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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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Investigation of the DnaI–stearoDnaB interaction by analytical gel filtration. (A) The gel filtration profile of a mixture of DnaI and stearoDnaB (5 μM each) is shown on the left and of the DnaI protein alone on the right. Samples from the peaks were analyzed by SDS–PAGE analysis. The numbers of the lanes in the gels correspond to the areas marked with the same numbers in the elution profiles. The complex is detected in fractions 1–5 in the early peak whilst free DnaI is found in fractions 6–8 and 9–11 in the later peak. (B) The same experiment was carried out with Nd and stearoDnaB (5 μM each). The complex is detected in fractions 1–4 in the early peak whereas Nd on its own elutes in the later peak, fractions 5–7 and 8–10.
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fig1: Investigation of the DnaI–stearoDnaB interaction by analytical gel filtration. (A) The gel filtration profile of a mixture of DnaI and stearoDnaB (5 μM each) is shown on the left and of the DnaI protein alone on the right. Samples from the peaks were analyzed by SDS–PAGE analysis. The numbers of the lanes in the gels correspond to the areas marked with the same numbers in the elution profiles. The complex is detected in fractions 1–5 in the early peak whilst free DnaI is found in fractions 6–8 and 9–11 in the later peak. (B) The same experiment was carried out with Nd and stearoDnaB (5 μM each). The complex is detected in fractions 1–4 in the early peak whereas Nd on its own elutes in the later peak, fractions 5–7 and 8–10.

Mentions: DnaI interacts with stearoDnaB [(11) and Figure 1A] and B.subtilis DnaC (16), forming stable complexes that can be isolated by gel filtration. StearoDnaB eluted at ∼9.8 ml and DnaI at 14.5 ml. The precise elution volume depends on the actual gel matrix and the size of the particular column, but the resolution power of the Superdex S200 column is not sufficient to clearly separate complexes above 200 kDa in size. However, the clear shift of some DnaI into the earlier peak in the presence of stearoDnaB indicates the formation of a complex (Figure 1A). An interaction between Nd and stearoDnaB could also be detected by gel filtration (Figure 1B). While Nd eluted at ∼18.2 ml in the presence of stearoDnaB some Nd appeared in the earlier peak indicating the formation of a complex. The interactions of DnaI and Nd with stearoDnaB were also verified by yeast two hybrid experiments (Figure 2A). By comparison, Cd exhibited a very weak interaction in these experiments (Figure 2B and C) and no complexes with stearoDnaB or Nd were detected by gel filtration (data not shown). These data were confirmed with quantitative ONPG assays (Figure 2B). Several yeast clones carrying Cd were tested and although none exhibited significant DnaB-interactions, weak signals just above the background were detectable (Figure 2B and C).


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)

Investigation of the DnaI–stearoDnaB interaction by analytical gel filtration. (A) The gel filtration profile of a mixture of DnaI and stearoDnaB (5 μM each) is shown on the left and of the DnaI protein alone on the right. Samples from the peaks were analyzed by SDS–PAGE analysis. The numbers of the lanes in the gels correspond to the areas marked with the same numbers in the elution profiles. The complex is detected in fractions 1–5 in the early peak whilst free DnaI is found in fractions 6–8 and 9–11 in the later peak. (B) The same experiment was carried out with Nd and stearoDnaB (5 μM each). The complex is detected in fractions 1–4 in the early peak whereas Nd on its own elutes in the later peak, fractions 5–7 and 8–10.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1636449&req=5

fig1: Investigation of the DnaI–stearoDnaB interaction by analytical gel filtration. (A) The gel filtration profile of a mixture of DnaI and stearoDnaB (5 μM each) is shown on the left and of the DnaI protein alone on the right. Samples from the peaks were analyzed by SDS–PAGE analysis. The numbers of the lanes in the gels correspond to the areas marked with the same numbers in the elution profiles. The complex is detected in fractions 1–5 in the early peak whilst free DnaI is found in fractions 6–8 and 9–11 in the later peak. (B) The same experiment was carried out with Nd and stearoDnaB (5 μM each). The complex is detected in fractions 1–4 in the early peak whereas Nd on its own elutes in the later peak, fractions 5–7 and 8–10.
Mentions: DnaI interacts with stearoDnaB [(11) and Figure 1A] and B.subtilis DnaC (16), forming stable complexes that can be isolated by gel filtration. StearoDnaB eluted at ∼9.8 ml and DnaI at 14.5 ml. The precise elution volume depends on the actual gel matrix and the size of the particular column, but the resolution power of the Superdex S200 column is not sufficient to clearly separate complexes above 200 kDa in size. However, the clear shift of some DnaI into the earlier peak in the presence of stearoDnaB indicates the formation of a complex (Figure 1A). An interaction between Nd and stearoDnaB could also be detected by gel filtration (Figure 1B). While Nd eluted at ∼18.2 ml in the presence of stearoDnaB some Nd appeared in the earlier peak indicating the formation of a complex. The interactions of DnaI and Nd with stearoDnaB were also verified by yeast two hybrid experiments (Figure 2A). By comparison, Cd exhibited a very weak interaction in these experiments (Figure 2B and C) and no complexes with stearoDnaB or Nd were detected by gel filtration (data not shown). These data were confirmed with quantitative ONPG assays (Figure 2B). Several yeast clones carrying Cd were tested and although none exhibited significant DnaB-interactions, weak signals just above the background were detectable (Figure 2B and C).

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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