The right half of the Escherichia coli replication origin is not essential for viability, but facilitates multi‐forked replication

Abstract

SummaryReplication initiation is a key event in the cell cycle of all organisms and oriC, the replication origin in Escherichia coli, serves as the prototypical model for this process. The minimal sequence required for oriC function was originally determined entirely from plasmid studies using cloned origin fragments, which have previously been shown to differ dramatically in sequence requirement from the chromosome. Using an in vivo recombineering strategy to exchange wt oriCs for mutated ones regardless of whether they are functional origins or not, we have determined the minimal origin sequence that will support chromosome replication. Nearly the entire right half of oriC could be deleted without loss of origin function, demanding a reassessment of existing models for initiation. Cells carrying the new DnaA box-depleted 163 bp minimal oriC exhibited little or no loss of fitness under slow-growth conditions, but were sensitive to rich medium, suggesting that the dense packing of initiator binding sites that is a hallmark of prokaryotic origins, has likely evolved to support the increased demands of multi-forked replication.

Relationship between Fork Progression and Initiation of Chromosome Replication in E. coli

“…Consistent with this, the presence of dnaA defective alleles, dnaA(Sx), suppresses the detrimental effect on DNA replication observed in mutants that have problems with the progression of forks due to the presence of defective subunits of DNA polymerase III coded by the dnaX gene (Gines-Candelaria et al, 1995;Blinkova et al, 2003;Skovgaard & Lobner-Olesen 2005). Furthermore, a lower availability of wild type DnaA protein induced by the presence of extra copies of the datA sequence alleviates replication problems in both the dnaX (Skovgaard & Lobner-Olesen, 2005) and the nrdA101 mutant (this work), whilst initiation defects caused by deletion of DnaA box R4 suppress replication elongation defects (Stepankiw et al, 2009). These observations, together with our data, are consistent with the idea that the progression of replication forks is not merely responsive to elongation factors (dNTP pools or proteins engaged in elongation) but also to the number of forks running along the chromosome.…”

“…In addition, the growth of cells in poor carbon source media is not known to affect nrdAB gene expression, and a decrease in replication rate in wild type cells has been observed under poor media conditions (Michelsen et al, 2003). Furthermore, it has been reported that deletion of DnaA box R4 suppresses replication elongation defects in gyrB mutant strains as a consequence of the lowering of initiation frequency (Stepankiw et al, 2009) indicating that no transcriptional factor is required to increase the replication rate. Hence the first proposition, in which faster replication forks are responsible for there being fewer forks, is difficult to justify.…”

The initiator protein DnaA, chromosomal origin oriC and their interaction to generate origin recognition complex and pre-replication complex like nucleoprotein structures in Escherichia coli

“…A recent finding established that the right half of the DARDnaA sub-complex is important for stimulating DnaB helicase loading 30 , while the DUE flanking left half produces a DnaA-oriC sub-complex competent in DUE unwinding and DnaA-DnaC assisted loading of helicase protein DnaB onto single stranded DUE 30 . Interestingly, it has been observed that deletion of the right half of E. coli oriC does not affect function of the origin present in bacterial cells growing on minimal media 36 . This is due to the fact that the time needed for the completion of chromosomal replication is shorter than the cell generation time 36 .…”

“…Interestingly, it has been observed that deletion of the right half of E. coli oriC does not affect function of the origin present in bacterial cells growing on minimal media 36 . This is due to the fact that the time needed for the completion of chromosomal replication is shorter than the cell generation time 36 . However in rich media, the generation time of fast growing cells is approximately one third to the time required to complete the replication process and therefore the requirement of a full length oriC to support initiation on a multi-forked chromosome 36 .…”

“…However in rich media, the generation time of fast growing cells is approximately one third to the time required to complete the replication process and therefore the requirement of a full length oriC to support initiation on a multi-forked chromosome 36 . Indeed the growth conditions for E. coli determine the cell mass, DNA content and more particularly the number of DnaA recognition boxes required to form adequate nucleoprotein structures respectively at single versus multi-forked chromosomes so as to compensate with the pace of initiation of replication 36 .…”