Specific genomic sequences of E. coli promote replicational initiation by directly reactivating ADP-DnaA

Abstract

In Escherichia coli, ATP-DnaA, unlike ADP-DnaA, can initiate chromosomal replication at oriC. The level of cellular ATP-DnaA fluctuates, peaking at around the time of replication initiation. However, it remains unknown how the ATP-DnaA level increases coordinately with the replication cycle. In this study, we show that two chromosomal intergenic regions, herein termed DnaA-reactivating sequence 1 (DARS1) and DnaA-reactivating sequence 2 (DARS2), directly promote regeneration of ATP-DnaA from ADP-DnaA by nucleotide exchange, resulting in the promotion of replication initiation in vitro and in vivo. Coordination of initiation with the cell cycle requires DARS activity and its regulation. Oversupply of DARSs results in increase in the ATP-DnaA level and enhancement of replication initiation, which can inhibit cell growth in an oriC-dependent manner. Deletion of DARSs results in decrease in the ATP-DnaA level and inhibition of replication initiation, which can cause synthetic lethality with a temperature-sensitive mutant dnaA and suppression of overinitiation by the lack of seqA or datA, negative regulators for initiation. DARSs bear a cluster of DnaA-binding sites. DnaA molecules form specific homomultimers on DARS1, which causes specific interactions among the protomers, reducing their affinity for ADP. Our findings reveal a novel regulatory pathway that promotes the initiation of chromosomal replication via DnaA reactivation.[Keywords: DnaA reactivation; nucleotide exchange; initiation regulation; functional DNA sequence; protein complex; replication cycle] Supplemental material is available at http://www.genesdev.org.

Transition from a plasmid to a chromosomal mode of replication entails additional regulators

“…Protein remodeling upon interaction with DNA is well known (22)(23)(24). It has been shown also for the two initiators of the iteron-bearing plasmids (23,25) and for the E. coli initiator (26). Increased handcuffing could explain the potency of 39-mer-carrying fragments in reducing oriII copy number (Fig.…”

DnaA binding locus datA promotes DnaA-ATP hydrolysis to enable cell cycle-coordinated replication initiation

“…The E. coli chromosome contains two such sequences, DARS1 and DARS2, each of which has a DnaA box cluster that binds multiple ADP-DnaA molecules and yields ATP-DnaA by nucleotide exchange. Consistent with this, ADPDnaA resulting from hydrolysis of ATP-DnaA by RIDA is reactivated in vitro by DARS1 (31). We therefore asked whether ADP-DnaA generated from ATP-DnaA by DDAH could be reactivated for replication in a DARS-dependent manner (Fig.…”

“…In vitro reconstitution of DARS1 reaction was performed as previously described (31). Minichromosome replication was performed using M13KEW101 and crude extracts, as previously described (31).…”

“…In E. coli, reactivation of ADP-DnaA by the DARS-(DnaA reactivating sequence) dependent exchange of ADP for ATP is crucial for timely replication initiation coordinated with the cell cycle (31) (Fig. 1A).…”

“…Deletion of oriC represses lethal overinitiation caused by RIDA inactivation, whereas deletion of the rnhA gene encoding RNaseH I activates alternative origins, allowing the growth of cells lacking oriC (38). DnaA was isolated by immunoprecipitation from the lysates of 32 P-labeled exponentially growing cells, and DnaA-bound nucleotides were quantified using TLC, as previously described (16,17,31). In KW262-5 cells, ATP-DnaA levels were low (27%) (Fig.…”