Role of M. tuberculosis error-prone DNA polymerase DnaE2 in mutagenesis and drug resistance

结核分枝杆菌易错 DNA 聚合酶 DnaE2 在诱变和耐药性中的作用

• 批准号: 9262376
• 负责人: Kathleen A McDonough
• 金额: $ 24.94万
• 依托单位: WADSWORTH CENTER
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-12-01 至 2018-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9262376
• 关键词: Bacteria; Bacterial DNA; Biochemical; Biological; Biological Assay; Cells; Closure by clamp; Complex; DNA; DNA Damage; DNA Polymerase III; DNA biosynthesis; DNA replication fork; DNA-Directed DNA Polymerase; Disease; Drug resistance; Drug resistance in tuberculosis; Environment; Evolution; Family; Fluoroquinolones; Foundations; Frequencies; Future; Genes; Genetic; Genetic Predisposition to Disease; Genome; Genomics; Goals; In Vitro; Incidence; Individual; Induced Mutation; Infection; Intervention; Mediating; Modeling; Molecular; Molecular Genetics; Multienzyme Complexes; Mus; Mutagenesis; Mutation; Mycobacterium tuberculosis; Nucleotides; Pathogenicity; Plasmids; Polymerase; Process; Property; Proteins; Research; Resistance; Role; Site; Slide; Stress; Testing; Therapeutic; Tuberculosis; Two-Hybrid System Techniques; UV induced; Work; Yeasts; base; clinically relevant; combat; global health; improved; inhibitor/antagonist; light effects; novel; prevent; protein complex; protein protein interaction; reconstitution; resistance gene; resistance mechanism; response; stressor; success; therapeutic target; tool; tuberculosis drugs

An increasing challenge in the control of tuberculosis (TB) worldwide is the emergence and spread of drug resistance in Mycobacterium tuberculosis (Mtb), the causative agent of TB. Evolution of drug resistance in Mtb is associated with chromosomal mutations, not with the acquisition of resistance plasmids or transferred resistance genes. DnaE2 is required for induced mutagenesis in Mtb and belongs to the C-family of bacterial DNA polymerases that are responsible for genome replication. dnaE2 is a component of the imuA-imuB-dnaE2 cassette and all three genes are essential for DNA damage-induced mutagenesis. The DnaE2-ImuA-ImuB protein complex is proposed to associate with the sliding clamp processivity factor (the β-subunit of DNA pol III) through interactions with the ImuB subunit, allowing DnaE2 access to sites of replication where it can perform error-prone DNA synthesis. However, this enzyme complex has not been well characterized. We hypothesize that error prone DNA synthesis by the DnaE2-ImuA-ImuB polymerase complex contributes to drug resistance during TB infection. Here, we will (Aim 1) evaluate the importance of the complex for Mtb survival, mutagenesis and drug resistance in host-associated stress conditions, and (Aim 2) characterize the assembly, catalytic activity and mutational properties of this putative error-prone polymerase. These studies are essential steps towards understanding the mechanistic bases of drug resistance emergence in Mtb and the potential use of this error-prone DNA polymerase complex as a target for novel adjunctive therapies to combat drug- resistance in Mtb.

结核病控制中的一个日益严峻的挑战是药物的出现和传播 结核病的病原体结核分枝杆菌（Mtb）的耐药性。结核分枝杆菌耐药性的演变 与染色体突变有关，而与获得耐药质粒或转移无关。 抗性基因DnaE 2是结核分枝杆菌诱变所必需的，属于细菌C家族。 负责基因组复制的DNA聚合酶。dnaE 2是imuA-imuB-dnaE 2的组分， 盒和所有三个基因是DNA损伤诱导的诱变所必需的。DnaE2-ImuA-ImuB 蛋白质复合物被认为与滑动钳合成因子（DNA聚合酶的β亚基）有关 III）通过与ImuB亚基的相互作用，允许DnaE 2进入复制位点， 进行容易出错的DNA合成。然而，这种酶复合物还没有得到很好的表征。我们 假设通过DnaE 2-ImuA-ImuB聚合酶复合物进行易错DNA合成有助于药物 结核病感染期间的耐药性。在这里，我们将（目标1）评估该复合体对结核病生存的重要性， 诱变和耐药性，以及（目的2）表征组装体， 催化活性和突变特性的这种假定的易错聚合酶。这些研究至关重要 了解结核分枝杆菌耐药性出现的机制基础和潜在用途的步骤 这种容易出错的DNA聚合酶复合物作为新的连续治疗的目标，以打击药物， 抗结核菌