A Role for Nonessential Domain II of Initiator Protein, DnaA, in Replication Control

Abstract

The initiation of replication in bacteria is regulated via the initiator protein DnaA. ATP-bound DnaA binds to multiple sequences at the origin of replication, oriC, unwinding the DNA and promoting the binding of DnaB helicase. From an Escherichia coli mutant highly perturbed for replication control, obgETTn5-EZ seqAD, we isolated multiple spontaneous suppressor mutants with enhanced growth and viability. These suppressors suppressed the replication control defects of mutants in seqA alone and genetically mapped to the essential dnaA replication initiator gene. DNA sequence analysis of four independent isolates revealed an identical deletion of the DnaA-coding region at a repeated hexanucleotide sequence, causing a loss of 25 amino acids in domain II of the DnaA protein. Previous work has established no function for this region of protein, and deletions in the region, unlike other domains of the DnaA protein, do not produce lethality. Flow cytometric analysis established that this allele, dnaAD 96-120 , ameliorated the over-replication phenotype of seqA mutants and reduced the DNA content of wild-type strains; virtually identical effects were produced by loss of the DnaA-positive regulatory protein DiaA. DiaA binds to multiple DnaA subunits and is thought to promote cooperative DnaA binding to weak affinity DNA sites through interactions with DnaA in domains I and/or II. The dnaAD 96-120 mutation did not affect DiaA binding in pull-down assays, and we propose that domain II, like DiaA, is required to promote optimal DnaB recruitment to oriC.

Regulating DnaA complex assembly: it is time to fill the gaps

“…For DnaA to span the distance between high and low affinity recognition sites, the flexible domain 2 linker [44] may be required to extend DnaA protomers attached by domain 1 interactions (Figure 3). Interestingly, it was recently shown that deletions in domain 2 reduce initiation efficiency [45]. Regulators such as DiaA may also promote pre-RC assembly at this stage [41 • ] by stabilizing the interaction between the anchoring and extended DnaA (Figure 3).…”

Helicase Loading at Chromosomal Origins of Replication

“…molgen.mpg.de/ messer/), domain 2 (residues 90-130 of E. coli DnaA) is not conserved in amino acid sequence or length. For E. coli DnaA, the removal of residues 96 -120 in domain 2 leads to reduced activity in DNA replication, but deletion of other consecutive sequences from this region has no apparent effect on activity (Nozaki and Ogawa 2008;Molt et al 2009). These observations suggest that domain 2 acts as a flexible link between domains 1 and 3.…”

Regulation of DnaA Assembly and Activity: Taking Directions from the Genome

“…Deletions placed in domain II can repress initiation (61), but a GFP-tag can be inserted into this domain without loss of viability (6). While Domain II has not been assigned any specific role, the linker region may accommodate the need to position Domain I over the wide range of distances that separate DnaA recognition sites within oriC , or allow some flexibility in positioning DnaB on single-stranded DNA.…”