PerA Helicase and Replication of Drug Resistance Plasmid

描述(由申请人提供)： 在革兰氏阳性菌中普遍存在以滚环(RC)机制复制的质粒，200多个滚环复制(RCR)质粒中的绝大多数属于四个主要家族。许多RCR质粒携带抗生素耐药基因，有证据表明，RCR质粒的水平转移相当常见。许多RCR质粒也含有与质粒动员和转移有关的基因。虽然一些RCR质粒只能在它们的原生宿主中稳定复制，但许多RCR质粒的宿主范围很广。RC复制涉及前导链的合成，需要PcrA解旋酶、单链DNA结合蛋白和DNA聚合酶III。滞后链复制需要宿主RNA聚合酶合成引物RNA，随后由DNA Pol I和DNA Pol III进行复制。PcrA解旋酶是质粒RC复制和革兰氏阳性菌生存所必需的。该R21应用程序的目的是检验一种假设，即质粒启动子蛋白和PcrA解旋酶之间的相互作用对于有效的质粒复制至关重要，并可能决定RCR质粒的窄宿主范围复制与宽宿主范围复制。尚未就此问题进行研究，当前应用程序的探索/开发性质与R21格式一致。在我们的研究中，我们计划利用一些寄主范围相对较窄的质粒和几个寄主范围较宽的质粒。我们将研究这些质粒在金黄色葡萄球菌、枯草杆菌、蜡状芽孢杆菌和肺炎链球菌中的复制情况。在特定宿主中复制有缺陷的RCR质粒将由其同源PcrA基因的克隆拷贝来补充。我们将从上述革兰氏阳性菌中提纯PcrA解旋酶，并研究它们与质粒启动子蛋白的相互作用。我们还将研究在存在和不存在各种PcrA解旋酶的情况下，上述质粒在金黄色葡萄球菌无细胞提取液中的体外复制。这些研究应该揭示PcrA是否在决定RCR质粒的有效复制和宿主范围方面起关键作用。这些研究可能有助于针对PcrA解旋酶和/或RCR质粒启动子的抗菌药物的开发。

DESCRIPTION (provided by applicant): Plasmids that replicate by a rolling-circle (RC) mechanism are ubiquitous in Gram-positive bacteria and a vast majority of over 200 rolling-circle replicating (RCR) plasmids belong to four major families. Many RCR plasmids carry antibiotic resistance genes and evidence suggests that horizontal transfer of RCR plasmids is quite common. Many RCR plasmids also contain genes that are involved in plasmid mobilization and transfer. While some RCR plasmids are able to replicate stably only in their native hosts, many have a broad host range. RC replication involves synthesis of the leading strand that requires the PcrA helicase, single strand DNA binding protein and DNA polymerase III. Lagging strand replication requires primer RNA synthesis by the host RNA polymerase, and subsequently replication is carried out by DNA Pol I and DNA Pol III. PcrA helicase is required for plasmid RC replication as well as survival of Gram-positive organisms. The goal of this R21 application is to test the hypothesis that interaction between plasmid initiator proteins and the PcrA helicases is critical for efficient plasmid replication and may determine narrow versus broad host range replication ofRCR plasmids. No studies have been performed dealing with this issue and the exploratory/development nature of the current application is consistent with the R21 format. We plan to utilize a few plasmids with a relatively narrow host range and a few with broad host range in our study. We will study the replication of these plasmids in S. aureus, B. subtilis, B. cereus and S. pneumoniae. RCR plasmids defective in replication in a particular host will be complemented by a cloned copy of their cognate pcrA gene. We will purify the PcrA helicases from the above Gram-positive organisms and study their interactions with the plasmid initiator proteins. We will also study the in vitro replication of the above plasmids in cell free extracts made from S. aureus in the presence and absence of the various PcrA helicases. These studies should reveal whether PcrA is critical in determining the efficient replication and host range of RCR plasmids. These studies may facilitate the development of antimicrobial drugs targeting the PcrA helicase and/or the initiators of RCR plasmids.