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The bacterial DnaA-trio replication origin element specifies single-stranded DNA initiator binding

An Erratum to this article was published on 29 June 2016


DNA replication is tightly controlled to ensure accurate inheritance of genetic information. In all organisms, initiator proteins possessing AAA+ (ATPases associated with various cellular activities) domains bind replication origins to license new rounds of DNA synthesis1. In bacteria the master initiator protein, DnaA, is highly conserved and has two crucial DNA binding activities2. DnaA monomers recognize the replication origin (oriC) by binding double-stranded DNA sequences (DnaA-boxes); subsequently, DnaA filaments assemble and promote duplex unwinding by engaging and stretching a single DNA strand3,4,5. While the specificity for duplex DnaA-boxes by DnaA has been appreciated for over 30 years, the sequence specificity for single-strand DNA binding has remained unknown. Here we identify a new indispensable bacterial replication origin element composed of a repeating trinucleotide motif that we term the DnaA-trio. We show that the function of the DnaA-trio is to stabilize DnaA filaments on a single DNA strand, thus providing essential precision to this binding mechanism. Bioinformatic analysis detects DnaA-trios in replication origins throughout the bacterial kingdom, indicating that this element is part of the core oriC structure. The discovery and characterization of the novel DnaA-trio extends our fundamental understanding of bacterial DNA replication initiation, and because of the conserved structure of AAA+ initiator proteins these findings raise the possibility of specific recognition motifs within replication origins of higher organisms.

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Figure 1: Genetic analysis of the oriC DNA unwinding element reveals a critical region required for initiation activity.
Figure 2: DnaA filaments are loaded from DnaA-boxes onto a specific single-strand sequence within the initially unwound region.
Figure 3: Analysis of the key origin unwinding region provides evidence for functional trinucleotide repeats.
Figure 4: Identification of the DnaA-trio motif.

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We thank J. Errington and W. Vollmer for reviewing the manuscript. We thank G. Scholefield for preliminary data, A. Koh for research assistance and I. Selmes for technical assistance. Research support was provided to H.M. by a Royal Society University Research Fellowship and a Biotechnology and Biological Sciences Research Council Research Grant (BB/K017527/1), and to O.H. by an Iraqi Ministry of Higher Education and Scientific Research Studentship.

Author information

Authors and Affiliations



H.M. and T.T.R. conceived and designed experiments; H.M., T.T.R. and O.H. constructed plasmids and strains; H.M. and O.H. performed growth and marker frequency analysis experiments; H.M. performed microscopy experiments; T.T.R. purified proteins, performed the open complex assay, and performed the DnaA filament formation assays; H.M. and T.T.R. interpreted results and wrote the paper.

Corresponding author

Correspondence to Heath Murray.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Structure of DnaA proteins.

a, Primary domain structure of DnaA. Key functions are listed below the relevant domain. b, Structure of Thermatoga maritima DnaA domain III, highlighting the single-strand binding residue Val176 (Ile190 B. subtilis) within the ISM (PDB accession number 2Z4S). c, Structure of E. coli DnaA domain IV bound to a DnaA-box (PDB accession number 1J1V). d, Structure of A. aeolicus DnaA domain III (blue shades) and domain IV (cyan shades) bound to a single DNA strand (orange), highlighting the single-strand binding residue Val156 (Ile190 B. subtilis) (PDB accession number 3R8F). e, Scheme used to construct mutants within the B. subtilis DNA replication origin. The green arrow highlights the location of a DnaA-box mutation.

Extended Data Figure 2 Characterization of the inducible repN/oriN replication initiation system.

Repression of repN expression inhibits DNA replication in a ΔoriC mutant. A large deletion was introduced into the B. subtilis replication origin using a strain harbouring the inducible oriN/repN construct. Strain growth was found to be dependent upon addition of the inducer IPTG. a, Strains streaked to resolve single colonies. b, A GFP-DnaN reporter was used to detect DNA replication after removal of IPTG from inducible oriN/repN strains. Scale bar, 5 μm. c, Genetic map indicating the location of oriN at the aprE locus in strain HM1108. d, Analysis of DNA replication initiation at oriC and oriN. Marker frequency analysis was used to measure the rate of DNA replication initiation in the presence and absence of IPTG (0.1 mM). Genomic DNA was harvested from cells during the exponential growth phase and the relative amount of DNA from either the endogenous replication origin (oriC) or the aprE locus (oriN) compared with the terminus (ter) was determined using qPCR (mean and s.d. of three technical replicates). Cell doubling times (in minutes) are shown above each data set.

Extended Data Figure 3 Wild-type DnaA assembles into filaments on 5′-tailed substrates.

DnaA filament formation using amine-specific crosslinking (BS3) on DNA scaffolds (represented by symbols above each lane). Protein complexes were resolved by SDS–PAGE and DnaA was detected by western blot analysis.

Extended Data Figure 4 DNA sequence of unwinding regions after mononucleotide and trinucleotide deletions.

Resulting sequences grouped in boxes are identical for more than one deletion.

Extended Data Figure 5 Crosslinking with BS3 captures a distinct DnaA oligomer.

DnaA was incubated with various DNA scaffolds and different crosslinking agents were added to capture distinct DnaA oligomers. a, Crosslinking with BMOE detects DnaA oligomers forming on both duplex and tailed substrates. b, Crosslinking with BS3 only detects DnaA oligomers forming on tailed substrates, revealing an interaction between DnaA and the first DnaA-trio motif located downstream of the GC-cluster.

Extended Data Figure 6 The nucleotide at the third position of the DnaA-trio is required to stabilize DnaA.

DNA scaffolds containing the first two nucleotides of a DnaA-trio either with or without a 5′-phosphate are unable to stabilize binding of an additional DnaA protomer, indicating that the nucleotide at the third position is required. Combined with the data shown in Fig. 4b where the position is abasic, the results suggest that the sugar at the third position plays a critical role in DnaA binding.

Extended Data Figure 7 Relationship between the DnaA-box and the DnaA-trios.

a, Sequence of the origin region used for constructing DNA scaffolds. Symbols below represent duplex DnaA-boxes (triangles), the GC-rich region (green rectangles), the two strands of the unwinding region (red or pink rectangles) and the AT-rich region (blue rectangle). b, Loading of the DnaA filament onto a single-stranded 5′-tail requires a DnaA-box and DnaA domains III–IV, but the DnaA-box position and orientation are flexible.

Extended Data Table 1 Bacterial replication origin regions in Fig. 4c
Extended Data Table 2 Oligonucleotides used for plasmid construction
Extended Data Table 3 Oligonucleotides used to assemble DNA scaffolds

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Richardson, T., Harran, O. & Murray, H. The bacterial DnaA-trio replication origin element specifies single-stranded DNA initiator binding. Nature 534, 412–416 (2016).

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