Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2012 Oct 26;287(44):37458-71.
doi: 10.1074/jbc.M112.372052. Epub 2012 Aug 31.

Differentiation of the DnaA-oriC subcomplex for DNA unwinding in a replication initiation complex

Shogo Ozaki  1 , Yasunori Noguchi, Yasuhisa Hayashi, Erika Miyazaki, Tsutomu Katayama
Affiliations

Differentiation of the DnaA-oriC subcomplex for DNA unwinding in a replication initiation complex

Shogo Ozaki et al. J Biol Chem. .

Abstract

In Escherichia coli, ATP-DnaA multimers formed on the replication origin oriC promote duplex unwinding, which leads to helicase loading. Based on a detailed functional analysis of the oriC sequence motifs, we previously proposed that the left half of oriC forms an ATP-DnaA subcomplex competent for oriC unwinding, whereas the right half of oriC forms a distinct ATP-DnaA subcomplex that facilitates helicase loading. However, the molecular basis for the functional difference between these ATP-DnaA subcomplexes remains unclear. By analyzing a series of novel DnaA mutants, we found that structurally distinct DnaA multimers form on each half of oriC. DnaA AAA+ domain residues Arg-227 and Leu-290 are specifically required for oriC unwinding. Notably, these residues are required for the ATP-DnaA-specific structure of DnaA multimers in complex with the left half of oriC but not for that with the right half. These results support the idea that the ATP-DnaA multimers formed on oriC are not uniform and that they can adopt different conformations. Based on a structural model, we propose that Arg-227 and Leu-290 play a crucial role in inter-ATP-DnaA interaction and are a prerequisite for the formation of unwinding-competent DnaA subcomplexes on the left half of oriC. These residues are not required for the interaction with DnaB, nucleotide binding, or regulatory DnaA-ATP hydrolysis, which further supports their important role in inter-DnaA interaction. The corresponding residues are evolutionarily conserved and are required for unwinding in the initial complexes of Thermotoga maritima, an ancient hyperthermophile. Therefore, our findings suggest a novel and common mechanism for ATP-DnaA-dependent activation of initial complexes.

