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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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A model for DnaI-mediated helicase loading in Bacillus. StearoDnaB consists of two domains, an Nd P17 and a Cd P33, while DnaI consists of an Nd and a Cd, as indicated. DnaI binds ATP in its Cd domain and exists in a ‘closed conformation'. The P33 domain of stearoDnaB interacts with the Nd domain of DnaI (11) and a conformational change switches DnaI into a DNA-interacting mode. The cryptic DNA binding site is exposed and the complex binds to the 5′-ssDNA tail. The ATPase activity is stimulated and ATP hydrolysis facilitates the ejection of DnaI from the complex leaving stearoDnaB bound to the DNA.
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fig9: A model for DnaI-mediated helicase loading in Bacillus. StearoDnaB consists of two domains, an Nd P17 and a Cd P33, while DnaI consists of an Nd and a Cd, as indicated. DnaI binds ATP in its Cd domain and exists in a ‘closed conformation'. The P33 domain of stearoDnaB interacts with the Nd domain of DnaI (11) and a conformational change switches DnaI into a DNA-interacting mode. The cryptic DNA binding site is exposed and the complex binds to the 5′-ssDNA tail. The ATPase activity is stimulated and ATP hydrolysis facilitates the ejection of DnaI from the complex leaving stearoDnaB bound to the DNA.

Mentions: We were unable to detect significant binding of DnaI to ssDNA even at high concentrations in the presence or absence of ADPNP, suggesting that the native protein does not bind ssDNA. Weak binding to fork and tail (5′ or 3′) DNA substrates was detected indicating a slight preference for ds-ssDNA junctions. The weak nature of this interaction makes its biological significance uncertain. Cd exhibited DNA-binding activity. The simplest interpretation of these data is that the Nd domain masks the DNA-binding site located on Cd. Nd acts as a ‘molecular switch’ that regulates the availability of the DNA-binding site. In the native protein, this site is buried but when the helicase binds to Nd a conformational change reveals the cryptic DNA-binding site. The helicase-loader can now deliver the helicase to the DNA but it can do so only when bound to ATP. This is consistent with the inability of the stearoDnaB–DnaI complex to produce a super-shifted complex on ssDNA in the absence of the ATP analogue ADPNP. Having eliminated misfolding problems, the partial insolubility of Cd is likely to indicate that certain hydrophobic patches on its surface are protected by the presence of Nd in the intact protein and exposed in its absence. It is likely that this allosteric control of domain movement in DnaI is an important functional property regulating helicase loading. Taking into account the combined data, we propose a model for the DnaI-mediated helicase loading (Figure 9). By comparison, the Gram-negative E.coli helicase loader DnaC has been reported to exhibit a cryptic ssDNA binding activity only when bound to the helicase (35) and to interact weakly to ssDNA in the absence of the helicase, with ATP strengthening this interaction (36). The Cd domain belongs to the AAA family of a diverse group of ATPases (37). They often consist of a non-ATPase Nd that acts in substrate recognition (in our case Nd that recognizes the helicase), followed by one or two AAA domains (in our case the Cd domain). This family also belongs to the classic AAA+ superfamily of chaperone-like ATPases that assist in the assembly and disassembly of protein complexes and the remodelling of protein–DNA complexes (38).


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)

A model for DnaI-mediated helicase loading in Bacillus. StearoDnaB consists of two domains, an Nd P17 and a Cd P33, while DnaI consists of an Nd and a Cd, as indicated. DnaI binds ATP in its Cd domain and exists in a ‘closed conformation'. The P33 domain of stearoDnaB interacts with the Nd domain of DnaI (11) and a conformational change switches DnaI into a DNA-interacting mode. The cryptic DNA binding site is exposed and the complex binds to the 5′-ssDNA tail. The ATPase activity is stimulated and ATP hydrolysis facilitates the ejection of DnaI from the complex leaving stearoDnaB bound to the DNA.
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Related In: Results  -  Collection

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fig9: A model for DnaI-mediated helicase loading in Bacillus. StearoDnaB consists of two domains, an Nd P17 and a Cd P33, while DnaI consists of an Nd and a Cd, as indicated. DnaI binds ATP in its Cd domain and exists in a ‘closed conformation'. The P33 domain of stearoDnaB interacts with the Nd domain of DnaI (11) and a conformational change switches DnaI into a DNA-interacting mode. The cryptic DNA binding site is exposed and the complex binds to the 5′-ssDNA tail. The ATPase activity is stimulated and ATP hydrolysis facilitates the ejection of DnaI from the complex leaving stearoDnaB bound to the DNA.
Mentions: We were unable to detect significant binding of DnaI to ssDNA even at high concentrations in the presence or absence of ADPNP, suggesting that the native protein does not bind ssDNA. Weak binding to fork and tail (5′ or 3′) DNA substrates was detected indicating a slight preference for ds-ssDNA junctions. The weak nature of this interaction makes its biological significance uncertain. Cd exhibited DNA-binding activity. The simplest interpretation of these data is that the Nd domain masks the DNA-binding site located on Cd. Nd acts as a ‘molecular switch’ that regulates the availability of the DNA-binding site. In the native protein, this site is buried but when the helicase binds to Nd a conformational change reveals the cryptic DNA-binding site. The helicase-loader can now deliver the helicase to the DNA but it can do so only when bound to ATP. This is consistent with the inability of the stearoDnaB–DnaI complex to produce a super-shifted complex on ssDNA in the absence of the ATP analogue ADPNP. Having eliminated misfolding problems, the partial insolubility of Cd is likely to indicate that certain hydrophobic patches on its surface are protected by the presence of Nd in the intact protein and exposed in its absence. It is likely that this allosteric control of domain movement in DnaI is an important functional property regulating helicase loading. Taking into account the combined data, we propose a model for the DnaI-mediated helicase loading (Figure 9). By comparison, the Gram-negative E.coli helicase loader DnaC has been reported to exhibit a cryptic ssDNA binding activity only when bound to the helicase (35) and to interact weakly to ssDNA in the absence of the helicase, with ATP strengthening this interaction (36). The Cd domain belongs to the AAA family of a diverse group of ATPases (37). They often consist of a non-ATPase Nd that acts in substrate recognition (in our case Nd that recognizes the helicase), followed by one or two AAA domains (in our case the Cd domain). This family also belongs to the classic AAA+ superfamily of chaperone-like ATPases that assist in the assembly and disassembly of protein complexes and the remodelling of protein–DNA complexes (38).

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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