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. 2009 May 15;23(10):1221-33.
doi: 10.1101/gad.1775809. Epub 2009 Apr 28.

Specific genomic sequences of E. coli promote replicational initiation by directly reactivating ADP-DnaA

Kazuyuki Fujimitsu  1 , Takayuki Senriuchi, Tsutomu Katayama
Affiliations

Specific genomic sequences of E. coli promote replicational initiation by directly reactivating ADP-DnaA

Kazuyuki Fujimitsu et al. Genes Dev. .

Abstract

In Escherichia coli, ATP-DnaA, unlike ADP-DnaA, can initiate chromosomal replication at oriC. The level of cellular ATP-DnaA fluctuates, peaking at around the time of replication initiation. However, it remains unknown how the ATP-DnaA level increases coordinately with the replication cycle. In this study, we show that two chromosomal intergenic regions, herein termed DnaA-reactivating sequence 1 (DARS1) and DnaA-reactivating sequence 2 (DARS2), directly promote regeneration of ATP-DnaA from ADP-DnaA by nucleotide exchange, resulting in the promotion of replication initiation in vitro and in vivo. Coordination of initiation with the cell cycle requires DARS activity and its regulation. Oversupply of DARSs results in increase in the ATP-DnaA level and enhancement of replication initiation, which can inhibit cell growth in an oriC-dependent manner. Deletion of DARSs results in decrease in the ATP-DnaA level and inhibition of replication initiation, which can cause synthetic lethality with a temperature-sensitive mutant dnaA and suppression of overinitiation by the lack of seqA or datA, negative regulators for initiation. DARSs bear a cluster of DnaA-binding sites. DnaA molecules form specific homomultimers on DARS1, which causes specific interactions among the protomers, reducing their affinity for ADP. Our findings reveal a novel regulatory pathway that promotes the initiation of chromosomal replication via DnaA reactivation.

