Validating a potential interaction between error-prone polymerases and SSB as a therapeutic target for Mycobacterium tuberculosis

项目摘要 由结核分枝杆菌（Mycobacterium tuberculosis，Mtb）引起的结核病仍然是严重的全球健康问题， 这一威胁每年在全世界造成100多万人死亡。虽然抗生素治疗已经存在， 但是，他们往往不成功。延长疗程的必要性和发展 在治疗过程中对常用抗生素的耐药性是需要克服的主要挑战 来改善病人的治疗效果 由于易错DNA聚合酶的作用而导致的染色体突变是导致染色体突变的主要驱动力。 Mtb中的突变以及因此抗生素抗性的出现。易错聚合酶是 参与DNA损伤耐受过程，称为跨损伤合成。通常，容易出错 聚合酶受到严格的调控，因此它们在DNA复制过程中没有活性。然而，他们成为 在应激条件下激活，例如在抗生素治疗期间经历的那些。在最近的工作中， 我们的实验室，我们证明，在模型细菌大肠杆菌，激活的错误- 倾向性聚合酶Pol IV需要与单链DNA结合蛋白（SSB）相互作用，其作用是 在DNA损伤位点附近局部浓缩Pol IV。重要的是，这种相互作用的消除基本上 减少Pol IV介导的跨损伤合成和诱变。在本提案中，我们将测试 Mtb中的三个Pol IV同源物与MtSSB相互作用。下一步，我们将确定消除这种假定的突变。 相互作用而不影响其他分子相互作用或聚合酶活性。最后，我们将介绍这些 突变到耻垢分枝杆菌，一种非致病性的Mtb模型系统，并测试这些突变是否 菌株对DNA损伤剂敏感。如果成功，这些研究将确定一种新的分子 相互作用可能在治疗上靶向抑制结核杆菌抗生素耐药性的出现。

Project Summary Tuberculosis, caused by the bacterium Mycobacterium tuberculosis (Mtb), remains a serious global health threat that kills over a million people per year worldwide. While antibiotic treatments have existed for some time, they are often not successful. The necessity for an extended treatment course and the development of resistance to commonly prescribed antibiotics during treatment are major challenges that need to be overcome to improve patient outcomes. Chromosomal mutations due to the action of error-prone DNA polymerases are a major driver of mutagenesis in Mtb and therefore the emergence of antibiotic resistance. Error-prone polymerases are involved in a DNA damage tolerance process known as translesion synthesis. Typically, error-prone polymerases are tightly regulated so that they are not active during DNA replication. However, they become activated under stress conditions, such as those experienced during antibiotic treatment. In recent work from our laboratory, we demonstrated that within the model bacterium Escherichia coli, the activation of the error- prone polymerase Pol IV requires an interaction with single-stranded DNA binding protein (SSB), which acts to locally concentrate Pol IV near sites of DNA damage. Importantly, ablation of this interaction substantially reduced Pol IV-mediated translesion synthesis and mutagenesis. In this proposal we will test whether the three Pol IV homologs in Mtb interact with MtSSB. Next, we will identify mutations that ablate this putative interaction without affecting other molecular interactions or polymerase activity. Finally, we will introduce these mutations into Mycobacterium smegmatis, a non-pathogenic model system of Mtb, and test whether these strains are sensitized to DNA damage agents. If successful, these studies will identify a novel molecular interaction to potentially target therapeutically to suppress the emergence of antibiotic resistance in Mtb.