Escherichia coli DnaA protein loads a single DnaB helicase at a DnaA box hairpin.
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.
Carr
Kevin M
KM
Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824-1319, USA.
Kaguni
Jon M
JM
D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002850:Chromatography, Gel; D004265:DNA Helicases / Q000737:chemistry / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D053488:DnaB Helicases; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D018741:Replication Origin
D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria; C071702:DnaC protein, E coli; D029968:Escherichia coli Proteins; D004265:DNA Helicases; D053488:DnaB Helicases