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Assembly of the primosome of DNA replication in Escherichia coli
1Department of Biochemistry, Beckman Center, Stanford University School of Medicine, California 94305.
Insights
The Escherichia coli primosome assembles using six proteins at a specific DNA site. This complex, once formed, becomes self-limiting and contains key enzymatic activities for DNA replication.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- The Escherichia coli primosome is essential for initiating DNA replication.
- Primosome assembly involves six proteins: PriA, PriB, PriC, DnaB, DnaC, and DnaT.
- Assembly occurs at a primosome assembly site (pas) on single-stranded DNA coated with SSB proteins.
Purpose of the Study:
- To elucidate the mechanism of primosome assembly in E. coli.
- To characterize the enzymatic activities within the assembled primosome.
- To understand the nucleotide requirements for primosome formation and maintenance.
Main Methods:
- In vitro assembly of the E. coli primosome using purified proteins and DNA.
- Biochemical assays to measure ATPase and GTPase activities.
- Analysis of isolated primosome composition and function.
Main Results:
- Primosome assembly is initiated by PriA and PriB interactions with DNA at the pas.
- The assembled primosome exhibits hyper-activated PriA dATPase activity and blocks further PriA recruitment.
- Nucleotide requirements involve ATP/dATP for assembly and ATP/GTP for maintenance, consistent with protein functions.
Conclusions:
- The E. coli primosome is a multi-protein complex with distinct enzymatic activities (PriA dATPase, DnaB GTPase, PriB replication).
- Primosome assembly is a regulated process that becomes self-limiting.
- Specific nucleotide hydrolysis by different proteins drives primosome assembly and function.
Abstract:
Assembly of the Escherichia coli primosome requires six proteins, PriA, PriB, PriC, DnaB, DnaC, and DnaT, acting at a primosome assembly site (pas) on an SSB-coated single-stranded (ss) DNA. Assembly is initiated by interactions of PriA and PriB with ssDNA and the pas. PriC, DnaB, DnaC, and DnaT then act on the PriA-PriB-DNA complex to yield the primosome. In the primosome, the dATPase (ATPase) of PriA becomes hyper-activated. In addition, the assembled primosome appears to block the pas, preventing it from activating additional PriA molecules. Either ATP alone or dATP in combination with GTP is sufficient for primosome assembly, while ATP or GTP provides for its maintenance during isolation. These nucleotide requirements can be reconciled with the need for ATP or dATP for DnaB-DnaC complex formation and hydrolysis of ATP or GTP by DnaB when it binds ssDNA. Such isolated primosomes contain a dATPase, the hallmark of PriA, and a GTPase indicative of DnaB. Further studies indicate that the isolated primosome contains the PriB replication activity in addition to PriA and DnaB.