Plasmid pT181 Replication and PcrA Helicase of S. aureus

描述（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。金黄色葡萄球菌菌株对一些常用抗生素的耐药性越来越强。滚动圈复制（RCR）质粒在金黄色葡萄球菌和许多其他革兰氏阳性细菌中普遍存在。这些质粒编码耐药性，并在这些基因在自然界的水平传播中发挥重要作用。我们将继续研究研究最好的RCR质粒之一，金黄色葡萄球菌的pT181的复制。我们将生成pT181编码的启动蛋白RepC的突变体，并使用生化方法鉴定其参与终止pT181复制的结构域。在金黄色葡萄球菌和其他革兰氏阳性细菌中存在一种染色体编码的必需解旋酶，PcrA。PcrA是细胞生长和生存以及质粒滚圈（RC）复制所必需的。我们已经证明它可以促进repc依赖性pT181 DNA解绕和体外复制。我们将使用体外和体内方法来确定RepC和PcrA参与其相互作用的结构域。我们已经证明，金黄色葡萄球菌的PcrA是不寻常的，因为它具有同样强大的双极性5‘ - bbbb3 ’和3‘- bbbb5 ’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，以及DNA解绕）在质粒复制中重要的作用。位点导向和随机突变的pcr将产生，过表达，纯化和分析其生化活性。由组成启动子表达的PcrA突变体将被引入含有可诱导的野生型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pT181复制方面存在缺陷，但能够复制属于其他RCR家族的质粒，如pC194、pE194和pSN2。我们将测试各种pcrA突变体支持上述质粒复制的能力。这些研究将提供对特定RCR质粒家族复制至关重要的PcrA结构域的信息，并可能确定与各种质粒的启动蛋白相互作用的PcrA结构域。我们的研究可以为开发靶向RCR质粒的新药物，以及靶向金黄色葡萄球菌和其他革兰氏阳性细菌的必需解旋酶提供新的途径。

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.