Purification and Initial Biochemical Analysis of the P. aeruginosa ImuABC Error-Prone DNA Polymerase

摘要 由于抗药性细菌的日益出现，改进的治疗方法以及新的靶点 可以开发出新的治疗方法，这是迫切需要的。各种机制有助于 抗药性和病原体对其人类宿主的适应(致病适应)，包括改变 基因表达、新基因的获取和DNA突变。一个有希望的治疗靶点，必须 DATA在病理适应和药物获得中所起的作用受到的关注非常少。 低保真DNA聚合酶(POL)的抗性。这些POL在复制时会产生突变 DNA，或者当通过称为跨损伤DNA合成(TLS)的过程绕过受损碱基时。 大肠杆菌Pol IV(DinB)和Pol V(UmuDC)代表了研究最深入的细菌TLS Pol。虽然最好的是 被研究的人类病原体具有作用于TLS的Pol IV同源物，许多人缺乏Pol V，相反，它们编码 一种高度保守但未被充分研究的多亚基TLS Pol，有几个不同的名字，将是 在本提案中称为ImuABC复合体。ImuABC产生的突变有助于毒力， 坚持不懈，耐药。根据序列，ImuA(也称为ImuA‘)与ATPase具有同源性 (但可能缺乏催化活性)，而ImuB是Pol V UMUC催化亚基的同系物，缺乏 必需的活性部位残基，这意味着它可能没有POL活性。与这一结论一致的是，突变 由ImuABC催化依赖于IMUC的POL活性(也称为DNAE2)，它在结构上与 细菌Pol III复制酶的DneE1催化亚基。酵母双杂交实验的结果表明 ImuB是一种适配蛋白，与ImuA和IMUC以及b加工能力钳和DNAE1相互作用 Pol III的亚基。后一种相互作用可能协调ImuABC和Pol III的作用。 尽管ImuABC在催化突变方面的重要作用得到了明确的证明 由于ImuABC复合体具有耐药性、致病力和致病适应能力，因此研究非常少。 在撰写本文时，只有42篇发表的论文包含搜索词“imuA，imuB，imuc 或dna E2。“重要的是，这些工作都没有讨论ImuABC复合体的生化分析。这是一个目标 建议开发用于纯化ImuA、ImuB和IMUC蛋白的可溶性形式的方法 详细的体外机制研究。为此，我们将重点介绍铜绿假单胞菌ImuABC蛋白，因为我们已经 对铜绿假单胞菌B夹和Pol III复制酶有过多生产和成熟的纯化方法， 在今后旨在确定ImuB-b夹具和ImuB-Pol III的贡献的工作中将需要 与ImuABC功能/调节的相互作用。作为第二个目标，我们将开发几种必要的体外检测方法 有关ImuABC在诱变中的作用机制的详细生化剖析。

ABSTRACT Due to the growing emergence of drug-resistant bacteria, improved therapies, as well as novel targets against which new therapies can be developed, are desperately needed. A variety of mechanisms contribute to drug-resistance, and adaptation of pathogens to their human hosts (pathoadaptation), including alterations to gene expression, the acquisition of new genes, and DNA mutations. A promising therapeutic target that has to date received remarkably little attention is the role played in pathoadaptation and the acquisition of drug- resistance by low fidelity DNA polymerases (Pols). These Pols generate mutations when replicating undamaged DNA, or when bypassing damaged bases via a process termed translesion DNA synthesis (TLS). E. coli Pol IV (dinB) and Pol V (umuDC) represent the best-studied bacterial TLS Pols. While most well studied human pathogens possess a Pol IV homolog that acts in TLS, many lack a Pol V. Instead, they encode a highly conserved yet understudied multi-subunit TLS Pol that goes by a few different names, and will be referred to in this proposal as the ImuABC complex. Mutations generated by ImuABC contribute to virulence, persistence, and drug-resistance. Based on sequence, ImuA (also called ImuA’) has homology to an ATPase (but likely lacks catalytic activity), while ImuB is a homolog of the Pol V UmuC catalytic subunit that lacks the essential active site residues, meaning it is likely devoid of Pol activity. Consistent with this conclusion, mutations catalyzed by ImuABC depend on the Pol activity of ImuC (also called DnaE2), which is structurally related to the DnaE1 catalytic subunit of the bacterial Pol III replicase. Results of yeast-two-hybrid experiments suggest that ImuB is an adapter protein that interacts with ImuA and ImuC, as well as the b processivity clamp and the DnaE1 subunit of Pol III. These latter interactions may coordinate the actions of ImuABC with those of Pol III. Despite the clear demonstration of an important role for ImuABC in catalyzing mutations that underlie drug resistance, virulence, and pathoadaptation, the ImuABC complex is the subject of remarkably little research. At the time of this writing, there were only 42 published papers containing the search terms “imuA, imuB, imuC or dnaE2.” Importantly, none of these works discuss biochemical analysis of the ImuABC complex. A goal of this proposal is to develop methods for the purification of soluble forms of the ImuA, ImuB, and ImuC proteins for detailed in vitro mechanistic studies. For this, we will focus on the P. aeruginosa ImuABC proteins, as we already have overproducers and established purification methods for the P. aeruginosa b clamp and Pol III replicase, which will be required in future work aimed at determining the contribution of the ImuB-b clamp and ImuB-Pol III interactions to ImuABC function/regulation. As a second goal, we will develop several in vitro assays necessary for detailed biochemical dissection of the mechanism underlying ImuABC function in mutagenesis.