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. 2014 Dec 1;42(21):13134-49.
doi: 10.1093/nar/gku1051. Epub 2014 Nov 6.

Timely binding of IHF and Fis to DARS2 regulates ATP-DnaA production and replication initiation

Kazutoshi Kasho  1 , Kazuyuki Fujimitsu  1 , Toshihiro Matoba  1 , Taku Oshima  2 , Tsutomu Katayama  3
Affiliations

Timely binding of IHF and Fis to DARS2 regulates ATP-DnaA production and replication initiation

Kazutoshi Kasho et al. Nucleic Acids Res. .

Abstract

In Escherichia coli, the ATP-bound form of DnaA (ATP-DnaA) promotes replication initiation. During replication, the bound ATP is hydrolyzed to ADP to yield the ADP-bound form (ADP-DnaA), which is inactive for initiation. The chromosomal site DARS2 facilitates the regeneration of ATP-DnaA by catalyzing nucleotide exchange between free ATP and ADP bound to DnaA. However, the regulatory mechanisms governing this exchange reaction are unclear. Here, using in vitro reconstituted experiments, we show that two nucleoid-associated proteins, IHF and Fis, bind site-specifically to DARS2 to activate coordinately the exchange reaction. The regenerated ATP-DnaA was fully active in replication initiation and underwent DnaA-ATP hydrolysis. ADP-DnaA formed heteromultimeric complexes with IHF and Fis on DARS2, and underwent nucleotide dissociation more efficiently than ATP-DnaA. Consistently, mutant analyses demonstrated that specific binding of IHF and Fis to DARS2 stimulates the formation of ATP-DnaA production, thereby promoting timely initiation. Moreover, we show that IHF-DARS2 binding is temporally regulated during the cell cycle, whereas Fis only binds to DARS2 in exponentially growing cells. These results elucidate the regulation of ATP-DnaA and replication initiation in coordination with the cell cycle and growth phase.

