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. 2012 Feb;40(4):1648-65.
doi: 10.1093/nar/gkr832. Epub 2011 Nov 3.

Highly Organized DnaA-oriC Complexes Recruit the Single-Stranded DNA for Replication Initiation

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Highly Organized DnaA-oriC Complexes Recruit the Single-Stranded DNA for Replication Initiation

Shogo Ozaki et al. Nucleic Acids Res. .
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Abstract

In Escherichia coli, the replication origin oriC consists of two functional regions: the duplex unwinding element (DUE) and its flanking DnaA-assembly region (DAR). ATP-DnaA molecules multimerize on DAR, unwinding DUE for DnaB helicase loading. However, DUE-unwinding mechanisms and functional structures in DnaA-oriC complexes supporting those remain unclear. Here, using various in vitro reconstituted systems, we identify functionally distinct DnaA sub-complexes formed on DAR and reveal novel mechanisms in DUE unwinding. The DUE-flanking left-half DAR carrying high-affinity DnaA box R1 and the ATP-DnaA-preferential DnaA box R5, τ1-2 and I1-2 sites formed a DnaA sub-complex competent in DUE unwinding and ssDUE binding, thereby supporting basal DnaB loading activity. This sub-complex is further subdivided into two; the DUE-distal DnaA sub-complex formed on the ATP-DnaA-preferential sites binds ssDUE. Notably, the DUE-flanking, DnaA box R1-DnaA sub-complex recruits DUE to the DUE-distal DnaA sub-complex in concert with a DNA-bending nucleoid protein IHF, thereby promoting DUE unwinding and binding of ssDUE. The right-half DAR-DnaA sub-complex stimulated DnaB loading, consistent with in vivo analyses. Similar features are seen in DUE unwinding of the hyperthermophile, Thermotoga maritima, indicating evolutional conservation of those mechanisms.

