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

基本信息

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
项目类别：
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

项目摘要

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.

抽象的由于耐药细菌的不断出现、治疗方法的改进以及新靶标的出现迫切需要多种机制来开发新疗法。耐药性和病原体对其人类宿主的适应（路径适应），包括改变基因表达、新基因的获得和 DNA 突变是一个有前景的治疗靶点。令人惊讶的是，令人惊讶的是，它在病理适应和获得药物中所发挥的作用却很少受到关注。低保真度 DNA 聚合酶 (Pols) 产生抗性，这些 Pols 在复制未受损的情况下会产生突变。 DNA，或者通过称为跨损伤 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) 代表了研究最充分的细菌 TLS Pols。研究的人类病原体拥有在 TLS 中起作用的 Pol IV 同源物，许多缺乏 Pol V。相反，它们编码高度保守但尚未充分研究的多亚基 TLS Pol，有几个不同的名称，并将在该提案中称为 ImuABC 复合体由 ImuABC 产生的突变有助于毒力，根据序列，ImuA（也称为 ImuA’）与 ATP 酶具有同源性。（但可能缺乏催化活性），而 ImuB 是 Pol V UmuC 催化亚基的同源物，缺乏重要的活性位点残基，这意味着它可能缺乏 Pol 活性，与这一结论一致，突变。 ImuABC 的催化依赖于 ImuC（也称为 DnaE2）的 Pol 活性，其结构与细菌 Pol III 复制酶的 DnaE1 催化亚基。酵母双杂交实验的结果表明： ImuB 是一种接头蛋白，可与 ImuA 和 ImuC 以及 b 持续钳和 DnaE1 相互作用 Pol III 的亚基。后面的这些相互作用可能协调 ImuABC 与 Pol III 的作用。尽管已清楚地证明 ImuABC 在催化突变方面发挥着重要作用，但耐药性、毒力和病理适应等方面，ImuABC 复合物的研究却少得惊人。截至撰写本文时，仅有 42 篇发表的论文包含搜索词“imuA、imuB、imuC 重要的是，这些作品都没有讨论 ImuABC 复合物的生化分析。建议开发可溶形式的 ImuA、ImuB 和 ImuC 蛋白的纯化方法为此，我们将重点关注铜绿假单胞菌 ImuABC 蛋白，正如我们已经进行的那样。拥有过量生产者并建立了铜绿假单胞菌 b 钳和 Pol III 复制酶的纯化方法，这将是未来工作中需要的，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测定。对 ImuABC 在诱变中的功能机制进行详细的生化剖析。