Plasmid pT181 Replication and PcrA Helicase of S. aureus

描述（由申请人提供）：金黄色葡萄球菌是一种主要的人类病原体，可引起多种疾病。链球菌菌株金黄色葡萄球菌对许多常用抗生素的耐药性越来越强。滚环复制（RCR）质粒在沙门氏菌中普遍存在。金黄色葡萄球菌和许多其他革兰氏阳性菌。这些质粒编码耐药性，并在这些基因在自然界中的水平传播中发挥重要作用。我们将继续对研究得最好的RCR质粒之一，S. pT 181进行复制研究。金黄色。我们将产生突变体的pT 181编码的起始蛋白，RepC，并确定其结构域参与终止的pT 181复制使用生化方法。染色体编码的必需解旋酶PcrA存在于S.金黄色葡萄球菌和其他革兰氏阳性菌。PcrA是细胞生长和活力以及质粒滚环（RC）复制所必需的。我们已经表明，它促进RepC依赖性的pT 181 DNA解旋和体外复制。我们将使用在体外和体内的方法来确定RepC和PcrA的结构域参与其相互作用。我们证明了S.金黄色葡萄球菌PcrA是不寻常的，因为它具有同样稳健的双极5' -> 3'和3 '->5'解旋酶活性。我们计划研究PcrA的结构-功能关系，以确定其结构域和酶活性（ssDNA易位，5 '->3'和3 '->5'解旋酶和DNA解旋），这些在质粒复制中很重要。将产生、过表达、纯化PcrA的定点突变体和随机突变体，并分析其生化活性。将从组成型启动子表达的PcrA突变体引入含有诱导型野生型pcrA基因的菌株中，并测试其在非诱导条件下支持质粒RC复制的能力。PcrAS突变体在支持pT 181复制方面有缺陷，但在属于其他RCR家族的质粒如pC 194、pE 194和pSN 2的复制中有能力。我们将测试各种pcrA突变体支持上述质粒复制的能力。这些研究应提供的信息，对特定的RCR质粒家族的复制是至关重要的PcrA域，并可能确定PcrA域与各种质粒的起始蛋白相互作用。我们的研究为靶向RCR质粒以及靶向S.金黄色葡萄球菌和其他革兰氏阳性菌。

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.