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出现日益增长，改善的疗法以及新的靶标迫切需要开发新疗法。多种机制有助于病原体对人类宿主的耐药性和适应性（病原体）的适应基因表达，新基因的获取和DNA突变。一个有前途的治疗靶受到的日期很少受到关注，这在pathAdaptation和获得药物中所起的作用 - 低忠诚度DNA聚合酶（POLS）的抗性。这些pol在复制未分娩时会产生突变 DNA，或者通过称为translesion DNA合成（TLS）的过程绕过损坏的碱基时。大肠杆菌pol IV（Dinb）和Pol V（UUDC）代表最佳研究的细菌TLS POLS。虽然很好研究菌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，它们编码一个高度构成的，但知识的多工资TLS POL，它以几个不同的名称呈现，并且将是在本提案中称为IMUABC综合体。 imuabc产生的突变有助于病毒，持久性和抗药性。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是缺乏催化亚基的同源物基本的主动部位保留，这意味着它可能没有POL活性。与这个结论一致，突变由imuabc催化的取决于IMUC的POL活性（也称为DNAE2），这在结构上与 pol III复制酶的DNAE1催化亚基。酵母2杂交实验的结果表明 IMUB是一种与IMUA和IMUC相互作用的衔接蛋白，以及B加工性夹具和DNAE1 Pol III的亚基。这些后来的相互作用可能会将imuabc的作用与Pol III的作用进行协调。尽管清楚地证明了imuabc在催化突变中的重要作用抗药性，病毒性和途径适应，IMUABC复合物是很少研究的主题。在撰写本文时，只有42篇文章包含搜索词“ IMUA，IMUB，IMUC 或DNAE2。“重要的是，这些作品都没有讨论IMUABC综合体的生化分析。建议是为纯化IMUA，IMUB和IMUC蛋白的固体形式纯化的方法详细的体外机械研究。为此，我们将重点放在铜绿假单胞菌imuabc蛋白上，就像我们已经具有过度生产剂和建立的铜绿假单胞菌B夹和Pol III复制酶的纯化方法，这是旨在确定IMUB-B夹具和IMUB-POL III的贡献的未来工作中所必需的与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有关诱变中IMUABC功能的机制的详细生化剖析。