Suppressors of DnaA^ATP imposed overinitiation in Escherichia coli

Abstract

SummaryChromosome replication in Escherichia coli is limited by the supply of DnaA associated with ATP. Cells deficient in RIDA (Regulatory Inactivation of DnaA) due to a deletion of the hda gene accumulate suppressor mutations (hsm) to counteract the overinitiation caused by an elevated DnaA ATP level. Eight spontaneous hda suppressor mutations were identified by whole-genome sequencing, and three of these were analysed further. Two mutations (hsm-2 and hsm-4) mapped in the dnaA gene and led to a reduced ability to initiate replication from oriC. One mutation (hsm-1) mapped to the seqA promoter and increased the SeqA protein level in the cell. hsm-1 cells had prolonged origin sequestration, reduced DnaA protein level and reduced DnaA-Reactivating Sequence (DARS)-mediated rejuvenation of DnaA ADP to DnaA ATP , all of which could contribute to the suppression of RIDA deficiency. Despite of these defects hsm-1 cells were quite similar to wild type with respect to cell cycle parameters. We speculate that since SeqA binding sites might overlap with DnaA binding sites spread throughout the chromosome, excess SeqA could interfere with DnaA titration and thereby increase free DnaA level. Thus, in spite of reduction in total DnaA, the amount of DnaA molecules available for initiation may not be reduced.

Regulating DNA Replication in Bacteria

“…RIDA is crucial for DnaA inactivation and thereby effectively supports once-per-generation initiation (Kurokawa et al 1999;Camara et al 2005;Riber et al 2009). The hda gene is required for promoting cell proliferation, decreasing cellular ATP-DnaA levels and repressing overinitiation (Kato and Katayama 2001;Fujimitsu et al 2008;Charbon et al 2011). Incubation of temperature-sensitive hda mutant cells at the restrictive temperature leads to overinitiation of replication and induces inhibition of cell division, producing filamentous cells (Fujimitsu et al 2008).…”

Replication initiation at the Escherichia coli chromosomal origin

“…In support that this region does not have a discrete function, ablation of contiguous segments within domain II did not affect cell viability [28,29]. However, deletion of residues 96–120 within domain II reduces the activity of DnaA in initiation [30,31], so this region is not entirely dispensable. Domain III (130–347) contains the sensor I, II (box VIII), and box VII motifs of the AAA + protein family (reviewed in [32]), and functions in ATP binding, ATP hydrolysis, and in DnaA oligomerization [11,33].…”

Stable nucleotide binding to DnaA requires a specific glutamic acid residue within the AAA+ box II motif

“…A nucleoprotein complex consisting of the DNA-loaded clamp subunit and ADP-bound Hda, an AAA+ protein, promote DnaA-ATP hydrolysis to yield inactive ADPbound DnaA (Keyamura and Katayama, 2011;Nakamura and Katayama, 2010;Su'etsugu et al, 2005Su'etsugu et al, , 2008. Cells that are defective in RIDA exhibit increased levels of ATP-DnaA and over-initiation of chromosomal replication (Fujimitsu et al, 2008;Kato and Katayama, 2001;Camara et al, 2005;Riber et al, 2006;Charbon et al, 2011). Two chromosomal DNA regions, termed DnaA-reactivating sequence 1 (DARS1) and DARS2, function to increase ATPbound DnaA levels by promoting ADP/ATP exchange on DnaA, an event that is crucial to the timely initiation of chromosomal replication during the cell cycle (Fujimitsu et al, 2009).…”