。