Stoichiometry of P1 plasmid partition complexes.
The P1 plasmid prophage is faithfully partitioned by a high affinity nucleoprotein complex assembled at the centromere-like parS site. This partition complex is composed of P1 ParB and Escherichia coli integration host factor (IHF), bound specifically to parS. We have investigated the assembly of ParB at parS and its stoichiometry of binding. Measured by gel mobility shift assays, ParB and IHF bind tightly to parS and form a specific complex, called I + B1. We observed that as ParB concentration was increased, a second, larger complex (I + B2) formed, followed by the formation of larger complexes, indicating that additional ParB molecules joined the initial complex. Shift Western blotting experiments indicated that the I + B2 complex contained twice as much ParB as the I + B1 complex. Using mixtures of ParB and a larger polyhistidine-tagged version of ParB (His-ParB) in DNA binding assays, we determined that the initial I + B1 complex contains one dimer of ParB. Therefore, one dimer of ParB binds to its recognition sequences that span an IHF-directed bend in parS. Once this complex forms, a second dimer can join the complex, but this assembly requires much higher ParB concentrations.
Bouet
J Y
JY
Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
Surtees
J A
JA
Funnell
B E
BE
D001426:Bacterial Proteins / Q000378:metabolism; D017101:Bacteriophage P1 / Q000235:genetics*; D001483:Base Sequence; D002455:Cell Division; D002503:Centromere; D018983:DNA Footprinting; D004261:DNA Replication; D004269:DNA, Bacterial; D004279:DNA, Viral; D019281:Dimerization; D004926:Escherichia coli / Q000166:cytology*; D018628:Gene Dosage; D034982:Integration Host Factors; D008969:Molecular Sequence Data; D009698:Nucleoproteins / Q000378:metabolism; D010957:Plasmids / Q000235:genetics*; D011533:Proviruses / Q000235:genetics*; D014764:Viral Proteins / Q000378:metabolism*; D014779:Virus Replication
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004279:DNA, Viral; D034982:Integration Host Factors; D009698:Nucleoproteins; C121785:ParB protein, Enterobacteria phage P1; D014764:Viral Proteins