CONJUGATIVE MOBILIZATION OF PLASMID R1162

本提案中概述的实验旨在实现 理解过程中发生的DNA加工反应 广宿主质粒R1162的接合转移。模特是 提出其中转移原点(ORIT)由两个域组成， 在转移前对DNA进行初始划痕所需的一个，以及 其他用于随后重新循环已转移但重叠的 大的开放阅读框(ORFI)的区域。突变将被隔离 在ORIT中识别和映射不同的功能域。蛋白质 ORFT片段参与不同的ORIT加工事件 将被提纯，并在体外测试它们的生化活性。 蛋白质-DNA松弛复合体将被表征以识别 与此结构相关的加工中间体。 DNA合成发生在细胞提取液中的ORIT中，并依赖于 将质粒基因进行转移。这一点的起源和方向 合成将被确定。ORFI与Rep2重叠，Rep2是一种需要 质粒DNA复制。Rep2与OrfI的遗传关系 将被阐明，以及Rep2区域参与DNA合成 来自奥立特的将被评估。ORIT的突变也将进行测试，以查看 合成是否需要刻痕。另一种动员蛋白， Mobi，在体内补充OrFI的Rep2区，并抑制 单链ORIT DNA的加工。摩拜将接受支持测试 从ORIT合成DNA，这种蛋白质的目标将是 已确认身份。最后，在此过程中转移的R1162 DNA链 通过捕获感染性DNA中的这种DNA来鉴定接合作用 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.