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

Abstract

New rounds of bacterial chromosome replication are triggered during each cell division cycle by the initiator protein, DnaA. For precise timing, interactions of DnaA-ATP monomers with the replication origin, oriC, must be carefully regulated during formation of complexes that unwind origin DNA and load replicative helicase. Recent studies in Escherichia coli suggest that high and low affinity DnaA recognition sites are positioned within oriC to direct staged assembly of bacterial pre-replication complexes, with DnaA contacting low affinity sites as it oligomerizes to 'fill the gaps' between high affinity sites. The wide variability of oriC DnaA recognition site patterns seen in nature may reflect myriad gap-filling strategies needed to couple oriC function to the lifestyle of different bacterial types.

Figure 1. Structure of DnaA

DnaA contains four domains, labeled with Roman numerals. Key amino acids, responsible for the indicated functions, are marked (details and references included in text).

Figure 2. Mechanisms that prohibit DnaA-ATP access to oriC after initiation

Three mechanisms, coupled to DNA replication, prevent untimely re-initiation by decreasing levels of available DnaA-ATP or by blocking DnaA binding sites in oriC. A) In E. coli, hydrolysis of DnaA-ATP bound to the chromosome is assisted by Hda associated with the β-clamp. The nascent DNA is red, and the parental strand is blue. B) Duplication of chromosomal loci, such as datA in E. coli, creates additional DnaA binding sites (marked by black rectangles) that titrate DnaA and decrease the level of available protein. In B. subtilis, YabA associated with the clamp plays a similar titrating role, by binding DnaA and limiting its access to oriC (not shown). C) Chromosome replication creates hemimethylated GATC sequences (small black circles), some of which are located in low affinity DnaA binding sites (black rectangles) in oriC or the dnaA promoter. SeqA binding to the hemimethylated GATCs blocks DnaA from rebinding to these sites, while DnaA can rebind to sites that do not contain a GATC.

Figure 3. Model of pre-RC assembly by filling gaps between occupied high affinity DnaA binding sites

After initiation of chromosome replication, DnaA rebinds to high affinity R1, R2 and R4 sites. Extension of DnaA oligomers from these anchor sites to weak sites in the gap regions is prevented (indicated by blocked arrows) by inhibitors SeqA and Fis (top cartoon). In early pre-RC assembly (stage 1), after sequestration ends, DnaA bound to R1, R2 and R4 recruits additional DnaA protomers via domain 1 interactions, stabilized by binding to weak sites in oriC and by association with DiaA. As DnaA-ATP levels rise in the cell (stage 2), closely spaced low affinity DnaA binding sites position DnaA protomers, facilitating domain 3 interactions (lines joining DnaA molecules) and stable gap-filling. Extension from R2 and R4 into the right gap region causes Fis displacement, allowing IHF to bind and bend oriC so that R1 and R5 interact, completing pre-RC assembly and strand separation by filling the left gap. DnaA-ATP binds to the single-stranded unwound region via domain 3 and domain 4 interactions.