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Helicase binding to DnaI exposes a cryptic DNA-binding site during helicase loading in Bacillus subtilis.

Ioannou C, Schaeffer PM, Dixon NE, Soultanas P - Nucleic Acids Res. (2006)

Bottom Line: DnaI binds ATP and exhibits ATPase activity that is not stimulated by ssDNA, because the DNA-binding site on Cd is masked by Nd.Therefore, Nd acts as a molecular 'switch' regulating access to the ssDNA binding site on Cd, in response to binding of the helicase.DnaI is sufficient to load the replicative helicase from a complex with six DnaI molecules, so there is no requirement for a dual helicase loader system.

View Article: PubMed Central - PubMed

Affiliation: Centre for Biomolecular Sciences, School of Chemistry, University of Nottingham, University Park, Nottingham NG7 2RD, UK.

ABSTRACT
The Bacillus subtilis DnaI, DnaB and DnaD proteins load the replicative ring helicase DnaC onto DNA during priming of DNA replication. Here we show that DnaI consists of a C-terminal domain (Cd) with ATPase and DNA-binding activities and an N-terminal domain (Nd) that interacts with the replicative ring helicase. A Zn2+-binding module mediates the interaction with the helicase and C67, C70 and H84 are involved in the coordination of the Zn2+. DnaI binds ATP and exhibits ATPase activity that is not stimulated by ssDNA, because the DNA-binding site on Cd is masked by Nd. The ATPase activity resides on the Cd domain and when detached from the Nd domain, it becomes sensitive to stimulation by ssDNA because its cryptic DNA-binding site is exposed. Therefore, Nd acts as a molecular 'switch' regulating access to the ssDNA binding site on Cd, in response to binding of the helicase. DnaI is sufficient to load the replicative helicase from a complex with six DnaI molecules, so there is no requirement for a dual helicase loader system.

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Binding of the helicase loader (DnaI)-helicase (stearoDnaB) complex to ssDNA. Gel shifts showing binding of increasing concentrations of stearoDnaB (lanes 1–6, 0.25, 0.5, 1, 2, 4 and 8 μM, respectively) to a 50mer single-stranded oligonucleotide in the presence of 1 mM ADPNP (A), in the absence of ADPNP (B), in the presence of 1 mM ATP (C) and in the presence (A–C, right segments) of 8 μM DnaI or absence of DnaI (A–C, left segments), as indicated. Arrows indicate the shifted bands.
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fig7: Binding of the helicase loader (DnaI)-helicase (stearoDnaB) complex to ssDNA. Gel shifts showing binding of increasing concentrations of stearoDnaB (lanes 1–6, 0.25, 0.5, 1, 2, 4 and 8 μM, respectively) to a 50mer single-stranded oligonucleotide in the presence of 1 mM ADPNP (A), in the absence of ADPNP (B), in the presence of 1 mM ATP (C) and in the presence (A–C, right segments) of 8 μM DnaI or absence of DnaI (A–C, left segments), as indicated. Arrows indicate the shifted bands.

Mentions: Although DnaI binds to stearoDnaB, the stability of the complex is not affected by ADPNP in the absence of DNA (11). In the presence of 1 mM ADPNP stearoDnaB binds better to ssDNA, as shown before (33), whilst in the presence of 8 μM DnaI and increasing concentrations of stearoDnaB (0.25–28 μM) a super-shifted band was detected representing binding of the DnaI–stearoDnaB complex to DNA (compare left and right segments in Figure 7A). DnaI does not bind to ssDNA as shown in Figure 6A. In the absence of ADPNP no super-shifted complex was observed (compare left and right segments in Figure 7B). In the presence of 1 mM ATP the super-shifted band disappeared (Figure 7C). More efficient loading was observed at higher DnaI:stearoDnaB molar ratios, 32:1, 16:1, 8:1 (Figure 7A, lanes 1–3 right segment) compared to lower molar ratios 4:1, 2:1, 1:1 (lanes 4–6), indicating that under these experimental conditions excess of DnaI is required to ensure formation of a stable loading complex. These data indicate that binding of the DnaI–stearoDnaB complex to ssDNA is stimulated by ATP-binding and ATP hydrolysis facilitates the dissociation of the complex leaving the helicase on the DNA.


