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. 2001 Mar 15;20(6):1469-76.
doi: 10.1093/emboj/20.6.1469.

Mechanism of Origin Unwinding: Sequential Binding of DnaA to Double- And Single-Stranded DNA

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Free PMC article

Mechanism of Origin Unwinding: Sequential Binding of DnaA to Double- And Single-Stranded DNA

C Speck et al. EMBO J. .
Free PMC article

Abstract

The initiator protein DnaA of Escherichia coli binds to a 9mer consensus sequence, the DnaA box (5'-TT(A/T)TNCACA). If complexed with ATP it adopts a new binding specificity for a 6mer consensus sequence, the ATP-DnaA box (5'-AGatct). Using DNase footprinting and surface plasmon resonance we show that binding to ATP-DnaA boxes in the AT-rich region of oriC of E.coli requires binding to the 9mer DnaA box R1. Cooperative binding of ATP-DnaA to the AT-rich region results in its unwinding. ATP-DnaA subsequently binds to the single-stranded region, thereby stabilizing it. This demonstrates an additional binding specificity of DnaA protein to single-stranded ATP-DnaA boxes. Binding affinities, as judged by the DnaA concentrations required for site protection in footprinting, were approximately 1 nM for DnaA box R1, 400 nM for double-stranded ATP-DnaA boxes and 40 nM for single-stranded ATP-DnaA boxes, respectively. We propose that sequential recognition of high- and low-affinity sites, and binding to single-stranded origin DNA may be general properties of initiator proteins in initiation complexes.

Figures

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Fig. 1. DNase I footprint of the AT-rich region. DNase I footprinting was performed using a 5′-end-labeled 428 bp PCR DNA fragment. Increasing amounts of ATP- or ADP-DnaA were added, giving final concentrations of 150, 300, 350, 400, 450, 500, 550 and 600 nM. Incubation was for 10 min at 37°C. DnaA box R1, the AT cluster and the three 13mers are indicated by bars; the lower strands are shown.
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Fig. 2. Binding reactions of ATP- and ADP-DnaA protein to the AT-rich region of oriC as measured by SPR. The fragment used in these measurements is shown at the top. DnaA box R1 is indicated in light gray, ATP-DnaA boxes are in bold. The three 13mer repeats are boxed. The stoichiometry of the complexes obtained at a protein concentration of 75 nM is indicated in the figure as x:1 (protein:DNA). ATP- and ADP-DnaA protein binding is shown for the wild type (R1), a DnaA box R1 mutant (M) and a fragment that contains an inverted DnaA box R1 (indicated as 1R). One resonance unit corresponds to a change in mass of 1 pg/mm2. Within each sensorgram, individual curves were obtained with protein concentrations (bottom to top) of 1.2, 2.3, 4.7, 9.4, 18.8, 37.5 and 75 nM.
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Fig. 3. Binding reactions of ATP- and ADP-DnaA protein to an artificial bubble as measured by SPR. At the top, a version of the artificial bubble DNA is indicated. The DnaA box R1 is indicated in gray, the three 13mers are boxed and the ATP-DnaA boxes are shown in bold. The upper strand corresponds to the E.coli sequence. In the lower strand, the M and R 13mers were replaced by the corresponding region from B.subtilis oriC (Krause and Messer, 1999), thereby avoiding hybridization in this segment. The stoichiometry of the complexes obtained at a protein concentration of 75 nM is indicated in the figure as x:1 (protein:DNA). Fragments that contain scrambled sequences in the lower or upper strand of the bubble or in the flanking sequence are indicated by N′s. One resonance unit corresponds to a change in mass of 1 pg/mm2. Within each sensorgram, individual curves were obtained with protein concentrations (bottom to top) of 1.2, 2.3, 4.7, 9.4, 18.8, 37.5 and 75 nM.
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Fig. 4. Nuclease P1 footprint of the artificial bubble substrate and ATP-DnaA. Nuclease PI footprinting was performed using the DNA fragment shown in Figure 3 (top). The DNA was 5′-end-labeled. Increasing amounts of ATP-DnaA were added, giving final concentrations of 10, 40, 75, 100 and 150 nM. Incubation was for 10 min at 37°C. DnaA box R1, the AT cluster, the three 13mers (L, M and R) and the single-stranded region (B) are indicated by bars.
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Fig. 5. A model describing the DnaA-dependent unwinding reaction at oriC. The DnaA box R1 and the three 13mers are labeled. The six ATP-DnaA boxes are indicated by small gray boxes. Initially, DnaA protein binds with a high affinity to DnaA box R1. This complex serves as an anchor for cooperative binding of ATP-DnaA to the ATP-DnaA boxes, positioning the protein to the region of unwinding. The formation of this complex needs high concentrations of ATP-DnaA protein and can be considered, therefore, as the rate-limiting reaction in unwinding. Single-stranded DNA resulting from unwinding will then be stabilized by cooperative binding of ATP-DnaA. The affinity of ATP-DnaA is higher to single-stranded ATP-DnaA boxes than to double-stranded ATP-DnaA boxes.

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