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

铜绿假单胞菌 ImuABC 易错 DNA 聚合酶的纯化和初步生化分析

• 批准号: 9891550
• 负责人: MARK D. SUTTON
• 金额: $ 7.56万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-02-03 至 2022-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9891550
• 关键词: ATP phosphohydrolase; Active Sites; Adaptor Signaling Protein; Adjuvant Therapy; Antibodies; Attention; Biochemical; Bypass; Catalysis; Catalytic Domain; Centers for Disease Control and Prevention (U.S.); Clinical; Closure by clamp; Collection; Complex; Culture Media; DNA; DNA Sequence Alteration; DNA biosynthesis; DNA replication fork; DNA-Directed DNA Polymerase; Development; Dissection; Drug resistance; Engineering; Escherichia coli; Exonuclease; Funding; Future; Gene Expression; Gene Mutation; Genes; Goals; Growth; Hand; Health; Homologous Gene; Human; In Vitro; Individual; Infection; Lead; Measures; Methods; Molecular; Monitor; Mutagenesis; Mutation; Mycobacterium tuberculosis; Names; Nucleic Acids; Paper; Plasmids; Play; Polymerase; Process; Proteins; Pseudomonas aeruginosa; Pseudomonas aeruginosa infection; Publishing; Regulation; Research; Role; Solubility; Structure; Time; Virulence; Work; Writing; Yeasts; base; combat; design; drug resistant bacteria; experimental study; gene product; genetic analysis; high throughput screening; human pathogen; improved; in vitro Assay; insight; mouse model; multidrug-resistant Pseudomonas aeruginosa; novel; novel therapeutics; pathogen; pathogenic bacteria; pathogenic microbe; protein expression; replicase; resistance mutation; small molecule inhibitor; therapeutic target; yeast two hybrid system

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.

摘要 由于耐药细菌的不断出现，改进的治疗方法以及新的靶点 我们迫切需要开发新的治疗方法。各种机制有助于 耐药性和病原体对人类宿主的适应（病理适应），包括 基因表达、新基因的获得和DNA突变。一个有希望的治疗靶点， 日期收到显着很少注意的是在病理适应和药物的获得中发挥的作用， 低保真DNA聚合酶（Pols）。这些Pol在未受损复制时产生突变 DNA，或者通过称为translesion DNA合成（TLS）的过程绕过受损的碱基。 E. coli Pol IV（dinB）和Pol V（umuDC）代表了研究得最好的细菌TLS Pol。虽然大多数人 研究的人类病原体具有在TLS中起作用的Pol IV同源物，许多缺乏Pol V。 一种高度保守但研究不足的多亚基TLS Pol，有几个不同的名字， 在本提案中称为ImuABC复合体。由ImuABC产生的突变有助于毒力， 持久性和耐药性。基于序列，ImuA（也称为ImuA '）与ATP酶具有同源性。 (but可能缺乏催化活性），而ImuB是Pol V UmuC催化亚基的同系物，其缺乏 必需的活性位点残基，这意味着它可能缺乏Pol活性。与这一结论相一致，突变 由ImuABC催化的蛋白质的合成依赖于ImuC（也称为DnaE 2）的Pol活性，其在结构上与免疫球蛋白的合成相关。 细菌Pol III复制酶的DnaE 1催化亚基。酵母双杂交实验的结果表明， Imu B是与ImuA和ImuC以及B持续合成钳和DnaE 1相互作用的衔接蛋白 Pol III亚基。这些后者的相互作用可以协调的行动ImuABC与Pol III。 尽管明确证明了ImuABC在催化突变中的重要作用， 尽管ImuABC复合物具有耐药性、毒力和病理适应性，但其研究却非常少。 在撰写本文时，只有42篇发表的论文包含检索词“imuA，imuB，imuC 或dnaE 2。”重要的是，这些作品都没有讨论ImuABC复合物的生物化学分析。这个目标 一个提议是开发用于纯化可溶形式的ImuA、ImuB和ImuC蛋白的方法， 详细的体外机制研究。为此，我们将重点关注铜绿假单胞菌ImuABC蛋白，因为我们已经 具有铜绿假单胞菌B夹和Pol III复制酶的过量生产者和已建立的纯化方法， 这将在未来的工作中需要，旨在确定ImuB-b夹和ImuB-Pol III的贡献 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试验， 以详细了解ImuABC在突变中功能的机制。