Building the bacterial orisome: high-affinity DnaA recognition plays a role in setting the conformation of oriC DNA.
During assembly of the E. coli pre-replicative complex (pre-RC), initiator DnaA oligomers are nucleated from three widely separated high-affinity DnaA recognition sites in oriC. Oligomer assembly is then guided by low-affinity DnaA recognition sites, but is also regulated by a switch-like conformational change in oriC mediated by sequential binding of two DNA bending proteins, Fis and IHF, serving as inhibitor and activator respectively. Although their recognition sites are separated by up to 90 bp, Fis represses IHF binding and weak DnaA interactions until accumulating DnaA displaces Fis from oriC. It remains unclear whether high-affinity DnaA binding plays any role in Fis repression at a distance and it is also not known whether all high-affinity DnaA recognition sites play an equivalent role in oligomer formation. To examine these issues, we developed origin-selective recombineering methods to mutate E. coli chromosomal oriC. We found that, although oligomers were assembled in the absence of any individual high-affinity DnaA binding site, loss of DnaA binding at peripheral sites eliminated Fis repression, and made binding of both Fis and IHF essential. We propose a model in which interaction of DnaA molecules at high-affinity sites regulates oriC DNA conformation.
Department of Biological Sciences, Florida Institute of Technology, 150 West University Boulevard, Melbourne, FL, 32901, USA.
Gulpreet
G
Kaur
Mansi P
MP
Vora
Christopher A
CA
Czerwonka
Tania A
TA
Rozgaja
Julia E
JE
Grimwade
Alan C
AC
Leonard
D001426:Bacterial Proteins / Q000378:metabolism*; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000378:metabolism; D035021:Factor For Inversion Stimulation Protein / Q000378:metabolism; D034982:Integration Host Factors / Q000378:metabolism; D051738:Origin Recognition Complex; D011485:Protein Binding; D055503:Protein Multimerization
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria; D029968:Escherichia coli Proteins; D035021:Factor For Inversion Stimulation Protein; C501034:Fis protein, E coli; D034982:Integration Host Factors; C114650:OriC chromosomal replication origin; D051738:Origin Recognition Complex; C440502:integration host factor, E coli