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Figures

Figure 1.
Figure 1.
A minimal DARS2 and roles for IHF and Fis. (A) Overall structure of the E. coli chromosome and oriC. The upper figure indicates the relevant loci on the circular chromosome. The lower figure indicates the basic structure of oriC (open rectangle). Filled, gray and dotted arrowheads indicate the high affinity DnaA boxes (R1 and R4), moderate affinity site (R2) and low affinity sites, respectively. The three AT-rich repeats in the duplex unwinding element (AT-rich) and the IBS are also shown by purple bars and a green bar, respectively. (B) Schematic presentation of the regulatory cycle of DnaA. ATP–DnaA initiates replication, resulting in activation of replisomes and RIDA. In addition, DDAH is activated by timely binding of IHF to datA. DARS1 and DARS2 regenerate ATP–DnaA from ADP–DnaA; invivo, DARS2 plays the predominant role in this process. (C) DARS2 deletion analysis. The open rectangle indicates the whole DARS2 region. Filled bars indicate regions carried on vector pACYC177. Cells bearing these plasmids were grown at 37°C in supplemented M9-ampicillin medium, and then subjected to flow cytometry analysis (see Supplementary Figure S1A for data). +, active in stimulation of initiation; -, inactive. (D) Sequence of the minimal DARS2 region on pOA67. Consensus binding sequences of IHF (green letters) and Fis (blue letters) are TAAnnnnTTGATW (where W is A or T) and GnnYAnnnnnTRnnC (Y = C or T; R = A or G; n is any nucleotide) (6,19), respectively. The red box represents the critical 21-mer. (E) DARS2 reaction using IHF-defective crude protein extracts. [3H]ADP–DnaA (2 pmol) and 5 fmol of pOA61 (DARS2, +) or pACYC177 (DARS2, −) were co-incubated at 30°C for 15 min with a crude protein extract (Fr II) of YH014 (WT) or YH014-I (ihfA::Tn10). See also Supplementary Figure S2A. (F) DARS2 reaction using Fis-defective crude protein extracts. Similar experiments were performed using a crude protein extract of MG1655 (WT) or KMG-2 (Δfis). See also Supplementary Figure S2B. (G) In vitro complementation experiments for IHF. Similar experiments were performed using pOA61 and purified IHF with no extract (None) or with a crude extract (Fr II, 5 μg) from YH014 (WT) or YH014-I (ihfA::Tn10).(H) In vitro complementation experiments for Fis. Similar experiments were performed using pOA61 and purified Fis with no extract (None) or crude extract (Fr II, 5 μg) from MG1655 (WT) or KMG-2 (Δfis).
Figure 2.
Figure 2.
In vitro reconstitution of DARS2 reactions. (A) ADP dissociation from DnaA. [3H]ADP–DnaA (2 pmol), the indicated amounts of IHF and 5 fmol of pOA61 (•,▴) or pACYC177 (○) were co-incubated at 30°C for 15 min without (▴) or with 50 fmol of Fis (•,○). See also Supplementary Figure S2C and D. (B) ADP dissociation from DnaA. Similar experiments were performed using the indicated amounts of Fis without (▴) or with 50 fmol of IHF (•,○). (C) ADP dissociation from DnaA. Similar experiments were performed using the indicated amounts of pOA61 without (○) or with 50 fmol each of Fis (•,▪) and IHF (•,▴). (D) ATP–DnaA generation. ADP–DnaA (2 pmol), 1.5 μM [α-32P]ATP and the indicated amounts of pOA61 (•,▪,▴,♦) or pACYC177 (○,□,Δ,⋄) were co-incubated at 30°C for 15 min with or without (▴,Δ) 50 fmol each of IHF (•,♦,○,⋄) and Fis (•,▪,○,□). apo-DnaA (×) was incubated similarly with [32P]ATP, but without DNA, IHF and Fis. (E) Reconstitution of the DnaA cycle using DARS2 and RIDA. In the 1st stage, ADP–DnaA (•,○) or apo-DnaA (▴,Δ) was incubated as above with or without pOA61 (5 fmol) (DARS2) in buffer containing 1.5 μM [α-32P]ATP and 50 fmol each of IHF and Fis, followed by digestion of pOA61 with PciI. In the 2nd stage, portions of the resultant reactions were incubated at 30°C for 20 min with the DNA-loaded clamp (40 fmol as the clamp) and wild-type Hda (WT) (•,▴) or the Hda Q6A mutant as a negative control (○,Δ). The Hda Q6A mutant is defective in clamp binding and RIDA (26). (F) Reconstitution of the DnaA cycle using DARS2 and DDAH. Similar experiments were performed for the 1st stage. In the 2nd stage, the reactions were incubated at 30°C for 10 min with IHF (0.2 pmol) and 991-bp datA DNA (WT) (•,▴) or datA subDnaAbox2 mutant as a negative control (○,Δ). The datA DnaA box 2 is a crucial DnaA box for DDAH and the datA subDnaAbox2 is a mutant datA bearing a substituted DnaA box 2 sequence that is inactive in DDAH (17). (G) oriC replication coupled with DARS2 ATP–DnaA regeneration. ADP–DnaA (•,▴) or ATP–DnaA (○,Δ) (0.5 pmol [20 nM] each) was incubated at 30°C for 15 min with 5 fmol of pOA61 (•,○) or pACYC177 (▴,Δ) in buffer containing M13KEW101 oriC plasmid (600 pmol nucleotides) and replication proteins, including 2 pmol (80 nM) of IHF and the indicated amounts of Fis.