Figures

Figure 1.
Figure 1.
Proposed E. coli oriC sub-structures and determination of the minimum region required for DUE unwinding in the plasmid oriC. (A) The overall structure of oriC and its sub-structures proposed in this study are shown at the top of the panel. The AT-rich motifs within the DUE, IHF-binding site and Fis-binding site are indicated by black, yellow and orange bars, respectively. DnaA binding motifs are indicated by arrowheads (blue for high-affinity sites R1 and R4 and red for low-affinity sites). The proposed DAR substructures are indicated above of the overall structure. The oriC regions used for deletion analysis (open bars) are shown below the oriC structure. The supercoiled form of pBSoriC, or its derivatives (3.4 nM), ATP-DnaA (0–60 nM) and IHF (55 nM) were used for the P1 nuclease assay. The unwinding activity at 60 nM ATP–DnaA is shown below the heading ‘dsDUE unwinding’. +, wild-type level; –, inactive. (B–D) The number of P1 nuclease-digested oriC DNA molecules per that of input DNA was analyzed by 1% agarose gel electrophoresis and ethidium bromide staining and shown as a percentage [dsDUE unwinding (%)] (B and C). The gel images are shown in panel D and Supplementary Figure S1.
Figure 2.
Figure 2.
ssDUE digestion assay using oriC fragments for analysis of DnaA–DAR complexes. (A) The plasmid, pOZoriCEC3, was digested using HincII and EcoRI, yielding DNA fragments of vector DNA (vector) and ori-EcoRI (EC3-WT). These fragments were end-labeled and incubated with ATP–DnaA and IHF, followed by analysis using P1 nuclease and polyacrylamide gel electrophoresis. The postulated structure of the DnaA multimers complexed with ori-EcoRI is also illustrated. An alternative structural model is also conceivable but it is not shown here for the simplicity (see ‘Discussion’ section). The DnaA domains III and IV and IHF are indicated by orange, pink and yellow balls, respectively. The DnaA B/H-motifs within domain III are indicated by small red balls. For simplicity, DnaA domains I–II are not shown. The DUE region is indicated by thick black bars. (B and C) The P1 nuclease sensitivity of the ori-EcoRI (EC3-WT) (1.3 nM) was analyzed in the presence of IHF (55 nM) and the indicated concentrations of either ATP–DnaA (ATP) or ADP–DnaA (ADP). Reaction mixtures were incubated for 5 min at 38°C, followed by incubation with P1 nuclease and polyacrylamide gel electrophoresis. A polyacrylamide gel containing the reaction products is shown (B). The ori-EcoRI (EC3-WT) and the vector remained were quantified, and the relative numbers of the remaining molecules of ori-EcoRI to that of the vector were deduced. The number obtained in the absence of DnaA was defined as 100% and the relative numbers are plotted as DnaA-dependent digestion (C). (D) The P1 nuclease sensitivity of ori-EcoRI (EC3-WT) was analyzed as above using 80 nM of wild-type ATP–DnaA (WT), ATP–DnaA V211A (V211A) or ATP–DnaA R245A (R245A) in the presence of IHF (55 nM). (E and F) The mutant derivatives of pOZoriEC3 were constructed and digested with EcoRI and HincII. The resultant ori-EcoRI derivatives are shown using black and gray bars, which indicate regions bearing wild-type sequences and base substitutions, respectively (E). In (F), only the DNA constructs between the DUE M and DnaA box R1 of ori-EcoRI derivatives are shown. The wild-type sequences and base substitutions are indicated by upper- and lower case, respectively. In (E) and (F), those fragments (1.3 nM) and vector fragments were incubated as above in the presence of IHF (55 nM) and ATP–DnaA (80 nM), followed by incubation with P1 nuclease. Relative levels in DnaA-dependent digestion of EC3-WT derivatives were deduced as above (see Supplementary Figure S2 for details). (G) EC3-WT (WT) and EC3-ibs (ibs) were incubated as above with the indicated concentrations of IHF in the presence (+) or absence (−) of ATP–DnaA (80 nM), followed by incubation with P1 nuclease. The relative molecular number of remaining ori-EcoRI was quantified as above, and that obtained in the absence of both DnaA and IHF was defined as 100%. This was used to deduce the relative levels of IHF-dependent digestion (shown as percentages, ‘IHF-dependent digestion’).
Figure 3.
Figure 3.
EMSA for ssDUE-binding activity of ATP–DnaA–DAR complexes. (A) Schematic of the assay. ATP–DnaA was incubated with DAR and end-labeled ssDUE, followed by EMSA. The symbols for DnaA are the same as those used in Figure 2A. 32P-labeled M28 DNA (32P-ssDUE), back bar; DAR derivatives, green bar. An alternative structural model is also conceivable but it is not shown here for the simplicity (see ‘Discussion’ section). (B–F) The motifs within DAR are shown using the same symbols as in Figure 1. (B) ATP–DnaA (0–60 nM) and 32P-ssDUE (2.5 nM) were incubated in the presence or absence (None) of the indicated DAR derivatives (5 nM), followed by EMSA. Representative gel images are shown (C), where – and free ssDUE indicate no DnaA and protein-free ssDUE, respectively. The amounts of ssDUE–ATP–DnaA–DAR complexes (Complex) were quantified using the data shown in (C) and Supplementary Figure S3 and the relative amounts of the complexes to the input ssDUE were plotted as ssDUE binding (%) (D–F). The values obtained using 60 nM ATP–DnaA are shown in (B) (‘ssDUE binding’). Accordingly, the relative activity levels are highlighted using differently grayed bars to indicate the DAR derivatives (closed, 100–80% of the wild-type level; shaded, 50–30% of the wild-type level; open, <25% of the wild-type level) (B).
Figure 4.
Figure 4.
ssDUE recruitment activity by DnaA box R1 residing in cis to the DUE. (A) A schematic of the assay. Symbols are the same as those used in Figure 3A, except that 32P-labeled ssDUE-R1 and ssDUE conjugated to dsDNA bearing DnaA box R1 (32P-ss-dsDNA) are indicated. An alternative structural model is also conceivable but it is not shown here for the simplicity (see ‘Discussion’ section). (B–E) DAR derivatives, ssDUE-R1 and ssDUE-non are illustrated schematically (R1, R1 box; N, non-sense box) (B). DAR derivatives (5 nM) and ATP–DnaA (0–60 nM) were incubated with 2.5 nM of either 32P-ssDUE-non (C) or ssDUE-R1 (D), followed by EMSA. Representative gel images are shown (E). The amounts of ss-dsDNA–ATP–DnaA–DAR complexes (Complex) were quantified using the data shown in (E) and Supplementary Figure S4 and the relative amounts of the complexes to the input ss-dsDNA were plotted as ss-dsDNA binding (%) (C and D). The values obtained using 60 nM ATP–DnaA are shown in (B) as ss-dsDNA binding. The relative activity levels are highlighted using differently grayed bars to indicate the DAR derivatives (closed, 100–60% of the wild-type level; open, <40% of the wild-type) (B). The ratios of the binding activities of ssDUE-R1 to ssDUE-non (R1/non) are also indicated.
