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

基本信息

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
项目类别：
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

项目摘要

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.

全球结核病控制面临的日益严峻的挑战是药物的出现和传播结核分枝杆菌 (Mtb) 的耐药性，结核病的病原体 Mtb 的耐药性演变。与染色体突变有关，与抗性质粒的获得或转移无关 DnaE2 是 Mtb 诱导突变所必需的，属于细菌 C 家族。负责基因组复制的 DNA 聚合酶是 imuA-imuB-dnaE2 的组成部分。盒和所有三个基因对于 DNA 损伤诱导的突变都是必需的。蛋白质复合物被认为与滑动钳持续性因子（DNA聚合体的β亚基）相关联。 III) 通过与 ImuB 亚基相互作用，允许 DnaE2 访问其可以复制的位点然而，这种酶复合物尚未得到很好的表征。研究发现 DnaE2-ImuA-ImuB 聚合酶复合物容易出错的 DNA 合成有助于药物的合成在这里，我们将（目标 1）评估复合物对结核分枝杆菌存活的重要性，宿主相关应激条件下的诱变和耐药性，以及（目标 2）表征组装，这种假定的易错聚合酶的催化活性和突变特性是至关重要的。逐步了解 Mtb 耐药性出现的机制基础及其潜在用途这种容易出错的 DNA 聚合酶复合物作为新型辅助疗法的靶点来对抗药物 Mtb 的抵抗力。