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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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Interaction of the stearoDnaB–DnaI complex with immobilized ssDNA, studied by SPR. (A) Investigation of the stearoDnaB–DnaI interaction by SPR. StearoDnaB 175 nM (red) or DnaI 1.75 μM (light blue) shows no binding to the immobilized oligonucleotide (3′-biotinylated dT35). Binding was observed when the two proteins were mixed. Injections of stearoDnaB (175 nM) with increasing amounts of DnaI (black, 85 nM; dark blue, 175 nM; green, 350 nM) exhibited binding responses with a fast dissociation phase. (B) DnaI dissociates from the ternary complex in the fast dissociation phase. A mixture of stearoDnaB (175 nM) and DnaI (1.75 μM) shows a biphasic binding response. A second injection of DnaI at 1.75 μM produces a second response, confirming that DnaI can rebind to reform the ternary complex. This shows that it is DnaI that dissociates with a fast off-rate once the stearoDnaB–DnaI complex is loaded on the ssDNA.
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fig8: Interaction of the stearoDnaB–DnaI complex with immobilized ssDNA, studied by SPR. (A) Investigation of the stearoDnaB–DnaI interaction by SPR. StearoDnaB 175 nM (red) or DnaI 1.75 μM (light blue) shows no binding to the immobilized oligonucleotide (3′-biotinylated dT35). Binding was observed when the two proteins were mixed. Injections of stearoDnaB (175 nM) with increasing amounts of DnaI (black, 85 nM; dark blue, 175 nM; green, 350 nM) exhibited binding responses with a fast dissociation phase. (B) DnaI dissociates from the ternary complex in the fast dissociation phase. A mixture of stearoDnaB (175 nM) and DnaI (1.75 μM) shows a biphasic binding response. A second injection of DnaI at 1.75 μM produces a second response, confirming that DnaI can rebind to reform the ternary complex. This shows that it is DnaI that dissociates with a fast off-rate once the stearoDnaB–DnaI complex is loaded on the ssDNA.

Mentions: An assay for DnaI-mediated loading of stearoDnaB onto ssDNA using SPR (BIAcore) was devised in a similar manner to that described for the interaction between the C-domain of E.coli DnaG and E.coli DnaB (31). A 3′-biotinylated (dT)35 was first loaded onto a streptavidin chip, exposing the 5′ end. Then either stearoDnaB alone or a mixture of stearoDnaB and DnaI were injected, in the presence of Mg-ATP. No interaction of 175 nM stearoDnaB or 1.75 μM DnaI, alone with DNA was observed (Figure 8A; red and light blue). Binding was detected when the two proteins were combined, and the response depended on the concentration of DnaI. Therefore, formation of a complex of DnaI with stearoDnaB is necessary for helicase loading onto ssDNA. The response consistently decreased by 40% over 2 min to a stable value regardless of [DnaI]. The simplest explanation for this behaviour is that the stearoDnaB–DnaI complex first loads, and then one of the components dissociates. We proceeded to discover which component dissociates from the ternary complex.


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)

Interaction of the stearoDnaB–DnaI complex with immobilized ssDNA, studied by SPR. (A) Investigation of the stearoDnaB–DnaI interaction by SPR. StearoDnaB 175 nM (red) or DnaI 1.75 μM (light blue) shows no binding to the immobilized oligonucleotide (3′-biotinylated dT35). Binding was observed when the two proteins were mixed. Injections of stearoDnaB (175 nM) with increasing amounts of DnaI (black, 85 nM; dark blue, 175 nM; green, 350 nM) exhibited binding responses with a fast dissociation phase. (B) DnaI dissociates from the ternary complex in the fast dissociation phase. A mixture of stearoDnaB (175 nM) and DnaI (1.75 μM) shows a biphasic binding response. A second injection of DnaI at 1.75 μM produces a second response, confirming that DnaI can rebind to reform the ternary complex. This shows that it is DnaI that dissociates with a fast off-rate once the stearoDnaB–DnaI complex is loaded on the ssDNA.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1636449&req=5

fig8: Interaction of the stearoDnaB–DnaI complex with immobilized ssDNA, studied by SPR. (A) Investigation of the stearoDnaB–DnaI interaction by SPR. StearoDnaB 175 nM (red) or DnaI 1.75 μM (light blue) shows no binding to the immobilized oligonucleotide (3′-biotinylated dT35). Binding was observed when the two proteins were mixed. Injections of stearoDnaB (175 nM) with increasing amounts of DnaI (black, 85 nM; dark blue, 175 nM; green, 350 nM) exhibited binding responses with a fast dissociation phase. (B) DnaI dissociates from the ternary complex in the fast dissociation phase. A mixture of stearoDnaB (175 nM) and DnaI (1.75 μM) shows a biphasic binding response. A second injection of DnaI at 1.75 μM produces a second response, confirming that DnaI can rebind to reform the ternary complex. This shows that it is DnaI that dissociates with a fast off-rate once the stearoDnaB–DnaI complex is loaded on the ssDNA.
Mentions: An assay for DnaI-mediated loading of stearoDnaB onto ssDNA using SPR (BIAcore) was devised in a similar manner to that described for the interaction between the C-domain of E.coli DnaG and E.coli DnaB (31). A 3′-biotinylated (dT)35 was first loaded onto a streptavidin chip, exposing the 5′ end. Then either stearoDnaB alone or a mixture of stearoDnaB and DnaI were injected, in the presence of Mg-ATP. No interaction of 175 nM stearoDnaB or 1.75 μM DnaI, alone with DNA was observed (Figure 8A; red and light blue). Binding was detected when the two proteins were combined, and the response depended on the concentration of DnaI. Therefore, formation of a complex of DnaI with stearoDnaB is necessary for helicase loading onto ssDNA. The response consistently decreased by 40% over 2 min to a stable value regardless of [DnaI]. The simplest explanation for this behaviour is that the stearoDnaB–DnaI complex first loads, and then one of the components dissociates. We proceeded to discover which component dissociates from the ternary complex.

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