Figure 5.
Figure 5.
DnaB loading activity of ATP-DnaA complexes formed on the truncated DAR. (A and B) DnaB loading activity was assessed by the form I* assay. The oriC derivatives used in this assay are shown schematically (A). The form I* assay was performed by incubating pBS-WT, or its derivatives (1.6 nM) at 30°C for 15 min in the presence of ATP–DnaA (20 nM) and 0–26 nM of DnaB–DnaC complexes. Gel images and the DNA migration positions of the supercoiled form (I), form I* (I*), the open circular form (II) and linear form (linear) are indicated (B). The relative amounts of form I* and total DNA were quantified and the relative amount of form I* DNA to total DNA was deduced using data obtained using 26 nM DnaBC. The resultant value for the wild-type DNA (pBS-WT) was defined as 1.0 and the relative values for the pBS-WT derivatives are shown in (A) (DnaB loading). Accordingly, the relative activity levels are indicated by grayed bars for the derivatives. (closed, wild-type; shaded, 45–60% of the wild-type level; open, <30% of the wild-type). (C–E) DnaB-binding activity to DnaA–oriC complexes was assessed by a pull-down assay. The biotinylated oriC derivatives used in this pull-down assay are shown schematically (C). ATP–DnaA (1 µM, 10 pmol), HisDnaB (0.5 µM) and DnaC (0.5 µM) were incubated in the presence of the biotinylated oriC derivatives (10 nM), followed by the pull-down assay. Proteins bound to DNA were analyzed by SDS-11% PAGE and silver staining (D). The recovered amounts of proteins (DnaA, DnaB and DnaC) in (D) were determined using standard curves and the values from the negative control (biovector) were subtracted. The recovered amounts of DNA were deduced as described in ‘Materials and Methods’ section. The relative amount of recovered DnaB for the bioWT recovered was defined as 1.0 and those for the bioWT derivatives recovered were deduced and shown as DnaB binding (C). Accordingly, the relative activity levels for the derivatives are indicated by grayed bars as above. Also, the mean numbers and their standard deviations for the recovered protein molecules per the recovered DNA were indicated (E).
Figure 6.
Figure 6.
Specific roles for DnaA boxes in T. maritima oriC. (A) DNase I footprint. ATP– or ADP–tmaDnaA (0–800 nM) and 32P-labeled tma-oriC fragments (10 nM) were incubated with DNase I, followed by sequencing gel analysis. Strongly or weakly protected sites by ATP–tmaDnaA are indicated by closed or open rectangles, respectively. The positions of the DUE (AT-2 and AT-3) and tmaDnaA boxes 1–5 are indicated. (B) EMSA using tmaDnaA, tmaDAR and tma-ssDUE. Various amounts of the wild-type ATP–tmaDnaA (WT) or ATP-forms of tmaDnaA V176A or K209A were incubated with 10 nM of DUE-deleted tma-oriC (tmaDAR WT) DNA, followed by incubation with 2 nM of 32P -labeled tma-ssDUE and electrophoresis on 4% polyacrylamide gels. The relative amounts in the DUE derivatives bound to the tmaDnaA–tmaDAR complexes to those input were deduced as described in Figure 3 and plotted as ‘DUE-bound complex (%)’. For tmaDAR WT, see below. (C) The structures of the DUE derivatives used for EMSA. The DUE derivatives used carry the ssDUE or dsDUE with, or without (Δ), tmaDnaA box1 (WT) or a non-sense box (mutant). Upper-strand, black bar; lower strand, light gray bar; tmaDnaA box 1, open box; non-sense box, shaded box; DUE AT-2 and 3, closed box. (D–F) EMSA using tmaDnaA and derivatives of tmaDAR and tmaDUE. Black and shaded bars indicate DAR wild-type sequences and base substitutions, respectively (D). The DUE derivatives shown in (C) were analyzed as above in the presence of various amounts of wild-type ATP– tmaDnaA and tmaDAR-delA (E) or its derivatives (F). The relative activities for DUE–tmaDnaA–DAR complex formation were deduced as above and plotted. The relative activities (++, >50%; +, 20–50%; −, <20%) for DUE–tmaDnaA–DAR complex formation at 80 nM ATP–tmaDnaA are indicated from experiments using the indicated DAR and DUE derivatives (D). See Supplementary Figure S5 for details. (G) Sequence comparison of an oriC region carrying the DUE and the flanking DnaA box. The arrowed boxes indicate the position and the orientation of the cognate DnaA box flanking the DUE. The unwinding motifs determined in vitro using potassium permanganate modification (dark gray) or P1 nuclease digestion (light gray) are indicated (40–42). Bold lines indicate the number of nucleotides (Length) from the center of the DnaA box to the unwinding motif.
Figure 7.
Figure 7.
Model for oriC unwinding, ssDUE recruitment and helicase loading. (A) Summary figure. Black and gray bars indicate sites bearing predominant and supportive roles, respectively. (B) Model for open complex. The DnaA domains (I, III and IV) and oriC domains (DUE, DF, LL, RL and RE) are distinguished using different colors. The DnaA multimer formed on DAR binds ssDUE. SsDUE is recruited by IHF-dependent DNA bending and interaction of DnaA molecules bound to the DF and LL regions. See text for details. (C) Alternative model for open complex. The DnaA multimer formed on ssDUE interacts with the DnaA multimer formed on DAR. The DnaA domain III within the DnaA–ssDUE complex are colored in gray. Only the DUE-LL regions are shown and the DnaA domains I and II are omitted for the simplicity. (D) Model for helicase loading. DnaA domain I which has a crucial binding site to DnaB is connected to domain II which is a flexible linker to domain III, the AAA+ domain (19). These structural features would support flexible interaction modes and conformational change in oriC–DnaA–DnaB–DnaC complexes. DnaC is omitted in this figure for the simplicity. Similar mechanisms would be possible by the alternative model (C). See text and Supplementary Figure S6 for details.

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