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. 2017 Dec 1;45(21):12354-12373.
doi: 10.1093/nar/gkx914.

Regulatory Dynamics in the Ternary DnaA Complex for Initiation of Chromosomal Replication in Escherichia Coli

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Regulatory Dynamics in the Ternary DnaA Complex for Initiation of Chromosomal Replication in Escherichia Coli

Yukari Sakiyama et al. Nucleic Acids Res. .
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Abstract

In Escherichia coli, the level of the ATP-DnaA initiator is increased temporarily at the time of replication initiation. The replication origin, oriC, contains a duplex-unwinding element (DUE) flanking a DnaA-oligomerization region (DOR), which includes twelve DnaA-binding sites (DnaA boxes) and the DNA-bending protein IHF-binding site (IBS). Although complexes of IHF and ATP-DnaA assembly on the DOR unwind the DUE, the configuration of the crucial nucleoprotein complexes remains elusive. To resolve this, we analyzed individual DnaA protomers in the complex and here demonstrate that the DUE-DnaA-box-R1-IBS-DnaA-box-R5M region is essential for DUE unwinding. R5M-bound ATP-DnaA predominantly promotes ATP-DnaA assembly on the DUE-proximal DOR, and R1-bound DnaA has a supporting role. This mechanism might support timely assembly of ATP-DnaA on oriC. DnaA protomers bound to R1 and R5M directly bind to the unwound DUE strand, which is crucial in replication initiation. Data from in vivo experiments support these results. We propose that the DnaA assembly on the IHF-bent DOR directly binds to the unwound DUE strand, and timely formation of this ternary complex regulates replication initiation. Structural features of oriC support the idea that these mechanisms for DUE unwinding are fundamentally conserved in various bacterial species including pathogens.

