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

在世界范围内控制结核病的一个日益严峻的挑战是药物的出现和传播 结核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，而不是与获得抗性质粒或转移有关 抗性基因。DNAE2是Mtb诱变所必需的，属于细菌的C-家族 负责基因组复制的DNA聚合酶。Dna E2是imuA-imuB-dna E2的一种成分 Cassette和所有这三个基因对于DNA损伤诱导的突变是必不可少的。DNAE2-ImuA-ImuB 蛋白质复合体被认为与滑动钳制加工因子(DNAPoll的β亚单位)有关 Iii)通过与ImuB亚单位的相互作用，允许DNAE2在其可能的情况下访问复制位点 执行容易出错的DNA合成。然而，这种酶的复合体还没有得到很好的表征。我们 假设DNAE2-ImuA-ImuB聚合酶复合体容易出错的DNA合成与药物有关 结核病感染期间的耐药性。在这里，我们将(目标1)评估复合体对结核杆菌生存的重要性， 在宿主相关的应激条件下的诱变和耐药性，以及(目的2)表征该组装， 这个假定的容易出错的聚合酶的催化活性和突变特性。这些研究是必不可少的 了解结核分枝杆菌耐药性产生的机制基础及其潜在用途的步骤 将这种容易出错的DNA聚合酶复合体作为对抗药物的新辅助疗法的靶点- 结核分枝杆菌的抗性。

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.