The P1 plasmid partition complex at parS. The influence of Escherichia coli integration host factor and of substrate topology.
The P1 ParB protein is required for active partition and thus stable inheritance of the plasmid prophage. ParB and the Escherichia coli protein integration host factor (IHF) participate in the assembly of a partition complex at the centromere-like site parS. In this report the role of IHF in the formation of the partition complex has been explored. First, ParB protein was purified for these studies, which revealed that ParB forms a dimer in solution. Next, the IHF binding site was mapped to a 29-base pair region within parS, including the sequence TAACTGACTGTTT (which differs from the IHF consensus in two positions). IHF induced a strong bend in the DNA at its binding site. Versions of parS which have lost or damaged the IHF binding site bound ParB with greatly reduced affinity in vitro and in vivo. Measurements of binding constants showed that IHF increased ParB affinity for the wild-type parS site by about 10,000-fold. Finally, DNA supercoiling improved ParB binding in the presence of IHF but not in its absence. These observations led to the proposal that IHF and superhelicity assist ParB by promoting its precise positioning at parS, a spatial arrangement that results in a high affinity of ParB for parS.
Funnell
B E
BE
Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002853:Chromatography, Liquid; D016172:DNA Fingerprinting; D004269:DNA, Bacterial / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D034982:Integration Host Factors; D008969:Molecular Sequence Data; D010957:Plasmids
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D034982:Integration Host Factors; C052883:chromosome partition proteins, bacterial