Helicase binding to DnaI exposes a cryptic DNA-binding site during helicase loading in Bacillus subtilis.

Ioannou C, Schaeffer PM, Dixon NE, Soultanas P - Nucleic Acids Res. (2006)

Binding of the helicase loader (DnaI)-helicase (stearoDnaB) complex to ssDNA. Gel shifts showing binding of increasing concentrations of stearoDnaB (lanes 1–6, 0.25, 0.5, 1, 2, 4 and 8 μM, respectively) to a 50mer single-stranded oligonucleotide in the presence of 1 mM ADPNP (A), in the absence of ADPNP (B), in the presence of 1 mM ATP (C) and in the presence (A–C, right segments) of 8 μM DnaI or absence of DnaI (A–C, left segments), as indicated. Arrows indicate the shifted bands.
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fig7: Binding of the helicase loader (DnaI)-helicase (stearoDnaB) complex to ssDNA. Gel shifts showing binding of increasing concentrations of stearoDnaB (lanes 1–6, 0.25, 0.5, 1, 2, 4 and 8 μM, respectively) to a 50mer single-stranded oligonucleotide in the presence of 1 mM ADPNP (A), in the absence of ADPNP (B), in the presence of 1 mM ATP (C) and in the presence (A–C, right segments) of 8 μM DnaI or absence of DnaI (A–C, left segments), as indicated. Arrows indicate the shifted bands.
Mentions: Although DnaI binds to stearoDnaB, the stability of the complex is not affected by ADPNP in the absence of DNA (11). In the presence of 1 mM ADPNP stearoDnaB binds better to ssDNA, as shown before (33), whilst in the presence of 8 μM DnaI and increasing concentrations of stearoDnaB (0.25–28 μM) a super-shifted band was detected representing binding of the DnaI–stearoDnaB complex to DNA (compare left and right segments in Figure 7A). DnaI does not bind to ssDNA as shown in Figure 6A. In the absence of ADPNP no super-shifted complex was observed (compare left and right segments in Figure 7B). In the presence of 1 mM ATP the super-shifted band disappeared (Figure 7C). More efficient loading was observed at higher DnaI:stearoDnaB molar ratios, 32:1, 16:1, 8:1 (Figure 7A, lanes 1–3 right segment) compared to lower molar ratios 4:1, 2:1, 1:1 (lanes 4–6), indicating that under these experimental conditions excess of DnaI is required to ensure formation of a stable loading complex. These data indicate that binding of the DnaI–stearoDnaB complex to ssDNA is stimulated by ATP-binding and ATP hydrolysis facilitates the dissociation of the complex leaving the helicase on the DNA.

Bottom Line: DnaI binds ATP and exhibits ATPase activity that is not stimulated by ssDNA, because the DNA-binding site on Cd is masked by Nd.Therefore, Nd acts as a molecular 'switch' regulating access to the ssDNA binding site on Cd, in response to binding of the helicase.DnaI is sufficient to load the replicative helicase from a complex with six DnaI molecules, so there is no requirement for a dual helicase loader system.

View Article: PubMed Central - PubMed

Affiliation: Centre for Biomolecular Sciences, School of Chemistry, University of Nottingham, University Park, Nottingham NG7 2RD, UK.

ABSTRACT
The Bacillus subtilis DnaI, DnaB and DnaD proteins load the replicative ring helicase DnaC onto DNA during priming of DNA replication. Here we show that DnaI consists of a C-terminal domain (Cd) with ATPase and DNA-binding activities and an N-terminal domain (Nd) that interacts with the replicative ring helicase. A Zn2+-binding module mediates the interaction with the helicase and C67, C70 and H84 are involved in the coordination of the Zn2+. DnaI binds ATP and exhibits ATPase activity that is not stimulated by ssDNA, because the DNA-binding site on Cd is masked by Nd. The ATPase activity resides on the Cd domain and when detached from the Nd domain, it becomes sensitive to stimulation by ssDNA because its cryptic DNA-binding site is exposed. Therefore, Nd acts as a molecular 'switch' regulating access to the ssDNA binding site on Cd, in response to binding of the helicase. DnaI is sufficient to load the replicative helicase from a complex with six DnaI molecules, so there is no requirement for a dual helicase loader system.

Show MeSH
Related in: MedlinePlus