CONJUGATIVE MOBILIZATION OF PLASMID R1162

本提案中概述的实验旨在 了解DNA加工过程中发生的反应， 广泛宿主范围质粒R1162的接合转移。 模型是 提出了其中转移的起源（oriT）由两个域组成， 一个是转移前DNA初始切口所需的， 另一种是对已转移但重叠的 大开放阅读框（ORFI）的区域。 突变将被隔离 在oriT中识别和映射不同的功能域。 蛋白 参与oriT不同加工事件的ORFT片段 将其纯化，并在体外测试其生化活性。 蛋白质-DNA松弛复合物将被表征以鉴定 与此结构相关的加工中间体。 DNA合成发生在细胞提取物中的oriT内，并且依赖于 质粒基因转移。 这一切的起源和方向 将确定合成。 ORFI与rep 2重叠，rep 2是一个基因， 质粒DNA复制。 rep 2与ORFI的遗传关系 将被澄清，并参与的rep 2区域在DNA合成 将被评估。 oriT中的突变也将被检测， 合成是否需要切口。 另一种动员蛋白， MobI在体内补充ORFI的rep 2区域，并抑制ORFI的表达。 单链oriT DNA的加工。 MobI将进行支持测试 oriT的DNA合成，这种蛋白质的目标将是 鉴定 最后，R1162 DNA链在转移过程中 将通过在感染性细胞中捕获该DNA来鉴定接合。 粒子

The experiments outlined in this proposal are directed toward an understanding of the DNA processing reactions that occur during conjugative transfer of the broad host-range plasmid R1162. A model is proposed in which the origin of transfer (oriT) consists of two domains, one required for initial nicking of the DNA prior to transfer, and the other for subsequent recircularization of the transferred but overlapping regions of a large open reading frame (ORFI). Mutations will be isolated in oriT to identify and map the different functional domains. Proteins fragments of ORFT that are involved in different processing events of oriT will be purified, and their biochemical activities tested in vitro. Protein-DNA relaxation complex will be characterized to identify the processing intermediate related to this structure. DNA synthesis occurs from within oriT in cell extracts and is dependent on the plasmid genes for transfer. The origin and direction of this synthesis will be determined. ORFI overlaps rep2, a gene required for plasmid DNA replication. The genetic relationship between rep2 and ORFI will be clarified, and the involvement of the rep2 region in DNA synthesis from oriT will be assessed. Mutations in oriT will also be tested to see whether nicking is required for synthesis. Another mobilization protein, MobI, complements the rep2 region of ORFI in vivo, and inhibits the processing of single-stranded oriT DNA. MobI will be tested for support of DNA synthesis from oriT, and the target of this protein will be identified. Finally, the R1162 DNA strands that are transferred during conjugation will be identified by capturing this DNA in infectious particles.