Escherichia coli prereplication complex assembly is regulated by dynamic interplay among Fis, IHF and DnaA.
Initiator DnaA and DNA bending proteins, Fis and IHF, comprise prereplication complexes (pre-RC) that unwind the Escherichia coli chromosome's origin of replication, oriC. Loss of either Fis or IHF perturbs synchronous initiation from oriC copies in rapidly growing E. coli. Based on dimethylsulphate (DMS) footprinting of purified proteins, we observed a dynamic interplay among Fis, IHF and DnaA on supercoiled oriC templates. Low levels of Fis inhibited oriC unwinding by blocking both IHF and DnaA binding to low affinity sites. As the concentration of DnaA was increased, Fis repression was relieved and IHF rapidly redistributed DnaA to all unfilled binding sites on oriC. This behaviour in vitro is analogous to observed assembly of pre-RC in synchronized E. coli. We propose that as new DnaA is synthesized in E. coli, opposing activities of Fis and IHF ensure an abrupt transition from a repressed complex with unfilled weak affinity DnaA binding sites to a completely loaded unwound complex, increasing both the precision of DNA replication timing and initiation synchrony.
Department of Biological Sciences, Florida Institute of Technology, Melbourne, Florida 32901-6795, USA.
Valorie T
VT
Ryan
Julia E
JE
Grimwade
Johanna E
JE
Camara
Elliott
E
Crooke
Alan C
AC
Leonard
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002876:Chromosomes, Bacterial; D018983:DNA Footprinting; D004261:DNA Replication; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D035021:Factor For Inversion Stimulation Protein / Q000235:genetics / Q000378:metabolism*; D034982:Integration Host Factors / Q000235:genetics / Q000378:metabolism*; D046911:Macromolecular Substances; D008957:Models, Genetic; D011485:Protein Binding; D018741:Replication Origin
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria; D029968:Escherichia coli Proteins; D035021:Factor For Inversion Stimulation Protein; D034982:Integration Host Factors; D046911:Macromolecular Substances; C440502:integration host factor, E coli