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Figures

Figure 1.
Figure 1.
Structure and in vitro activity of DARS1. (A) Identification of DARS1. Open arrows indicate the coding regions. The right-angled arrow indicates the promoter. Black- and gray-filled arrowheads represent DnaA boxes that completely match the 9-mer consensus and that differ from the consensus by a single base, respectively. Numbers above the sequence of FK7-7 indicate the position from the stop codon of bioD. FK7-8, FK7-9, and FK7-13 contain nonsense sequence (AACTATATC; gray-filled square) (Schaper and Messer 1995) instead of DnaA box I (DB I), DnaA box II (DB II), and DnaA box III (DB III), respectively. [3H]ADP-DnaA (2 pmol) was incubated for 15 min at 30°C in buffer containing 2 mM ATP in the presence (50 fmol) or absence of the indicated DNA fragment (filled bars). DNA-dependent release of ADP is shown for each fragment (ADP release %). (B,C) ADP-releasing activity of DARS1 mutants. The indicated amounts of FK7-7 (○, ●) or its derivatives FK7-8 (◇, ♦), FK7-9 (△, ▲), and FK7-13 (□, ■) were incubated at 0°C (unfilled symbols) or 30°C (filled symbols) for 15 min with [3H]ADP-DnaA (2 pmol). (D) Nucleotide-exchanging activity of DARS1. ADP-DnaA (2 pmol) was incubated for 15 min at 0°C (○) or 30°C (●) with 1.5 μM [α-32P]ATP and the indicated amounts of FK7-7. [α-32P]ATP-DnaA (2 pmol) was similarly incubated at 0°C (◇). (▲) DnaA was not included. (E) Reactivation of DnaA by DARS1 for replication initiation. ADP-DnaA (○, ●) and ATP-DnaA (△, ▲) (0.4 pmol) were incubated (first reaction) for 15 min at 0°C (○, ▵) or 30°C (●, ▲) with 2 mM ATP and the indicated amounts of FK7-7. The samples were then further incubated for 20 min at 30°C in a minichromosome replication system. (■) minichromosome was not included; (♦) DNA synthesis by 1 pmol of ATP-DnaA.
Figure 2.
Figure 2.
Structure and in vitro activity of DARS2. (A) Identification of DARS2. Symbols are the same as those in Figure 1A, with the exception that the numbers above the sequence indicate the positions from the start codon of ygpD. The ADP-releasing activities were assessed using the indicated regions on pACYC177 (5 fmol), as described in Figure 1A (ADP release %), except that a crude extract (2 μg of protein) was included. (B,C) ADP-releasing activity of DARS2 in the absence of a crude protein extract. (B) [3H]ADP-DnaA (2 pmol) was incubated for 15 min at 30°C with 2 mM ATP and the indicated amounts of pACYC177 (Vec; ▲) or pOA54 (●). (C) Similar reactions were performed at 30°C using 40 fmol of each plasmid for the indicated incubation time. (D,E) ADP-releasing activity of DARS2 in the presence of a crude protein extract. (D) [3H]ADP-DnaA (2 pmol) was incubated for 15 min at 0°C (■) or 30°C with the indicated amounts of pACYC177 (Vec; ▵, ▲) or pOA54 (○, ●, ■), and the indicated amounts of crude protein extract (Ext) (0 μg, ○ and ▵; 2 μg, ●, ▲, and ■). (E) Similar experiments were performed by incubating the indicated amounts of crude extract and each plasmid (5 fmol) for 15 min at 30°C. (F) ADP-releasing activity of minimal DARS2. [3H]ADP-DnaA (2 pmol) was incubated for 15 min at 0°C (■) or 30°C in the buffer containing the indicated amounts of pACYC177 (Vec; ■) or pOA61 (○, ●, ■) and the indicated amounts of crude extract (0 μg, ○; or 2 μg, ●, ▲, ■). (G) Reactivation of DnaA in replication initiation activity by DARS2. ADP-DnaA (○, ●) or ATP-DnaA (▵, ▲) (0.4 pmol) was incubated with the indicated amounts of pOA61 in the presence of a crude protein extract (0.4 μg of protein) at 0°C (○, △) or 30°C (●, ▲) (first reaction), followed by the minichromosomal replication assay as described in Figure 1E.
Figure 3.
Figure 3.
