CONJUGATIVE MOBILIZATION OF PLASMID R1162

描述（改编自研究者摘要）：长期 该项目的目标是在分子水平上表征DNA 广泛宿主范围的配偶转移所需的处理步骤 质粒。 建议的工作将集中在多拷贝质粒 R1162，其编码其接合所需的三种蛋白质（MobA-C）， 动员。 基因启动与基因突变之间潜在的生化相似性 转移（双链体DNA的切口）和终止（双链体DNA的连接） 单个线性DNA链）将被鉴定和表征。 如何 MobA和MobC导致oriT DNA在细胞中的局部解链， 将研究松弛体。 MobA的羧基末端区域是 一种引发酶，也是单独合成的， R1162的营养体复制。 引发酶结构域在 将检查转移后的互补链合成。 的 MobB蛋白在结合动员中的功能目前尚不清楚。 将探讨这种蛋白质调节水平的可能性 可以启动一轮转移， 破坏oriT DNA和MobA蛋白之间的结合。 纯化 MobA蛋白在体外可切割和连接单链oriT DNA。 该反应的机理及其与 转让终止，将进行调查。 实验 本研究的方法将包括分离第二位点抑制基因 突变，松弛体的化学探测，以及重组MobA oriT DNA与纯化组分的体外相互作用。

DESCRIPTION (Adapted from the investigator's abstract): The long-term goal of this project is to characterize at the molecular level the DNA processing steps required for the conjugal transfer of broad host-range plasmids. The proposed work will be focussed on the multi-copy plasmid R1162, which encodes three proteins (MobA-C) required for its conjugal mobilization. Underlying biochemical similarities between initiation of transfer (nicking of duplex DNA) and termination (ligation of a single, linear DNA strand) will be identified and characterized. How MobA and MobC bring about the localized melting of oriT DNA in the relaxosome will be investigated. The carboxy-terminal region of MobA is a primase, also synthesized separately, that is required for the vegetative replication of R1162. The role of the primase domain in complementary strand synthesis after transfer will be examined. The function of MobB protein in conjugal mobilization is presently unknown. The possibility will be explored that this protein regulates the level of nicked molecules available to initiate a round of transfer by disrupting the association between oriT DNA and MobA protein. Purified MobA protein can cleave and ligate single-stranded oriT DNA in vitro. The mechanism of this reaction, and its compatibility with a model for the termination of transfer, will be investigated. Experimental approaches in this study will include isolating second-site suppressor mutations, chemical probing of the relaxosome, and reconstituting MobA oriT DNA interactions in vitro with purified components.