Figure 3.
Figure 3.
DARS2 IBS1–2 and FBS2–3 are required for DARS2 activation in vitro. (A, B) Electrophoretic mobility shift assay. IHF (A) or Fis (B) was incubated for 5 min at 30°C in buffer GS containing DARS2 DNA (455 bp, 100 fmol) and poly (dI-dC) (120 ng), followed by 4% PAGE. Well; gel well, Bound; protein-bound DNA, Free; protein-free DNA. Poly (dI-dC) remained in the gel well. (C, D) Footprint analysis. The upper or lower strand of [32P]-DARS2 (100 fmol) was incubated at 30°C for 10 min with DNase I and IHF (0, 1.2, 2.4, or 4.8 pmol) (C) or Fis (0, 0.12, 0.24, 0.48, 0.96, 1.9, 3.8, or 7.6 pmol) (D), followed by urea–PAGE and phosphorimaging. Green lines: IBS1–2. Blue lines: FBS1 FBS2–5, and FBS6. Blue dotted lines: low affinity Fis-binding sites. For detailed sequences, see also Supplementary Figure S3. (E, F) Roles for DARS2 IBS and FBS in ADP dissociation from DnaA. MG1655 Fr II (5 μg) (E) or 50 fmol each of IHF and Fis (F) was co-incubated at 30°C for 15 min with [3H]ADP–DnaA (2 pmol) in the presence of pOA61 (WT) (•), pKX35 (subIBS1–2) (▴), pKX36 (subFBS2–3) (♦), pKX37 (subFBS4–5) (▪) or pACYC177 (None) (○). See also Supplementary Figures S4 and S5.
Figure 4.
Figure 4.
Mechanistic analyses of DnaA oligomer formation on DARS2. (A) Analysis of DnaA mutants. Wild-type and mutant forms of [3H]ADP–DnaA were incubated as described for Figure 2C, with or without 5 fmol of pOA61. Average values and errors of ADP dissociation from DnaA [%] are shown. (B) Electrophoretic mobility shift assay using DnaA AID-2 mutant. Indicated amounts of the ADP forms of wild-type DnaA or the DnaA AID-2 mutant L290A were incubated at 30°C for 5 min in dissociation buffer containing DARS2ΔRight DNA (15 fmol) and poly (dI-dC) (25 ng), followed by 5% PAGE and GelStar staining. Well; gel well, Bound; protein-bound DNA, Free; protein-free DNA. Poly (dI-dC) remained in the gel well. (C) Dissociation of ATP or ADP from DnaA. [α-32P]ATP–DnaA (▴,Δ) or [3H]ADP–DnaA (•,○) (2 pmol) was incubated with pOA61 (•,▴) or pACYC177 (○, Δ) as described above. Two independent experiments were done for each assay, and both data and mean values are shown. (D) Pull-down assay. ADP–DnaA (5 pmol) and 5’-biotinylated DNA (0.5 pmol) of DARS2ΔRight or DARS2ΔRightΔCore were co-incubated with or without IHF and Fis. DNA-bound DnaA was collected using magnetic beads and was quantitatively analyzed by SDS-PAGE and silver staining. DARS2ΔRightΔCore was a DARS2ΔRight derivative bearing ΔCore (Figure 1C). Two independent experiments were done, and both data and the mean values are shown for the recovered DnaA. (E–H) Electrophoretic mobility shift assay using DARS2ΔRight/subFBS1. ADP/ATP–DnaA was incubated at 30°C for 5 min in dissociation buffer containing [32P]DARS2ΔRight/subFBS1 DNA (15 fmol) and poly (dI-dC) (25 ng), followed by 5% PAGE and phosphorimaging (E). Intensities of the signals corresponding to lower (C1–C2) (F), middle (C3) (G) and higher-order multimers (C6+) (H) on the gel image were quantified, and relative levels [% of total] were plotted. Two independent experiments were done, and both data and mean values are shown. (I–L) Electrophoretic mobility shift assay using DARS2ΔRight/subFBS1. Similar experiments were performed in the presence of IHF (0.6 pmol) and Fis (0.3 pmol) (I), followed by analysis of the signals (J–L). Two independent experiments were done, and both data and mean values are shown.
Figure 5.
Figure 5.
In vivo roles for IHF and Fis in DARS2 activation. (A) Transformation inhibition. Strains MG1655 (WT), KMG-5 (ΔihfA), KMG-6 (ΔihfB) and KMG-2 (Δfis) were transformed with pBR322 (vector), pKX11 (DARS2 WT) or pOA77 (DARS2ΔCore) and incubated at 37°C for 12 h on LB-ampicillin agar plates. Transformation efficiencies of pKX11 and pOA77, relative to those of pBR322, are shown. (B) Flow cytometry analysis using Δihf and Δfis cells. MG1655, KMG-2, KMG-5 and KMG-6 cells bearing pACYC177 (vector), pOA61 (DARS2) or pOA21 (DARS1) were grown at 37°C in supplemented M9-ampicillin medium, and then subjected to flow cytometry analysis as described for Figure 1C. The numbers inserted