Escherichia coli DnaA Protein Loads a Single DnaB Helicase at a DnaA Box Hairpin

Kevin M Carr,Jon M Kaguni

doi:10.1074/jbc.m205031200

Abstract

The molecular engine that drives bidirectional replication fork movement from the Escherichia coli replication origin (oriC) is the replicative helicase, DnaB. At oriC, two and only two helicase molecules are loaded, one for each replication fork. DnaA participates in helicase loading; DnaC is also involved, because it must be in a complex with DnaB for delivery of the helicase. Since DnaA induces a local unwinding of oriC, one model is that the limited availability of single-stranded DNA at oriC restricts the number of DnaB molecules that can bind. In this report, we determined that one DnaB helicase or one DnaB-DnaC complex is bound to a single-stranded DNA in a biologically relevant DNA replication system. These results indicate that the availability of single-stranded DNA is not a limiting factor and support a model in which the site of entry for DnaB is altered so that it cannot be reused. We also show that 2-4 DnaA monomers are bound on the single-stranded DNA at a specific site that carries a DnaA box sequence in a hairpin structure.

Highlights

The Escherichia coli chromosomal origin has two major roles
We developed assay conditions to measure the binding of DnaA protein to a 379-nucleotide-long single-stranded DNA (ssDNA) fragment carrying the DnaA box hairpin
One important conclusion was that only two DnaB hexamers were bound at oriC, one for each replication fork that moves in opposing directions

Summary

EXPERIMENTAL PROCEDURES

DNAs and Proteins—M13 A-site ssDNA [25], purified proteins, and antibodies have been described previously [4, 26]. After PCR amplification, the product was digested with HindIII endonuclease that cleaves within the latter primer and end-filled at this restriction site with [␣-32P]dATP and the large fragment of DNA polymerase I to label the viral strand ssDNA fragment. Primer Extension Assay—Reactions (25 ␮l) contained M13 A-site ssDNA annealed to the Ϫ40 universal primer (50 ng), SSB (1 ␮g), DnaA (45 ng), DnaB (50 ng), and DnaC (25 ng) as indicated and ATP␥S (0.1 mM) as indicated in ABC Buffer. Isolation of Prepriming Complexes—Reactions of prepriming complex formation (100 ␮l), a 50-fold scale up of a standard replication reaction in terms of DNA and replication protein components, contained M13 A-site ssDNA (5 ␮g), SSB (30 ␮g), DnaA (2.8 ␮g), DnaB (10 ␮g), and DnaC (5 ␮g) as indicated in ABC Buffer supplemented with 0.1 mM. Chemiluminescence (SuperSignal; Pierce) of immune complexes of horseradish peroxidase conjugated to the secondary antibody was analyzed with a Bio-Rad model GS505 molecular imager and associated software

RESULTS

DISCUSSION

DnaC n