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. 2009 Nov 3;106(44):18479-84.
doi: 10.1073/pnas.0909472106. Epub 2009 Oct 15.

Bacterial Origin Recognition Complexes Direct Assembly of Higher-Order DnaA Oligomeric Structures

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Free PMC article

Bacterial Origin Recognition Complexes Direct Assembly of Higher-Order DnaA Oligomeric Structures

Diana T Miller et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Eukaryotic initiator proteins form origin recognition complexes (ORCs) that bind to replication origins during most of the cell cycle and direct assembly of prereplication complexes (pre-RCs) before the onset of S phase. In the eubacterium Escherichia coli, there is a temporally similar nucleoprotein complex comprising the initiator protein DnaA bound to three high-affinity recognition sites in the unique origin of replication, oriC. At the time of initiation, this high-affinity DnaA-oriC complex (the bacterial ORC) accumulates additional DnaA that interacts with lower-affinity sites in oriC, forming a pre-RC. In this paper, we investigate the functional role of the bacterial ORC and examine whether it mediates low-affinity DnaA-oriC interactions during pre-RC assembly. We report that E. coli ORC is essential for DnaA occupation of low-affinity sites. The assistance given by ORC is directed primarily to proximal weak sites and requires oligomerization-proficient DnaA. We propose that in bacteria, DnaA oligomers of limited length and stability emerge from single high-affinity sites and extend toward weak sites to facilitate their loading as a key stage of prokaryotic pre-RC assembly.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Map of oriC. Locations and sequences of DnaA recognition sites are shown. The mutated sequences in oriC1,2,4/R5M are placed below the WT sequences. Positions of IHF, Fis, and the duplex unwinding region (DUE) are marked. The change in DMS modification pattern caused by DnaA binding is indicated by up (increased modification) or down (decreased modification) arrowheads.
Fig. 2.
Fig. 2.
A mutant oriC lacking high-affinity sites does not bind DnaA in vivo. (A) DnaC(ts) cells harboring either WT oriC or oriC1,2,4R5/M were grown exponentially or were held at nonpermissive temperature for 1 h to align them at the stage of initiation just before helicase loading. Cells were treated with DMS, and modification patterns were measured. Positions of DnaA-binding sites and bands representing the Gs at positions 2 and 4 are marked. (B and C) Relative intensities of DMS-modified guanosines in DnaA-binding sites were quantified from scans of footprinting gels. Quantitation from representative scans is shown.
Fig. 3.
Fig. 3.
Strong sites facilitate DnaA binding to low-affinity sites in oriC. (A) R1, R2, and R4 were replaced by R5M in oriC to make oriC1,2,4/R5M. DMS modification patterns were measured on WT oriC and oriC1,2,4/R5M after incubation with DnaA-ATP. Positions of DnaA-binding sites are marked, and bands representing the Gs at position 2 or 4 are indicated. (B and C) Relative intensities of DMS-modified guanosines in DnaA-binding sites were quantified from scans of footprinting gels. (B) WT oriC was incubated with 0, 20, 40, 80, and 100 nM DnaA. (C) oriC1,2,4/R5M was incubated with 0, 40, 80, 160, 240, and 320 nM DnaA. Quantitation from representative scans is shown.
Fig. 4.
Fig. 4.
Oligomerization-proficient DnaA is required for binding to low-affinity sites. (A) oriC was incubated with the indicated concentrations of WT DnaA or DnaA(W6A) and treated with DMS. Modification patterns are shown. Positions of DnaA-binding sites are marked, and bands representing the Gs at position 2 or 4 are indicated. (B and C) Relative intensities of DMS-modified guanosines in DnaA-binding sites were quantified from scans of footprinting gels.
Fig. 5.
Fig. 5.
oriC promotes formation of DnaA oligomers. (A) A DNA oligomer containing a single R4 box (Left) or an R4 box and an R2 box separated by 7 bp (Right) were incubated with DnaA-ATP at the indicated molar ratios of DnaA/DNA, and complexes were resolved on polyacrylamide gels. Complexes 1 and 2 are labeled, and arrows point to the unbound probe. (B) DnaA-ATP (80 nM) was incubated in the absence of DNA (lane 1) and with nonspecific DNA (NS; pACYC184; lane 2) or oriC (lane 3). After incubation, the cross-linking agent DSP was added. The reactions were loaded onto SDS/polyacrylamide gels, and DnaA was revealed by Western blot analyses. Locations of DnaA monomers, dimers, and trimers are marked. Positions of molecular weight markers are indicated on the left.
Fig. 6.
Fig. 6.
High-affinity sites facilitate DnaA binding only to proximal low-affinity sites. Mutant oriC plasmids containing only one high-affinity site were incubated with the indicated concentrations of DnaA-ATP and treated with DMS. (A–C) (Left) Scans of footprinting gels with the positions of DnaA-binding sites marked. Mutated sites are indicated by R1M, R2M, or R4M to mark that the respective site was converted to the R5M sequence. (Right) Graphs of the quantitation of relative intensities (arbitrary units) of the G4 in each site. (A) oriC2,4/R5M (R1 is the sole high-affinity site). (B) oriC1,4/R5M (R2 is the sole high-affinity site). (C) oriC1,2/R5M (R4 is the sole high-affinity site. (D) The increase in intensity of G4 in each site in the three single-site mutants after incubation of DnaA is shown as the ratio of G4 (80 nM DnaA)/G4 (0 nM DnaA).

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