PubMed Disclaimer

Figures

FIGURE 1.
FIGURE 1.
Model of an open complex and basic structures of E. coli oriC and DnaA. E. coli oriC consists of DUE, which has AT-rich repeats, and DAR, which carries an IHF binding site (light gray) and multiple DnaA-binding sites (i.e. R, I, τ, and C sites; open or dark gray symbols). The arrowheads indicate the orientation of R, I, and C sites. The highest affinity sites, R1 and R4, are indicated by open symbols. DAR can be divided into four functional regions (i.e. DF, LL, RL, and RE). E. coli DnaA domains (I, II, III, and IV) and IHF are also shown in different colors. The ATP-DnaA multimer formed on DAR binds ssDUE (heavy lines). The binding of ssDUE to the ATP-DnaA multimer formed on LL region is stimulated by IHF (yellow) and the DF (DnaA box R1)-bound ATP-DnaA. This stimulation is probably caused by the sharp DNA bending induced by IHF and the interaction between the DF-bound ATP-DnaA and the LL-bound ATP-DnaA multimer (i.e. ssDUE recruitment), as illustrated in Ref. .
FIGURE 2.
FIGURE 2.
Homology modeling and purification of mutant tmaDnaA proteins. A, the monomeric tmaDnaA AAA+ domain structures (Protein Data Bank entry 2Z4S) were superimposed onto an oligomeric structure of the A. aeolicus DnaA ortholog (Protein Data Bank entry 2HCB). Only two tmaDnaA protomers are shown for simplicity using ribbons and a schematic representation. Each protomer is colored differently. The residues investigated in this study are represented by a ball-and-stick model. B, possible interactions (dashed lines) between two tmaDnaA protomers are indicated using a linear map of the tmaDnaA AAA+ domain. Representative motifs of AAA+ domain and the residues investigated in this study are indicated. Corresponding E. coli DnaA residues are indicated in parentheses. WA, Walker A; IV, box IV, WB, Walker B; VII, box VII; SI, sensor 1; VIII, box VIII. C, sequence homology analysis. Protein sequences of ∼1000 DnaA orthologs were obtained from the Comprehensive Microbial Resource and aligned using the ClustalW program (available from the Max-Planck Institute for Developmental Biology Web site). The number of DnaA residues with the same chemical property (hydrophobic, acidic, or basic) at the position corresponding to the tmaDnaA Phe-115, Glu-169, Arg-192, Phe-205, Leu-256, or Lys-308, respectively, was indicated as a percentage (Conservation (%)). D, mutant tmaDnaA proteins were purified, and each protein (0.4 μg) in the final fraction was analyzed by SDS-12% PAGE and Coomassie Brilliant Blue staining.
FIGURE 3.
FIGURE 3.
tmaDnaA mutants defective in open complex formation. The WT or mutant tmaDnaA protein (F115A, E169A, R192A, F205A, L256A, and K308A) (1.5 μm) was preincubated with 3 μm ATP or ADP. The resultant ATP form (ATP) or ADP form (ADP) of tmaDnaA (0–300 nm) was incubated at 48 °C for 10 min in buffer containing the supercoiled form of the tma-oriC plasmid, pOZ14 (200 fmol or 4 nm), followed by digestion with P1 nuclease (10 units, 50 s at 48 °C). DNA products were purified and digested with AlwNI, followed by analysis using a 1% agarose gel and ethidium bromide staining. The gel images are shown (A–C). The number of P1 nuclease-digested oriC DNA molecules per molecule of input DNA is shown as a percentage (DUE unwinding (%)) (D–F).
FIGURE 4.
FIGURE 4.
Analysis of E. coli DnaA R227A and L290A in DUE unwinding and minichromosome replication. A and B, DUE unwinding assay. The WT or mutant DnaA protein (R227A and L290A) (1.5 μm) was preincubated with 3 μm ATP or ADP. The resultant ATP form (ATP) or ADP form (ADP) of DnaA was incubated at 38 °C for 3 min in buffer containing the supercoiled form of pBSoriC and IHF, followed by digestion with P1 nuclease. The number of P1 nuclease-digested oriC DNA molecules per molecule of input DNA was analyzed by 1% agarose gel electrophoresis and ethidium bromide staining (A) and is shown as a percentage (DUE unwinding (%)) (B). C, minichromosome replication assay. The ATP form (ATP) or ADP form (ADP) of WT or mutant DnaA protein (R227A and L290A) was incubated at 30 °C for 10 min in buffer containing the supercoiled form of an oriC plasmid M13KEW101 and purified replication proteins (29).
FIGURE 5.
FIGURE 5.
ssDUE binding analysis of E. coli DnaA R227A and L290A. A, schematic of the ssDUE binding assay. ATP-DnaA was incubated with DAR and end-labeled ss-dsDUE, followed by EMSA. For simplicity, only the DnaA domains III and IV are illustrated by blue and pink balls, respectively. The DnaA B/H motifs within domain III are indicated by small red balls. DARΔR1 (green bar) and 32P-labeled ssDUE (black line) conjugated to dsDNA bearing DnaA-binding site R1 (blue box) (ssDUE-dsR1) are also indicated. ss-dsDUE, ssDUE-dsR1, ssDUE-dsNon, or both. B and C, WT ATP-DnaA, ATP-DnaA R227A, or ATP-DnaA L290A (0–60 nm) was incubated with 5 nm DARDR1 and a 2.5 nm concentration of either 32P-labeled ssDUE-dsR1 or ssDUE-dsNon, followed by EMSA. ss-dsDUE, ssDUE-dsR1 or ssDUE-dsNon. ssDUE-dsNon is the same as ssDUE-dsR1, except that the R1 site is replaced with a sequence with no affinity for DnaA. Radioactivity of the resultant gel was visualized (B). The amount of ss-dsDUE-DnaA-DAR complexes was quantified, and the amount of the complexes relative to the input ss-dsDUE was plotted as ss-dsDUE-DnaA-DAR complex formation (%) (C). Certain amounts of the complex were unstable during electrophoresis, yielding smeared DNA bands with moderate retardation in mobility. Well, gel well; Free ss-dsDUE, protein-free ss-dsDUE.