DARS1 can reactivate ADP-DnaA resulting from RIDA in vitro. (A) DnaA-ATP hydrolysis by RIDA. [α-32P]ATP-DnaA (1 pmol) was incubated for 20 min at 30°C with the indicated amounts of Hda in the presence (+DNA-clamp) or absence (−DNA-clamp) of the DNA-loaded β sliding clamp (20 fmol of clamp). Nucleotides bound to DnaA were recovered and analyzed as described in the Materials and Methods. (B) Release of DnaA-bound ADP that was produced by RIDA. The RIDA reaction was first performed using [α-32P]ATP-DnaA (1 pmol), Hda (10 ng), and DNA-loaded β clamp (20 fmol of clamp) under the same conditions as in A. Products were further incubated for 15 min at 30°C with the indicated amounts of FK7-7 or FK7-21, followed by determination of DnaA-bound nucleotides. ADP-DnaA constituted 96% of ATP-DnaA/ADP-DnaA after the RIDA reaction. (C) DARS1-driven reactivation of RIDA-produced ADP-DnaA. In the first stage, ATP-DnaA or ADP-DnaA (0.4 pmol) was incubated for 20 min at 0°C or 30°C with Hda (4 ng) in the presence (+) or absence (−) of the DNA-loaded β clamp (8 fmol). In the second stage, the samples that had been incubated at 30°C with (red) or without (blue) the DNA-clamp complexes in the first stage were further incubated for 15 min at 30°C with the indicated amounts of FK7-7 or FK7-21. After the first or second stage, replication activity of DnaA was assessed in a minichromosomal replication system as described in Figure 1E. Incorporation of nucleotides was 6 pmol or 5 pmol in the absence of DnaA or minichromosome, respectively (data not shown).
Figure 4.
Figure 4.
Regeneration of ATP-DnaA and reactivation of DnaA by DARS1 or DARS2 multicopies in vivo. (A) KA474 (dnaN59) cells bearing pACYC177 (None) or pOA21 (DARS1) were incubated in a medium containing [32P]orthophosphate at 28°C until the optical density (A660) reached 0.2, and shifted to 42°C in the presence of chloramphenicol. After incubation for the indicated amount of time, DnaA was immunoprecipitated using anti-DnaA serum, and DnaA-bound nucleotides were analyzed by thin-layer chromatography. Error bars represent the standard deviation from three independent experiments. (pOA21) pACYC177 derivative carrying FK7-7. (B) KA474 (dnaN59) cells bearing pACYC177 (None) or pOA61 (DARS2) were similarly analyzed. (C) Flow cytometry analysis. MG1655 cells bearing pACYC177 (vector), pOA21 (DARS1), pOA76 (DARS1∷ΔDnaA boxes [DARS1∷ΔDBs]), pOA61 (DARS2), or pOA71 (DARS2∷ΔDnaA boxes [DARS2∷ΔDBs]) were grown at 37°C in M9 medium containing ampicillin, followed by incubation for 4 h in the presence of rifampicin and cephalexin. Cell size and DNA content were analyzed by flow cytometry. The numbers inserted in each histogram are relative ratios of mean cell mass to that of cells bearing pACYC177. (pOA76) pACYC177 derivative bearing FK7-21. (D) Relative ratios of the number of origins (ori) per cell mass in cells bearing the indicated plasmids (obtained from C) to those in cells bearing pACYC177. (E) Immunoblot analysis using anti-DnaA antiserum. Cells were grown under the same conditions as those described in C. Portions of the cultures equivalent to the total cell volume in 200 μL at an optical density (A660) of 0.1 were subjected to immunoblot analysis. Relative ratios of DnaA content of cells bearing the indicated plasmids to that of cells carrying pACYC177 are shown. Results are representative of three independent experiments. (F) oriC- and dnaA-dependent colony formation inhibition by DARS2 oversupply. Cells were transformed with pBR322 (Vector), pKX11 (pBR322-DARS2), and pOA77 (pBR322-DARS2∷ΔDnaA boxes [DARS2∷ΔDBs]) and incubated at 30°C (KH5402-1, 22 h; YT411, 26 h; KA451, KA429, and KA450; 36 h) on LB agar plates containing ampicillin. Colonies with a diameter of >1.0 mm were counted. Relative ratios of transformation efficiency by pKX11 and pOA77 to that by pBR322 are shown. Transformation efficiency of the indicated strains by pBR322 was 104 to 107 per microgram of DNA. (Am) Amber mutation; (Δ) del-1017.
Figure 5.
Figure 5.
