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 Aug;40(15):7375-83.
doi: 10.1093/nar/gks371. Epub 2012 May 11.

Topological characterization of the DnaA-oriC complex using single-molecule nanomanipuation

Sylvain Zorman  1 , H Seitz, B Sclavi, T R Strick
Affiliations

Topological characterization of the DnaA-oriC complex using single-molecule nanomanipuation

Sylvain Zorman et al. Nucleic Acids Res. 2012 Aug.

Abstract

In most bacteria, the timing and synchrony of initiation of chromosomal replication are determined by the binding of the AAA(+) protein DnaA to a set of high- and low-affinity sites found within the origin of chromosomal replication (oriC). Despite the large amount of information on the role and regulation of DnaA, the actual structure of the DnaA-oriC complex and the mechanism by which it primes the origin for the initiation of replication remain unclear. In this study, we have performed magnetic tweezers experiments to investigate the structural properties of the DnaA-oriC complex. We show that the DnaA-ATP-oriC complex adopts a right-handed helical conformation involving a variable amount of DNA and protein whose features fit qualitatively as well as quantitatively with an existing model based on the crystal structure of a truncated DnaA tetramer obtained in the absence of DNA. We also investigate the topological effect of oriC's DNA unwinding element.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
The E. coli origin of replication bears five 9-mer DnaA-binding sites (R1, R2, R3, R4 and R5) as well as three 13-mer binding sites included in an A+T-rich DNA unwinding element (DUE). Although the 9-mers show no differential specificity between DnaA-ATP and DnaA-ADP, the 13-mers specifically recruit DnaA-ATP. In addition to DnaA-binding sites, oriC hosts-specific binding sites for IHF, SeqA and FIS, three proteins that regulate the activity of DnaA.
Figure 2.
Figure 2.
Single DNA micromanipulations with magnetic tweezers. (A) The DnaA-ATP–oriC is modeled as a right-handed helical structure with DNA-binding domain (red stick) facing outward. The structural properties of the helix are characterized by its pitch, p, and its diameter, a. (B) Sketch of the experimental setup: a single ∼2 kb DNA is attached at one end to a magnetic bead and at the other to a functionalized glass surface. A pair of permanent magnets located above the sample generates a vertical extending force, F, on the DNA and constrains the rotation of the bead and hence DNA’s supercoiling. In all experiments presented here, the force F = 0.2 pN. The end-to-end extension, z, of the DNA is measured in real time (30 Hz) by monitoring the bead position with ∼5 nm accuracy. To account for microscope drift, the position of a second bead fixed to the surface (reference bead) is simultaneously monitored. (C) Typical rotation–extension curve with, superimposed, the corresponding DNA conformation and polynomial fit. The apical point of the curve is computed by polynomial fitting and is indicated by a blue cross on the curve. (D) Rotation–extension curves, and their fits, performed on the same single DNA molecule containing the oriC sequence in the absence of protein (green circles) and in the presence of 50 nM of DnaA-ATP (red squares). As shown on the superimposed sketch, the change in the DNA conformation imposed by the formation of a nucleoprotein complex (blue structure) results in a shift in the coordinates of the apex. For this particular experiment, the shifts in apex coordinates are Δz = −45 nm and Δr = 0.54 turns.
Figure 3.
Figure 3.
Shifts in apex coordinates (Δr, Δz) obtained after incubating nanomanipulated DNA with DnaA under various conditions and for a fixed extending force F = 0.2 pN. (A) Shift in apex coordinates for: (i) no DnaA present in the injected solution (green squares, n = 20), ⟨Δr⟩ = 0.04 ± 0.06 turns (SD = 0.25 turns) and ⟨Δz⟩ = 1 ± 3 nm (SD = 12 nm), (ii) DNA lacking oriC sequence (red circles, n = 13): ⟨Δr⟩ = 0.08 ± 0.04 turns (SD = 0.16 turns) and ⟨Δz⟩ = 1 ± 2 nm (SD = 6 nm) and (iii) when ADP is substituted to ATP in the reaction mix (purple triangles, n = 12): ⟨Δr⟩ = 0.09 ± 0.06 turns (SD = 0.21 turns) and ⟨Δz⟩ = −5 ± 3 nm (SD = 12 nm). (B) Shifts in apex coordinates under standard conditions: DNA bearing the oriC sequence, DnaA-ATP (blue circles, n = 57). Pink is the predicted area for a left-handed helical complex and the light-blue region is the predicted area for a right-handed helical complex. The corresponding projection on both axes is depicted in blue on both the upper histogram (projection along the axis of rotation) and right histogram (projection along the axis of extension). The orange bars correspond to the distribution of the combined controls from panel A. The dotted line corresponds to the maximum of likelihood for the Gaussian fit applied to the clustered points along the two axes as calculated in Supplementary Figure S3 (see main text for details).
Figure 4.
Figure 4.
Comparison with model for DnaA-ATP–oriC nucleoprotein filament. The shaded area reflects the experimental area accounted for by the structural model proposed by Erzberger, Mott et al. using the mechanical model introduced by Hegner et al. (22,33). In this case, the radius and pitch of the helix are as discussed in the Supplementary Data, and the shaded area is delimited at bottom by the case for a flexible helical filament (Ξ = 30 nm), and at top by the case for a rigid helical filament (Ξ = 180 nm). Empty blue circles: standard conditions (from Figure 3). Red squares: DNA bearing a mutated version of oriC lacking the DUE (oriCΔDUE, n = 15). Purple diamonds: DNA lacking oriC and incubated with DnaA in the absence of bulk competitor DNA (n = 16). The complete data set collected under standard condition are linearly fitted (solid line, y = Ax + B with A = −31 nm/turn and B = −20 nm). The blue cross represents the mean for points which distribute in a Gaussian fashion.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Giraldo R. Common domains in the initiators of DNA replication in Bacteria, Archaea and Eukarya: combined structural, functional and phylogenetic perspectives. FEMS Microbiol. Rev. 2003;26:533–554. - PubMed
    1. Georgescu RE, O'Donnell M. Structural biology. Getting DNA to unwind. Science. 2007;317:1181–1182. - PubMed
    1. Gaudier M, Schuwirth BS, Westcott SL, Wigley DB. Structural basis of DNA replication origin recognition by an ORC protein. Science. 2007;317:1213–1216. - PubMed
    1. Dueber ELC, Corn JE, Bell SD, Berger JM. Replication origin recognition and deformation by a heterodimeric archaeal orc1 complex. Science. 2007;317:1210–1213. - PubMed
    1. Kornberg A, Baker T. DNA Replication. 2nd edn. New York: W.H. Freeman and Company; 1991.

Publication types

MeSH terms