FIGURE 6.
FIGURE 6.
DNase I footprint analysis. Various concentrations (0–600 nm) of WT and mutant DnaA proteins were incubated for 10 min at 30 °C in buffer containing 32P-labeled oriC DNA (419 bp) and 3 mm ATP or ADP, followed by digestion with DNase I. The resultant DNA fragments were analyzed on a 5% sequencing gel and visualized using a BAS2500 imager. The positions of the oriC subregions (DF, LL, RL, and RE), DnaA-binding sequences (R1–R5, I1–I3, τ1 and τ2, and C1–C3), and DUE are indicated. A schematic of the minimum oriC region is illustrated above the gel image. Asterisks indicate bands that are quantified in intensity for Fig. 7.
FIGURE 7.
FIGURE 7.
Analysis of band intensities in DNase I footprint data. The intensities of the representative bands indicated by asterisks in Fig. 6 were quantified in each lane. The intensities obtained in the absence of DnaA were referred to as 1.0, and the relative values at 200, 400, and 600 nm DnaA are presented. A, LL region. B, RL-RE region.
FIGURE 8.
FIGURE 8.
Analysis of the DnaA complexes formed on the DF-LL and RL-RE regions. The DF-LL DNA (A and B) or the RL-RE DNA (C and D) (350 fmol or 35 nm) were incubated at 30 °C for 10 min in buffer (10 μl) containing the indicated amounts of WT or mutant ATP-DnaA and analyzed by 2% agarose gel electrophoresis and Gel-Star staining. Representative gel images are shown in a black and white inverted mode (A and C). The molar ratio of input DnaA/DNA fragment is indicated below each lane. Each lane was analyzed by densitometric scanning, and proportions of DNA complexed with DnaA multimers (DnaA-DNA complex) to the total DNA were quantified and shown as a percentage (Complex formation (%)) (B and D). Free DNA, protein-free DNA.
FIGURE 9.
FIGURE 9.
Activity in DnaB binding and RIDA. A–C, the oriC pull-down assay. A schematic of the assay is shown using the same symbols as in Fig. 1 in addition to symbols for the DnaB (green)-DnaC (dark blue) complex (A). WT ATP-DnaA, ATP-DnaA R227A, or ATP-DnaA L290A (5 pmol or 500 nm) and the bio-oriC DNA (419 bp, 100 fmol or 10 nm) were incubated on ice for 10 min in buffer (10 μl) containing various amounts (0, 1.3, 2.5, and 5 pmol) of His-DnaB-DnaC protein mixtures, followed by the pull-down assay, using streptavidin-coated magnetic beads. The bio-oriC-bound materials were analyzed by SDS-10% PAGE and silver staining (B). The recovered amounts of DnaA, HisDnaB, and DnaC in A were determined using standard curves of purified proteins. The recovered amounts of bio-oriC were deduced as described under “Experimental Procedures.” These values were used to deduce the number of each protein recovered per bio-oriC (C). D, RIDA activity. WT DnaA (circle), DnaA R227A (R227A; triangle), or DnaA L290A (L290A; square) (500 nm) was preincubated on ice for 15 min with [α-32P]ATP (0.8 μm), and portions of ATP-bound protein (0.5 pmol or 20 nm, final) were incubated at 30 °C for 20 min in buffer containing the indicated amounts of ADP-Hda and ADP (30 μm) in the presence (open symbols) or absence (closed symbols) of the DNA-loaded clamps (50 fmol or 2 nm as the clamp), followed by filter retention and thin layer chromatography. The ratio of ADP-DnaA to total ATP-/ADP-DnaA is shown as a percentage (ATP hydrolysis).
FIGURE 10.
FIGURE 10.
Model for open complex architecture activated for ssDUE binding. A model for an open complex including four distinct ATP-DnaA-DAR subcomplexes is illustrated using the same symbols as in Fig. 1A. Unlike other ATP-DnaA-DAR subcomplexes (b), the conformation of the ATP-DnaA-LL subcomplex is activated by inter-ATP-DnaA interactions mediated by the AID-1 and -2 motifs (a), which exposes the B/H motifs of the ATP-DnaA-LL subcomplex onto the pore surface, thereby increasing affinity for ssDUE. Plausible modes of inter-ATP-DnaA interactions are illustrated schematically using only the DnaA AAA+ domain for simplicity. See “Discussion” for details.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Bramhill D., Kornberg A. (1988) Duplex opening by dnaA protein at novel sequences in initiation of replication at the origin of the E. coli chromosome. Cell 52, 743–755 - PubMed
    1. Messer W. (2002) The bacterial replication initiator DnaA. DnaA and oriC, the bacterial mode to initiate DNA replication. FEMS Microbiol. Rev. 26, 355–374 - PubMed
    1. Duderstadt K. E., Berger J. M. (2008) AAA+ ATPases in the initiation of DNA replication. Crit. Rev. Biochem. Mol. Biol. 43, 163–187 - PubMed
    1. Ozaki S., Katayama T. (2009) DnaA structure, function, and dynamics in the initiation at the chromosomal origin. Plasmid 62, 71–82 - PubMed
    1. Leonard A. C., Grimwade J. E. (2010) Regulating DnaA complex assembly. It is time to fill the gaps. Curr. Opin. Microbiol. 13, 766–772 - PMC - PubMed

Publication types

MeSH terms

LinkOut - more resources