DARS1 and DARS2 are required for the regulation of cellular ATP-DnaA levels. (A) hda∷cat was transduced into MG1655 (wild type), MIT17 (ΔDARS1∷kan), MIT84 (ΔDARS2∷spec), MIT92 (ΔDARS1∷kan ΔDARS2∷spec), and KW262-5 (rnhA∷Tn3 ΔoriC∷Tn10: oriC-independent replication) by P1 phage, followed by incubation at 37°C for 20 h or 36 h (only for KW262-5) on LB agar plates containing chloramphenicol (20 μg/mL). Colonies with diameters of >1 mm were counted. Frequency of transduction represents the number of colonies per P1 phage particle. (B) KW262-5 (rnhA∷Tn3 ΔoriC∷Tn10), MK86 (rnhA∷Tn3 ΔoriC∷Tn10 Δhda∷cat), MIT47 (rnhA∷Tn3 ΔoriC∷Tn10 Δhda∷cat ΔDARS1∷kan), MIT86 (rnhA∷Tn3 ΔoriC∷Tn10 Δhda∷cat ΔDARS2∷spec), and MIT88 (rnhA∷Tn3 ΔoriC∷Tn10 Δhda∷cat ΔDARS1∷kan ΔDARS2∷spec) were incubated in medium containing [32P]orthophosphate at 37°C until the optical density (A660) reached 0.2. DnaA-bound nucleotides were recovered and determined as in Figure 4A. Error bars represent the standard deviation from three independent experiments.
Figure 6.
Figure 6.
DARS1 and DARS2 are required for the timely initiation of replication. (A) Enhancement of dnaA508 thermosensitivity by deletion of DARS. Cells were grown overnight at 30°C, serially diluted, spotted on LB plate, and incubated for 16 h at 30°C or for 11 h at 37°C, 39°C, or 42°C. (B) Flow cytometry analyses. Cells of MG1655 (wild type), MIT17 (ΔDARS1∷kan), MIT78 (ΔDARS2∷cat), and MIT80 (ΔDARS1∷kan ΔDARS2∷cat) were grown in M9 medium at 30°C, 37°C, or 42°C, followed by run-out replication, as described in Figure 4C. The numbers inserted in each histogram are ratios of mean cell mass to that of wild-type cells at each temperature. (C) Relative ratios of the number of origins (ori) per cell mass in the indicated cells (from B) to that of wild-type cells at the indicated temperatures.
Figure 7.
Figure 7.
Formation of highly ordered complexes on DARS1 is associated with the AAA+ domain and bound nucleotide. (A,B) Analysis of DARS1-dependent ADP-release activity of DnaA mutants. [3H]ADP-bound wild-type or mutant DnaA proteins (2 pmol) were incubated for 15 min at 30°C with 2 mM ATP and the indicated amounts (A) or 50 fmol (B) of FK7-7. In A, wild-type (●) and mutant DnaA (R399A, ▲; T435M, ■) proteins were assessed. In B, the amount of ADP released in a FK7-7-dependent manner is shown as a percentage of the total starting amount of DnaA-bound ADP. (III–IV) Truncated DnaA carrying only domain III (AAA+) and IV. The other DnaA proteins are full length, with amino acid substitutions in the AAA+ motifs indicated. (C) Complexes consisting of DARS1 and wild-type DnaA or DnaA D269N. Various amounts (0, 60, 150, 300, 600, and 1000 fmol) of wild-type DnaA (WT) or DnaA D269N (D269N) were incubated with 32P-labeled FK7-7 (30 fmol) and 2 mM ADP, followed by EMSA. (C I–IV) Complexes I–IV; (Free) protein-free FK7-7. Asterisks indicate the lanes scanned for D. (D) Intensities in the lanes indicated by asterisks in C were determined by densitometric scanning and are presented in arbitrary units. (Black line) Wild-type DnaA; (gray line) DnaA D269N. Migration positions for free DNA (Free) and complexes I–IV (C I–IV) are indicated. (E) Complexes consisting of DARS1 and ADP-DnaA or apo-DnaA. Various amounts (0, 60, 150, 300, 600, and 1000 fmol) of ADP-DnaA (ADP-form) or apo-DnaA (Apo) were incubated for 15 min on ice with 32P-labeled FK7-7 (30 fmol) in the presence or absence of 2 mM ADP, followed by EMSA. Asterisks indicate the lanes scanned for F. (F) Intensities in the lanes indicated by asterisks in E were determined and are presented similarly to D. (Black line) ADP-form; (gray line) Apo. Migration positions for free DNA (Free) and complexes I–IV (C I–IV) are indicated.
Figure 8.
Figure 8.
Model for the DnaA cycle in regulation of the replicational initiation. A considerable amount of ATP-DnaA is regenerated from ADP-DnaA by DARSs in a cell cycle-coordinated or constitutive manner, leading to replicational initiation. After this, ATP-DnaA is converted to ADP-DnaA by RIDA. See the text for details.
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