Figures

Figure 1.
Figure 1.
Determination of the key DnaA boxes required for oriC DUE unwinding. (A) Overall structure of oriC. The duplex-unwinding element (DUE; closed bar), DnaA-oligomerization region (DOR; open bar), and IHF-binding site (IBS; striped pattern box) are indicated. High-affinity DnaA boxes (R1 and R4) are indicated by closed triangles, and moderate-affinity (R2) and low-affinity sites (R5M, τ1–2, I1–3 and C1–3) are indicated by open triangles. The functions of left half and middle-right half of the DOR are indicated. The minimal region for DUE unwinding determined in this study is indicated by a gray bar (also see text). (B) DnaA protein domain structure. Domains of E. coli DnaA are indicated by open boxes with relevant amino acid numbering. The residues investigated in this study and their functions are indicated. (C) Proposed models of DUE-unwinding-complex structure. For simplicity, only DnaA domains III and IV are shown. DnaA domain III bound to ssDNA (and not dsDNA) is indicated by light-gray shading, and IHF by a purple hexagon. IHF binding bends DNA to >160°. ssDUE strands are shown in red. (D–G) DUE-unwinding assay. ATP–DnaA or ADP–DnaA, along with IHF (32 nM), were incubated for 3 min at 38°C with oriC DNA (1.32 nM) containing DnaA-box mutations, followed by incubation for 200 s at 38°C with P1 nuclease (4 U). Resultant DNAs were analyzed by gel electrophoresis shown in (D) and (F). The percentages of DNA digested by P1 nuclease out of the total input DNA are shown as ‘DUE unwinding (%)’ in (E) and (G). Two independent experiments were carried out, and both data, mean values, and representative gel images in a black-white inverted mode are shown.
Figure 2.
Figure 2.
DnaA bound to R5M, but not R1, is essential for DnaA assembly on the left-half DOR. (A–D) EMSA with the left-half DOR bearing R1non or R5Mnon mutations. Each DNA fragment (35 nM) was incubated with the indicated amount of DnaA in the absence (A) or presence (C) of 72 nM IHF, then analyzed by 2% agarose-gel electrophoresis. The amount of DNA complexed with DnaA relative to the input DNA is shown as ‘Complex formation (%)’ in (B) and (D). Two independent experiments were carried out, and both data, mean values, and representative gel images in a black-white inverted mode are shown. (E–G) DNase I-footprint assay with oriC fragments (346 bp) containing R1non or R5Mnon mutations. Indicated amounts of ATP–DnaA or ADP–DnaA were incubated for 10 min at 30°C with 2.4 nM 32P-end-labeled oriC and 3 mM ATP or ADP in the absence (E) or presence (F) of 150–600 nM IHF, followed by DNase I digestion and gel-electrophoresis analysis. Positions of DNA motifs are indicated. The sequence of the region protected by IHF binding is shown in (G). The IBS consensus sequence and DNase I digestion-protected sites are indicated by bold lettering and asterisks, respectively.
Figure 3.
Figure 3.
H/B motifs of DnaA bound to R1 and R5M are important for DUE unwinding. (A) Structures of E. coli DnaA (EcoDnaA), T. maritima DnaA (TmaDnaA), and chimeric DnaA (ChiDnaA). Domains of EcoDnaA and TmaDnaA are indicated by open and gray boxes, respectively. (B) Sequences of R1 and R5M boxes substituted with TmaDnaA box. (C) A schematic of DUE unwinding assay using ChiDnaA, EcoDnaA, and R5MTma oriC plasmid. ChiDnaA mutant (indicated by striped domain III and gray domain IV) was suggested to bind specifically to R5MTma site. The other DnaA boxes were occupied by EcoDnaAs (indicated by open shapes of domain III and IV). (D) DUE-unwinding assays with an R5MTma oriC plasmid. An oriC plasmid (1.32 nM) with the R5MTma substitution was incubated for 3 min at 38°C with wild-type ATP-EcoDnaA (EcoWT) and indicated amounts of ATP/ADP-chimeric DnaA (ChiWT) or its R285A mutant (ChiR285A), followed by incubation with P1 nuclease. Resultant DNAs were analyzed by gel electrophoresis. The percentages of DNA digested by P1 nuclease relative to the total input DNA are shown as ‘DUE unwinding (%)’. (E–G) DUE-unwinding assay with ssDUE-binding-variant ChiDnaA. oriC plasmid with (E and F) or without (G) the substitution of R1Tma (E) or R5MTma (F) were incubated as described above, except for the use of ATP-ChiDnaA V211A (ChiV) and ATP-ChiDnaA V211A R245A (ChiVR). For panels D–G, two independent experiments were carried out, and both data, mean values and representative gel images in a black–white inverted mode are shown.
Figure 4.
Figure 4.
Roles of DnaA bound to R5M in ssDUE binding to the DnaA–oriC complex. (A) A schematic of EMSA of ssDUE recruitment. ATP–DnaA was incubated with the DOR R5MI2 fragment and 32P-end-labeled ssDUE M28, followed by electrophoretic-mobility-shift assay. For simplicity, only DnaA domains III–IV are shown. ssDUE M28 is shown with thick black lines, 32P with a star, and the DOR R5MI2 DNA with thin black lines. ATP and Arg finger are also shown as in Figure 3C. (B and C) EMSA using the DOR R5MI2 and EcoDnaA or its truncated form bearing domain III-IV. Indicated amounts of the ATP or ADP form of DnaA were incubated for 10 min at 30°C with (+) or without (–) the DOR R5MI2 (35 nM) and λ phage DNA in the absence (B) and presence(C) of ssDUE M28 (T-rich strand) and ssDUE M28rev (A-rich strand), followed by 4% polyacrylamide-gel electrophoresis. Two independent experiments were carried out, and representative gel images are shown. The amounts of DOR R5MI2 complexed with DnaA, or ssDUE M28 and ssDUE M28rev bound to the DnaA–DOR R5MI2 complexes were quantified as ‘Complex (%)’ in (B) and ‘ssDUE-bound complex (%)’ in (C), and both data and mean values are shown. λ phage DNA included as a competitor remained near the gel wells, which is not included in Complex (%) and ssDUE-bound complex (%). (D and E) EMSA using DOR R5MI2 with R5MTma substitution (DOR R5MI2-R5MTma), EcoDnaA, ChiDnaA(ChiWT) or its derivatives (ChiV, ChiVR) in the absence (D) and presence(E) of ssDUE M28 as described above. Two independent experiments were carried out, representative gel images are shown, and both data, mean values and detected complexes (%) are also shown as described above. For panels B and D, representative gel images are shown in a black-white inverted mode.
