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. 2016 Jun 16;534(7607):412-6.
doi: 10.1038/nature17962. Epub 2016 Jun 8.

The Bacterial DnaA-trio Replication Origin Element Specifies Single-Stranded DNA Initiator Binding

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Free PMC article

The Bacterial DnaA-trio Replication Origin Element Specifies Single-Stranded DNA Initiator Binding

Tomas T Richardson et al. Nature. .
Free PMC article

Erratum in

Abstract

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 synthesis. In bacteria the master initiator protein, DnaA, is highly conserved and has two crucial DNA binding activities. 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 strand. 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.

Figures

Extended Data Figure 1
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 2Z4S). (c) Structure of Escherichia coli DnaA domain IV bound to a DnaA-box (PDB 1J1V). (d) Structure of Aquifex 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 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
Extended Data Figure 2. Characterisation 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 following 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 to the terminus (ter) was determined using qPCR (mean and s.d. of 3 technical replicates). Cell doubling times (min) are shown above each data set.
Extended Data Figure 3
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
Extended Data Figure 4. DNA sequence of unwinding regions following mononucleotide and trinucleotide deletions.
Resulting sequences grouped in boxes are identical for more than one deletion.
Extended Data Figure 5
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
Extended Data Figure 6. The nucleotide at the third position of the DnaA-trio is required to stabilise DnaA.
DNA scaffolds containing the first two nucleotides of a DnaA-trio either with or without a 5’-phosphate are unable to stabilise 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
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.
Figure 1
Figure 1. Genetic analysis of the oriC DNA unwinding element reveals a critical region required for initiation activity.
(a) B. subtilis oriC unwinding region. DnaA-box colouring indicates conservation (consensus 5′-TTATCCACA-3′ in black). (b) The oriC-independent strain used for constructing replication origin mutations. (c) Growth of an oriC deletion mutant is dependent upon oriN activity. (d) Deletions extending beyond the AT-cluster into the initially unwound region inhibit cell growth. (e) Sequences between the GC-rich and AT-rich clusters are essential for origin function. (f) Sequences proximal to DnaA-boxes are most important for origin function. Marker frequency analysis was used to measure the rate of DNA replication initiation (mean and s.d. of 3 technical replicates). (g) Open complex formation by DnaA requires the native sequence between the GC- and AT-clusters. DNA duplex unwinding was probed by KMnO4 and detected by primer extension.
Figure 2
Figure 2. DnaA filaments are loaded from DnaA-boxes onto a specific single-strand sequence within the initially unwound region.
(a) DnaA-boxes proximal to the unwinding region are most important for origin function. Marker frequency analysis was used to measure the rate of DNA replication initiation (mean and s.d. of 3 technical replicates). (b) Sequence of the origin region used for constructing DNA scaffolds in (c). (c) DnaA filament formation using cysteine-specific crosslinking on DNA scaffolds. DnaA complexes were resolved by SDS-PAGE and detected by Western blot analysis. (d) Crystal structure showing ssDNA (dA12) bound to the DnaA filament through the AAA+ domain (PDB: 3R8F). (e) Sequence of the origin region used for constructing DNA scaffolds in (f). (f) DnaA filament formation on tailed substrates is arrested by a poly(A) tract. Long oligomers highlighted within the dotted box are shown above with increased contrast.
Figure 3
Figure 3. Analysis of the key origin unwinding region provides evidence for functional 3-mer repeats.
(a) Mutagenesis identifies A:T base pairs spaced three nucleotides apart are most critical for origin activity. Marker frequency analysis was used to measure the rate of DNA replication initiation (mean and s.d. of 3 technical replicates). (b) Crystal structure showing the interaction of DnaA with sets of three nucleotides (PDB: 3R8F). Residues for A. aeolicus indicated above; B. subtilis below. (c) In vivo deletion analysis of the unwinding region. Isogenic deletions indicated in black. Marker frequency analysis was used to measure the rate of DNA replication initiation (mean and s.d. of 3 technical replicates). (d) Growth of mutants used in (c). (e) In vitro deletion analysis of tailed substrates.
Figure 4
Figure 4. Identification of the DnaA-trio motif.
(a) Varying the length of 5′-tailed substrates identifies the likely DnaA-trio sequence. Lane 2 shows DnaA filament formation on a duplex DNA scaffold (DnaA-box6, DnaA-box7, GC-rich cluster). Letters indicate the nucleotide sequentially added to the 5′-tail. (b) Targeted mutagenesis of the proposed DnaA-trio motif. (c) Bioinformatic analysis identifies DnaA-trio motifs adjacent to a DnaA-box throughout the bacterial kingdom. Underlined sequences indicate experimentally determined DnaA-dependent unwinding sites. (d) DnaA-trios sequence logo (WebLogo28). (e) A schematic of DnaA filament formation from double-stranded DnaA-boxes (triangles) onto a single strand containing the DnaA-trios.

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