Plasmid pT181 Replication and PcrA Helicase of S. aureus

金黄色葡萄球菌质粒 pT181 复制和 PcrA 解旋酶

• 批准号: 7883924
• 负责人: SALEEM A. KHAN
• 金额: $ 13.64万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-20 至 2010-12-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7883924
• 关键词: Antibiotics; Archaea; Biochemical; Chromosomes, Human, 1-3; DNA; DNA-Directed DNA Polymerase; Development; Disease; Drug Delivery Systems; Drug resistance; Family; Genes; Gram-Negative Bacteria; Gram-Positive Bacteria; Human; In Vitro; Nature; Plasmids; Play; Proteins; Resistance; Role; Site; Staphylococcus aureus; Structure-Activity Relationship; Testing; cell growth; helicase; in vivo; mutant; novel; overexpression; pathogen; promoter; Antibiotics; Archaea; Biochemical; Chromosomes, Human, 1-3; DNA; DNA-Directed DNA Polymerase; Development; Disease; Drug Delivery Systems; Drug resistance; Family; Genes; Gram-Negative Bacteria; Gram-Positive Bacteria; Human; In Vitro; Nature; Plasmids; Play; Proteins; Resistance; Role; Site; Staphylococcus aureus; Structure-Activity Relationship; Testing; cell growth; helicase; in vivo; mutant; novel; overexpression; pathogen; promoter

DESCRIPTION (provided by applicant): Staphylococcus aureus is a major human pathogen that causes a variety of diseases. Strains of S. aureus have increasingly become resistant to a number of commonly used antibiotics. Rolling-circle replicating (RCR) plasmids are ubiquitous in S. aureus and many other Gram-positive bacteria. These plasmids encode drug resistance and play a major role in the horizontal spread of these genes in nature. We will continue our studies on the replication of one of the best studied RCR plasmid, pT181 of S. aureus. We will generate mutants of the pT181-encoded initiator protein, RepC, and identify its domains involved in the termination of pT181 replication using biochemical approaches. A chromosome-encoded, essential helicase, PcrA, is present in S. aureus and other Gram-positive bacteria. PcrA is required for cell growth and viability, and plasmid rolling-circle (RC) replication. We have shown that it promotes RepC-dependent pT181 DNA unwinding and in vitro replication. We will use both in vitro and in vivo approaches to identify domains of RepC and PcrA that are involved in their interaction. We have shown that the S. aureus PcrA is unusual in that it has equally robust bipolar 5' -> 3' and 3'->5' helicase activities. We plan to study the structure-function relationship of PcrA to identify its domains and enzymatic activities (ssDNA translocation, 5'->3' and 3'->5' helicase, and DNA unwinding) that are important in plasmid replication. Site-directed and random mutants of PcrA will be generated, overexpressed, purified and analyzed for their biochemical activities. PcrA mutants expressed from constitutive promoters will be introduced into strains containing an inducible, wild-type pcrA gene and tested for their ability to support plasmid RC replication under non-inducing conditions. The PcrAS mutant is defective in supporting pT181 replication, but is competent in the replication of plasmids belonging to other RCR families such as pC194, pE194 and pSN2. We will test various pcrA mutants for their ability to support replication of the above plasmids. These studies should provide information on domains of PcrA that are critical for the replication of particular RCR plasmid families and may identify PcrA domains that interact with the initiator proteins of various plasmids. Our studies could provide new avenues for the development of novel drugs targeting RCR plasmids, as well as targeting an essential helicase in S. aureus and other Gram-positive bacteria.

描述（由申请人提供）：金黄色葡萄球菌是引起多种疾病的主要人类病原体。金黄色葡萄球菌的菌株越来越对许多常用的抗生素具有抵抗力。滚环复制（RCR）质粒在金黄色葡萄球菌和许多其他革兰氏阳性细菌中无处不在。这些质粒编码耐药性，并在这些基因在自然界的水平扩散中起主要作用。我们将继续研究研究最佳研究的RCR质粒之一，即金黄色葡萄球菌的PT181。我们将生成PT181编码的引发剂蛋白，RETC的突变体，并使用生化方法识别其与PT181复制终止的域。金黄色葡萄球菌和其他革兰氏阳性细菌中存在染色体编码的必需解旋酶PCRA。 PCRA是细胞生长和生存能力以及质粒滚环（RC）复制所必需的。我们已经表明，它促进了依赖RETC的PT181 DNA脱光并在体外复制。我们将同时使用体外和体内方法来识别与其相互作用涉及的RETC和PCRA领域。我们已经表明，金黄色葡萄球菌PCRA与众不同，因为它具有同样可靠的双极性5' - > 3'和3' - > 5'解旋酶活性。我们计划研究PCRA的结构功能关系，以鉴定其域和酶促活性（SSDNA易位，5' - > 3'和3' - > 5'旋转酶和DNA放松酶，以及DNA放松在质粒复制中很重要。将生成，过表达，纯化和分析其生化活性的位置定向和随机突变体。由本构启动子表达的PCRA突变体将被引入含有诱导，野生型PCRA基因的菌株中，并测试了其在非诱导条件下支持质粒RC复制的能力。 PCRA突变体在支持PT181复制方面有缺陷，但在属于其他RCR家族（例如PC194，PE194和PSN2）的质粒的复制方面具有胜任。我们将测试各种PCRA突变体的支持上述质粒复制的能力。这些研究应提供有关PCRA领域的信息，这对于对特定RCR质粒家族的复制至关重要，并可以鉴定与各种质粒的引发剂蛋白相互作用的PCRA结构域。我们的研究可以为靶向RCR质粒的新药物的开发提供新的途径，并针对金黄色葡萄球菌和其他革兰氏阳性细菌中的必不可少的解旋酶。