Figure 5.
Figure 5.
Roles of DnaA bound to R1 and R5M in ssDUE recruitment into the DnaA–oriC complex. (A) A schematic of EMSA of ssDUE recruitment using 32P-end-labeled ssDUE-conjugated dsDNA bearing DnaA box R1 (ssDUE-dsR1). ATP–DnaA was preincubated with DOR R5MI2, followed by co-incubation with ssDUE-dsR1. Possible complexes are illustrated as in Figure 3C. See text for details. (B and C) EMSA of ssDUE recruitment with DOR R5MI2 and DOR R5MI2-R5MTma. Indicated amounts of the ATP form of EcoDnaA and ChiDnaA (ChiWT) or its derivatives (ChiV, ChiVR) were pre-incubated for 5 min on ice with the DOR R5MI2 or DOR R5MI2-R5MTma (35 nM), followed by further incubation for 10 min at 30°C with ssDUE-dsR1 (16 nM). A representative gel image is shown. The amounts of ssDUE-dsR1 bound to the DnaA–DOR R5MI2 complexes were quantified as ‘ssDUE-dsR1−bound complex (%)’. Two independent experiments were carried out, and both data and mean values are shown in (C). λ phage DNA included as a competitor remained near the gel wells, which is not included in ssDUE-dsR1−bound complex (%). (DG) EMSA of ssDUE recruitment with ssDUE-dsR1 bearing the R1Tma substitution. Experiments were performed as described above, except for ssDUE-dsR1 (WT) and ssDUE-dsR1Tma (R1Tma). The ATP-form (D and E) or ADP-form (F and G) ChiDnaA wild-type and its derivatives were analyzed. Two independent experiments were carried out, and quantified as described above, and both data, mean values and ssDUE-dsR1−bound complex (%) are shown in (E) and (G), respectively.
Figure 6.
Figure 6.
Analysis of chromosomal oriC mutants. (A and B) Flow-cytometry analysis of a series of chromosomal oriC mutants bearing DnaA-box nonsense sequences. Relevant oriC genotypes of strains are shown as brackets. Cells were exponentially grown at 30°C in LB medium (A) or M9 Glc CAA medium (B), followed by further incubation for run-out replication. DNA contents were quantified with a flow cytometer, and are indicated with the equivalent chromosome numbers on the x axes. Cell sizes (mass) at the time of drug addition were quantified using a Coulter counter, and the mass and ori/mass ratio relative to those of NY20 cells and the doubling time (Td) of cells are indicated at the top right of each panel. (C) Spot test of diaA-null cells with oriC mutations. Serial dilutions of the full-growth cultures (∼109 cells/ml) at 30°C were spotted onto LB-agar plates and incubated for 9 h at 42°C or for 13 h at 30°C. (D) Flow-cytometry analysis of diaA-null cells with oriC mutations. Experiments were performed as described for panel A.
Figure 7.
Figure 7.
Roles of DnaA bound to R1 and R5M in vivo. (A) Spot test of cells with the chromosomal R5MTma mutation (or with wild-type oriC) and pECTM plasmid (pING1) containing the wild-type (WT) chimeric dnaA gene (pChidnaA) or its derivatives (pChidnaA V211A, R245A and V211A R245A). Serial dilutions of the full-growth cultures (∼109 cells/ml) at 42°C were spotted onto LB-agar plates and incubated for 9 h at 42°C or for 13 h at 30°C. (BD) Flow-cytometry analysis. Cells with the wild-type (WT) chromosomal oriC or with R1Tma or R5MTma mutations along with pChidnaA (WT) or its parent vector (pING1) or derivatives were grown at 30°C (B), 25°C (C) or 37°C (D) in LB medium, followed by further incubation for run-out replication. DNA contents were quantified with a flow cytometer, and are indicated with the equivalent chromosome numbers on the x axes. Cell sizes (mass) at the time of drug addition were quantified with a Coulter counter, and the mass and ori/mass ratio relative to those of NY20 cells with pING1 and the doubling time (Td) of cells are indicated at the top right of each panel.
Figure 8.
Figure 8.
Model of DUE-unwinding-complex formation. (AD) Model for oriC-complex dynamics. The R5M-bound and R4-bound DnaAs have a central role in ATP–DnaA assembly on each half of the DOR (A and B). Both ATP–DnaA and ADP–DnaA can bind to the R1 and R4 boxes for initiation of DNA replication (32). The Arg fingers of each DnaA molecule (indicated by small red arrows) are expected to be oriented inward within the DOR, stimulating ATP–DnaA complex formation in opposite directions, except for the R2-bound DnaA (33). In the DUE-unwinding process, binding of IHF causes DNA bending, enabling interaction between DnaA molecules bound to R1 and R5M boxes. The DUE is unwound, and the resultant T-rich strand of ssDUE (red line) is recruited into the DnaA protomers and binds to DnaA bound to R1 and R5M, resulting in stable DUE unwinding that can initiate replication (D). If the R1-bound DnaA is in the ATP form, unstable intermediates might be formed by binding of free ATP–DnaA to ssDUE via interaction with the R1-bound ATP–DnaA (C), As domains I-II are basically dispensable for DUE unwinding, those domains are omitted. (E) oriC sequences of representative bacteria. The DUE, IBS, and IHF binding region shown in Figure 2F and G are indicated by black, striped, and gray bars, respectively. DnaA boxes with complete match to the consensus sequence and with moderate similarity are indicated by closed and open triangles, respectively. DnaA box τ1 and its corresponding sites are indicated by dotted lines. DnaA boxes with left-ward orientation in possible left-half DORs are highlighted. Abbreviations: Eco, Escherichia coli K-12; Eca, Erwinia carotovora atroseptica SCRI1043; Kpn, Klebsiella pneumonia 342; Vpa, Vibrio parahaemolyticus RIMD 2210633 chromosome I; Vch, Vibrio cholerae O1 biovar eltor str. N16961 chromosome I; Ype, Yersinia pestis CO92; Pae, Pseudomonas aeruginosa PAO1; Lpn, Legionella pneumophila str. Paris; Sae, Staphylococcus aureus RF122; Bsu, Bacillus subtilis QB928; Tma, Thermotoga maritima. See Supplementary Table S1 for sequences.

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