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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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MANT-ATP binding to DnaI. (A) A Job plot for MANT-ATP binding to DnaI. The sum of the concentrations of MANT-ATP and DnaI was kept constant at [MANT-ATP] + [DnaI] = 1.1 μM. The fluorescence (arbitrary units) was plotted as a function of the molar fraction of [DnaI] relative to the total ([MANT-ATP] + [DnaI]) concentration. The intersection for the fitted lines gives a value close to 0.5, indicating a binding stoichiometry of MANT-ATP:DnaI of 1:1. (B) Determination of the apparent dissociation constant (KD) for MANT-ATP binding to DnaI. Experiments were carried out at four fixed concentrations of MANT-ATP, as indicated, and the fluorescence (arbitrary units) was plotted as a function of [DnaI]. The data were fitted to a single-site binding hyperbola and analysed by the GraphPad Prism programme to obtain KD values for each set of data. The dissociation constant is KD = 0.9 μM.
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fig4: MANT-ATP binding to DnaI. (A) A Job plot for MANT-ATP binding to DnaI. The sum of the concentrations of MANT-ATP and DnaI was kept constant at [MANT-ATP] + [DnaI] = 1.1 μM. The fluorescence (arbitrary units) was plotted as a function of the molar fraction of [DnaI] relative to the total ([MANT-ATP] + [DnaI]) concentration. The intersection for the fitted lines gives a value close to 0.5, indicating a binding stoichiometry of MANT-ATP:DnaI of 1:1. (B) Determination of the apparent dissociation constant (KD) for MANT-ATP binding to DnaI. Experiments were carried out at four fixed concentrations of MANT-ATP, as indicated, and the fluorescence (arbitrary units) was plotted as a function of [DnaI]. The data were fitted to a single-site binding hyperbola and analysed by the GraphPad Prism programme to obtain KD values for each set of data. The dissociation constant is KD = 0.9 μM.

Mentions: Binding of ATP to DnaI was investigated by fluorescence using MANT-ATP. The binding stoichiometry was determined using the continuous variation analysis from Job plots and the fitted lines gave an intersection at the stoichiometry 1:1 (Figure 4A). Binding was examined at four different [MANT-ATP] (90, 200, 300 and 500 nM) while the [DnaI] was varied and the fluorescence change was plotted as function of [DnaI], (Figure 4B). The binding curves were fitted to an one site binding hyperbola using GraphPad Prism and KD values were obtained for each curve (0.89, 0.78, 1.09 and 0.94 μM), indicating an average KD = 0.9 μM for binding of DnaI to MANT-ATP.


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)

MANT-ATP binding to DnaI. (A) A Job plot for MANT-ATP binding to DnaI. The sum of the concentrations of MANT-ATP and DnaI was kept constant at [MANT-ATP] + [DnaI] = 1.1 μM. The fluorescence (arbitrary units) was plotted as a function of the molar fraction of [DnaI] relative to the total ([MANT-ATP] + [DnaI]) concentration. The intersection for the fitted lines gives a value close to 0.5, indicating a binding stoichiometry of MANT-ATP:DnaI of 1:1. (B) Determination of the apparent dissociation constant (KD) for MANT-ATP binding to DnaI. Experiments were carried out at four fixed concentrations of MANT-ATP, as indicated, and the fluorescence (arbitrary units) was plotted as a function of [DnaI]. The data were fitted to a single-site binding hyperbola and analysed by the GraphPad Prism programme to obtain KD values for each set of data. The dissociation constant is KD = 0.9 μM.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1636449&req=5

fig4: MANT-ATP binding to DnaI. (A) A Job plot for MANT-ATP binding to DnaI. The sum of the concentrations of MANT-ATP and DnaI was kept constant at [MANT-ATP] + [DnaI] = 1.1 μM. The fluorescence (arbitrary units) was plotted as a function of the molar fraction of [DnaI] relative to the total ([MANT-ATP] + [DnaI]) concentration. The intersection for the fitted lines gives a value close to 0.5, indicating a binding stoichiometry of MANT-ATP:DnaI of 1:1. (B) Determination of the apparent dissociation constant (KD) for MANT-ATP binding to DnaI. Experiments were carried out at four fixed concentrations of MANT-ATP, as indicated, and the fluorescence (arbitrary units) was plotted as a function of [DnaI]. The data were fitted to a single-site binding hyperbola and analysed by the GraphPad Prism programme to obtain KD values for each set of data. The dissociation constant is KD = 0.9 μM.
Mentions: Binding of ATP to DnaI was investigated by fluorescence using MANT-ATP. The binding stoichiometry was determined using the continuous variation analysis from Job plots and the fitted lines gave an intersection at the stoichiometry 1:1 (Figure 4A). Binding was examined at four different [MANT-ATP] (90, 200, 300 and 500 nM) while the [DnaI] was varied and the fluorescence change was plotted as function of [DnaI], (Figure 4B). The binding curves were fitted to an one site binding hyperbola using GraphPad Prism and KD values were obtained for each curve (0.89, 0.78, 1.09 and 0.94 μM), indicating an average KD = 0.9 μM for binding of DnaI to MANT-ATP.

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