Sozhamannan S, Chattoraj D K
Laboratory of Biochemistry, National Cancer Institute, Bethesda, Maryland 20892.
J Bacteriol. 1993 Jun;175(11):3546-55. doi: 10.1128/jb.175.11.3546-3555.1993.
Binding of the P1-encoded protein RepA to the origin of P1 plasmid replication is essential for initiation of DNA replication and for autoregulatory repression of the repA promoter. Previous studies have shown defects in both initiation and repression in hosts lacking heat shock proteins DnaJ, DnaK, and GrpE and have suggested that these proteins play a role in the RepA-DNA binding required for initiation and repression. In this study, using in vivo dimethyl sulfate footprinting, we have confirmed the roles of the three heat shock proteins in promoting RepA binding to the origin. The defects in both activities could be suppressed by increasing the concentration of wild-type RepA over the physiological level. We also isolated RepA mutants that were effective initiators and repressors without requiring the heat shock proteins. These data suggest that the heat shock proteins facilitate both repression and initiation by promoting only the DNA-binding activity of RepA. In a similar plasmid, F, initiator mutants that confer heat shock protein independence for replication were also found, but they were defective for repression. We propose that the initiator binding involved in repression and the initiator binding involved in initiation are similar in P1 but different in F.
P1编码蛋白RepA与P1质粒复制起点的结合对于DNA复制的起始以及repA启动子的自动调节抑制至关重要。先前的研究表明，在缺乏热休克蛋白DnaJ、DnaK和GrpE的宿主中，起始和抑制均存在缺陷，并表明这些蛋白在起始和抑制所需的RepA-DNA结合中发挥作用。在本研究中，我们使用体内硫酸二甲酯足迹法，证实了这三种热休克蛋白在促进RepA与起点结合中的作用。通过将野生型RepA的浓度提高到生理水平以上，可以抑制这两种活性中的缺陷。我们还分离出了无需热休克蛋白即可有效起始和抑制的RepA突变体。这些数据表明，热休克蛋白仅通过促进RepA的DNA结合活性来促进抑制和起始。在类似的质粒F中，也发现了赋予复制热休克蛋白独立性的起始突变体，但它们在抑制方面存在缺陷。我们提出，P1中参与抑制的起始子结合与参与起始的起始子结合相似，但在F中不同。
