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. 2011 Oct;82(2):475-88.
doi: 10.1111/j.1365-2958.2011.07827.x. Epub 2011 Sep 14.

Two Oppositely Oriented Arrays of Low-Affinity Recognition Sites in oriC Guide Progressive Binding of DnaA During Escherichia Coli pre-RC Assembly

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Free PMC article

Two Oppositely Oriented Arrays of Low-Affinity Recognition Sites in oriC Guide Progressive Binding of DnaA During Escherichia Coli pre-RC Assembly

Tania A Rozgaja et al. Mol Microbiol. .
Free PMC article

Abstract

The onset of chromosomal DNA replication requires highly precise and reproducible interactions between initiator proteins and replication origins to assemble a pre-replicative complex (pre-RC) that unwinds the DNA duplex. In bacteria, initiator protein DnaA, bound to specific high- and low-affinity recognition sites within the unique oriC locus, comprises the pre-RC, but how complex assembly is choreographed to ensure precise initiation timing during the cell cycle is not well understood. In this study, we present evidence that higher-order DnaA structures are formed at oriC when DnaA monomers are closely positioned on the same face of the DNA helix by interaction with two oppositely oriented essential arrays of closely spaced low-affinity DnaA binding sites. As DnaA levels increase, peripheral high-affinity anchor sites begin cooperative loading of the arrays, which is extended by sequential binding of additional DnaA monomers resulting in growth of the complexes towards the centre of oriC. We suggest that this polarized assembly of unique DnaA oligomers within oriC plays an important role in mediating pre-RC activity and may be a feature found in all bacterial replication origins.

Figures

Fig. 1
Fig. 1
Arrays of DnaA contacts exist in each half of oriC. (A) Map of oriC with positions of DnaA, IHF, and Fis binding sites, as well as the DNA unwinding element (DUE), marked. The three high affinity sites are designated by large black squares, and the low affinity sites are marked by small black rectangles. Horizontal arrows indicate orientation of sites. C1, C2, and C3, and vertical arrows mark regions of unmapped DnaA contacts between R4 and R2. (B) In vitro DMS modification patterns of oriC, after incubating oriC plasmids with the indicated concentrations of DnaA prior to treatment with DMS. Two different primers were used to analyze modifications between R1 and R2 (left panel) and between R2 and R4 (right panel). Binding site positions are marked, with their orientations indicated by vertical arrow. Guanosine residues in position 2 or 4 are labeled, with up or down arrows indicating enhanced or diminished DMS sensitivity, respectively. (C) In vivo DMS modification patterns of oriC on purified chromosomal DNA, or DNA isolated from cells aligned at the stage of initiation just prior to helicase loading, measured by LMPCR. Guanosine residues in position 2 or 4 are labeled, with up or down arrows indicating enhanced or diminished DMS sensitivity, respectively. Filled circles indicate changes in the modification pattern in the Fis binding region. (D) Relative intensities of DMS modification at guanosine residues within DnaA binding sites.
Fig. 2
Fig. 2
Sequence of all DnaA interaction sites in oriC, based on placing hypersensitive G residues in position 4, and suppressed Gs in position 2.
Fig. 3
Fig. 3
DnaA contacts at specific low affinity 9mer sites are detected by EMSA. Double-stranded DNA oligonucleotides containing R4 and R2 boxes flanking the putative low affinity binding site C1 (A); C2 (B); or C3 (C) were incubated with DnaA-ATP at DnaA/DNA molar ratios of 0:1, 0.5:1, 1:1, 2:1, 5:1, 10:1, 20:1, and 50:1, and the resulting complexes were resolved on polyacrylamide gels. The position of the unbound probe, and complexes resulting from 1, 2 or 3 molecules of DnaA bound to the probe are marked. The last lane of each panel shows complexes formed by incubating DnaA-ATP (50:1 DnaA/DNA) with an equivalent probe, in which the putative site is scrambled (see text).
Fig. 4
Fig. 4
Cooperative binding between low affinity sites is dependent on site orientation, while strong sites in either orientation can donate DnaA. The EMSA system described in the text was used to examine DnaA-ATP binding to double-stranded DNA oligonucleotide probes containing R4 flanking two low affinity sites (C1 and R5M) in varying orientations, shown by the drawing below each panel (A–D). End-labeled probes were incubated with DnaA-ATP at DnaA/DNA molar ratios of 0:1, 20:1, and 50:1, and the resulting complexes were resolved on polyacrylamide gels.
Fig. 5
Fig. 5
Extension of DnaA requires precise spacing between low affinity sites. Double-stranded oligonucleotide probes containing R4, C1, and R5M were designed so that C1 was separated from R5M by 1bp (A), 2bp (B), 3 bp (C), or 4 bp (D). All sites were in the same orientation. The configuration of the probes is indicated below each panel. End-labeled probes were incubated with DnaA-ATP at DnaA/DNA molar ratios of 0:1, 20:1, and 50:1, and the resulting complexes (1, 2, and 3) were resolved on polyacrylamide gels.
Fig. 6
Fig. 6
Cooperative binding between high and low affinity sites can extend over a range of bases. Double-stranded oligonucleotide probes containing R4 and R5M were designed so that the sites were separated by 1bp, 2bp, 3 bp, or 5 bp, or 7 bp. All sites were in the same orientation. The configuration of the probes is indicated below each panel. End-labeled probes were incubated with DnaA-ATP at DnaA/DNA molar ratios of 0:1, 20:1, and 50:1, and the resulting complexes (1, 2, and 3) were resolved on polyacrylamide gels.
Fig. 7
Fig. 7
DnaA extends from high affinity anchor sites in a preferred direction. Wild-type oriC plasmids, or oriC plasmids that are mutated in one of the DnaA contact sites at the end of an array were incubated with the indicated concentrations of DnaA-ATP and treated with DMS. Binding sites are marked. (A) DMS modified nucleotide positions in the array between R1 and R2 are shown for wt oriC (left panel); scramble (scr) mutation in R5M (middle panel); scr mutation in I2 (right panel). (B) DMS modified nucleotide positions in the array between R2 and R4 are shown for wt oriC (left panel); scr C1 mutation (middle panel); scr C3 mutation (right panel). Graphs of relative intensities (arbitrary units) of the DMS modified G4 in each arrayed site (marked by arrows in gel scans) are shown on the right of each set of panels.
Fig. 8
Fig. 8
Model of pre-RC assembly based on a revised oriC map. In the original (1980s era) map of oriC (A), five R boxes were identified, with the remaining regions as “spacer sequences” without defined function. High affinity R boxes are shown in yellow, and low affinity boxes are blue. In panel B, a revised map of oriC is presented, with a model of pre-RC assembly. In the first stage of pre-RC assembly (top, showing the bacterial ORC), DnaA occupies the three high affinity sites, and Fis is bound. It is not known if these DnaA molecules must be in a particular nucleotide form. In the second stage, R1 and R4 act as nucleation sites for a DnaA oligomer which is extended by sequential binding of protomers to arrayed sites, toward R2 (shown by arrows). Some sites must bind DnaA-ATP (marked in blue). Oligomer extension displaces Fis (red rectangle), and IHF (green rectangle) binds in this stage, modulating the distance between strong and weak sites. The negative regulator SeqA (red pentagon) prevents oligomer extension and unwinding by blocking sites containing a GATC, and the positive regulator DiaA (green circle) is proposed to stabilize the DnaA interactions that are required to extend DnaA from the nucleation sites, and to connect the two converging oligomers at R2. After arrays are filled and joined, a compact helical filament of DnaA-ATP extends from R1 into the DUE, and DiaA may also stabilize this extension.

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