| Abstrakt: |
Replication of the chromosome of bacteriophage λ depends on the cooperative action of two phage-coded proteins and seven replication and heat shock proteins from its Escherichia colihost. As previously described, the first stage in this process is the binding of multiple copies of the λ O initiator to the λ replication origin (oriλ) to form the nucleosomelike O-some. The O-some serves to localize subsequent protein-protein and protein-DNA interactions involved in the initiation of λ DNA replication to oriλ. To study these interactions, we have developed a sensitive immunoblotting protocol that permits the protein constituents of complex nucleoprotein structures to be identified. Using this approach, we have defined a series of sequential protein assembly and protein disassembly events that occur at oriλduring the initiation of λ DNA replication. A second-stage oriλ•O (λ O protein)•P (λ P protein)•DnaB nucleoprotein structure is formed when O, P, and E. coliDnaB helicase are incubated with oriλDNA. In a third-stage reaction the E. coliDnaJ heat shock protein specifically binds to the second-stage structure to form an oriλ•O•P•DnaB•DnaJ complex. Each of the nucleoprotein structures formed in the first three stages was isolated and shown to be a physiological intermediate in the initiation of X DNA replication. The E. coliDnaK heat shock protein can bind to any of these early stage nucleoprotein structures, and in a fourth-stage reaction a complete oriλ•O-P•DnaB•DnaJ•DnaK initiation complex is assembled. Addition of ATP to the reaction enables the DnaK and DnaJ heat shock proteins to mediate a partial disassembly of the fourth-stage complex. These protein disassembly reactions activate the intrinsic helicase activity of DnaB and result in localized unwinding of the oriλtemplate. The protein diassembly reactions are described in the accompanying articles (Alfano, C. and McMacken, R. (1989) J. Biol. Chem. 264, 10709–10718; Dodson, M., McMacken, R. and Echols, H. (1989) J. Biol. Chem. 264, 10719–10725). |