in the histograms indicate mean cell mass and ori/mass relative to that of MG1655 cells bearing pACYC177. (C) Cellular ATP–DnaA levels. The following strains were grown at 37°C; MK86 (WT), MIT47 (DARS1ΔCore), MIT86 (DARS2ΔCore), KX97 (ΔihfB), KX101 (ΔihfB DARS1ΔCore), KX90 (ΔihfB DARS2ΔCore), KX83 (ΔdatA), KX179 (ΔdatA DARS1ΔCore), KX102 (ΔdatA DARS2ΔCore) and KX29 (Δfis). Error bars represent SD from three independent experiments except for KX29. Two independent experiments were done for KX29, and both data and mean values are shown. (D) Flow cytometry analysis using pOA61 derivatives. MG1655 cells bearing pACYC177 (vector), pOA61 (DARS2 WT) or the indicated pOA61-derivative DARS2 mutant plasmid were analyzed by flow cytometry as described for Figure 1C. Ratios of mean cell mass and ori/mass are shown in the histograms. (E) The ori/mass ratios of the chromosomal DARS2 mutant cells. Cells of the MG1655 (DARS1, WT; DARS2, WT) strain and its derivatives bearing the indicated DARS2 mutations in the presence or absence of the DARS1ΔCore mutation were analyzed by flow cytometry. Ratios of two or four oriC in each strain were calculated. The ori/mass ratios are shown as relative values to those of MG1655, which was used as a standard. (F) The initiation mass of DARS2 mutant cells. The initiation masses relative to that of MG1655 (WT) were deduced from the flow cytometry data obtained above. (G) Cellular ATP–DnaA levels of IBS and FBS mutants. Cells of the KX41 (rnhA::Tn3 ΔoriC Δhda) (WT) strain and its derivatives bearing the indicated DARS2 mutations were analyzed as described for Figure 5C. Two independent experiments were done, and both data and mean values are shown. (H) DARS2-medicated ATP-DnaA regeneration activity in vivo. KA474 (dnaN59) cells bearing pACYC177 (None), pOA61 (WT) or the indicated pOA61-derivative DARS2 mutant plasmid were incubated in TG-ampicillin medium containing [32P]orthophosphate at 28°C until the A660 reached 0.2, transferred to 42°C in the presence of 150 μg/ml chloramphenicol (Time 0) and further incubated for 20 min, followed by immunoprecipitation and thin-layer chromatography. Two independent experiments were done for each assay, and both data and mean values are shown.
Figure 6.
Figure 6.
Cell cycle- and growth phase-coordinated regulation of DARS2. (A, B) IHF-ChIP of DARS2. KYA018 (dnaC2) cells growing at 30°C were transferred to 38°C and incubated for 90 min. The cells were then transferred to 30°C (Time 0) and incubated for 5 min, followed by further incubation at 30°C (A) or 38°C (B). The relative DARS2 levels before (Input) and after (IHF-ChIP) IP using anti-IHF antiserum were determined using real-time qPCR, yielding the ChIP/Input ratio [expressed as%]. Error bars represent SD from at least three independent experiments. In addition, cellular levels of oriC, DARS2 and ter in the Input samples were quantified, and the relative ratios of oriC/ter and DARS2/ter are expressed relative to the ratio at 0 min (defined as 1). (C, D) Fis-ChIP of DARS2. MG1655 cells were incubated at 30 or 38°C in supplemented M9 medium. At the indicated A660, samples were withdrawn for determination of the Fis-ChIP/Input ratio [expressed as%] (C). Error bars represent SD from at least three independent experiments. O/N, overnight incubation. Growth curve at 38°C (D). Arrows: sampling times.
Figure 7.
Figure 7.
Regulated activation of DARS2 by IHF and Fis in the DnaA cycle. (A) A schematic view of the roles for IHF and Fis in regulatory systems for DARS2, oriC and datA. See text for details. (B) A model of the timely regulation of DARS2 by IHF and Fis. In exponential phase cells, Fis binds to DARS2. In the pre-initiation stage, the time when the ADP–DnaA level is high, IHF binds to oriC and DARS2 in a cell cycle-coordinated manner, and the DARS2–IHF–Fis complex increases the level of ATP–DnaA. Next, the elevated ATP–DnaA level allows the IHF-bound oriC to initiate replication, at which point IHF dissociates from DARS2. In the post-initiation stage, IHF binds to datA, activating DDAH. IHF then dissociates from datA in a cell cycle-coordinated manner, and re-binds to DARS2 and oriC for the next round of initiation. For simplicity, only single molecules of IHF and Fis are shown to bind to DARS2 in this figure. In growing cells, the pre-initiation stage overlaps the post-initiation stage of the previous round of initiation.
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