Limits...
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.

Show MeSH

Related in: MedlinePlus

DNA binding gel shift assays. (A) Full length DnaI does not bind to ssDNA in the presence or absence of ADPNP or ATP (top) whereas it binds weakly to a fork-DNA substrate (bottom). (B) Cd binds to ss (top) and fork (bottom) DNA substrates. In the presence of ADPNP or ATP its DNA binding activity is marginally reduced. DNA substrates are shown on top of the gels. Asterisks indicate the radioactively labelled 5′ end and arrows indicate the shifted bands. Increasing concentrations of DnaI are indicated by sloping bars, left to right 0.25, 0.5, 1, 2, 4 and 8 μM in the absence of ADPNP and 2, 4 and 8 μM in the presence of ADPNP or ATP. Lanes labelled C indicate controls without protein.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC1636449&req=5

fig6: DNA binding gel shift assays. (A) Full length DnaI does not bind to ssDNA in the presence or absence of ADPNP or ATP (top) whereas it binds weakly to a fork-DNA substrate (bottom). (B) Cd binds to ss (top) and fork (bottom) DNA substrates. In the presence of ADPNP or ATP its DNA binding activity is marginally reduced. DNA substrates are shown on top of the gels. Asterisks indicate the radioactively labelled 5′ end and arrows indicate the shifted bands. Increasing concentrations of DnaI are indicated by sloping bars, left to right 0.25, 0.5, 1, 2, 4 and 8 μM in the absence of ADPNP and 2, 4 and 8 μM in the presence of ADPNP or ATP. Lanes labelled C indicate controls without protein.

Mentions: The ssDNA-induced stimulation of Cd ATPase activity implies that this domain binds ssDNA. By comparison, the lack of stimulation of the full-length DnaI in the presence of ssDNA implies that either it does not bind ssDNA or ssDNA binding does not confer the conformational changes required for ATPase stimulation. To distinguish between these two possibilities, the DNA-binding ability of DnaI with single-strand and fork-DNA substrates using gel shift assays was examined (Figure 6A). Under our experimental conditions in the range 0.25–28 μM DnaI-binding to a single-strand 50mer oligonucleotide was undetectable whereas weak binding to fork-substrates was detected (Figure 6A). Binding to ssDNA could only be detected at concentrations higher than 0.9 mM DnaI (data not shown). DnaI exhibited weak binding to 3′- and 5′-tailed DNA substrates (data not shown). The presence of ADPNP or ATP had no effect on DNA binding. Thus, the lack of stimulation of DnaI ATPase activity is a reflection of its inability to bind ssDNA. Instead it exhibits a preference for ss-dsDNA junctions but binding is still weak.


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)

DNA binding gel shift assays. (A) Full length DnaI does not bind to ssDNA in the presence or absence of ADPNP or ATP (top) whereas it binds weakly to a fork-DNA substrate (bottom). (B) Cd binds to ss (top) and fork (bottom) DNA substrates. In the presence of ADPNP or ATP its DNA binding activity is marginally reduced. DNA substrates are shown on top of the gels. Asterisks indicate the radioactively labelled 5′ end and arrows indicate the shifted bands. Increasing concentrations of DnaI are indicated by sloping bars, left to right 0.25, 0.5, 1, 2, 4 and 8 μM in the absence of ADPNP and 2, 4 and 8 μM in the presence of ADPNP or ATP. Lanes labelled C indicate controls without protein.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1636449&req=5

fig6: DNA binding gel shift assays. (A) Full length DnaI does not bind to ssDNA in the presence or absence of ADPNP or ATP (top) whereas it binds weakly to a fork-DNA substrate (bottom). (B) Cd binds to ss (top) and fork (bottom) DNA substrates. In the presence of ADPNP or ATP its DNA binding activity is marginally reduced. DNA substrates are shown on top of the gels. Asterisks indicate the radioactively labelled 5′ end and arrows indicate the shifted bands. Increasing concentrations of DnaI are indicated by sloping bars, left to right 0.25, 0.5, 1, 2, 4 and 8 μM in the absence of ADPNP and 2, 4 and 8 μM in the presence of ADPNP or ATP. Lanes labelled C indicate controls without protein.
Mentions: The ssDNA-induced stimulation of Cd ATPase activity implies that this domain binds ssDNA. By comparison, the lack of stimulation of the full-length DnaI in the presence of ssDNA implies that either it does not bind ssDNA or ssDNA binding does not confer the conformational changes required for ATPase stimulation. To distinguish between these two possibilities, the DNA-binding ability of DnaI with single-strand and fork-DNA substrates using gel shift assays was examined (Figure 6A). Under our experimental conditions in the range 0.25–28 μM DnaI-binding to a single-strand 50mer oligonucleotide was undetectable whereas weak binding to fork-substrates was detected (Figure 6A). Binding to ssDNA could only be detected at concentrations higher than 0.9 mM DnaI (data not shown). DnaI exhibited weak binding to 3′- and 5′-tailed DNA substrates (data not shown). The presence of ADPNP or ATP had no effect on DNA binding. Thus, the lack of stimulation of DnaI ATPase activity is a reflection of its inability to bind ssDNA. Instead it exhibits a preference for ss-dsDNA junctions but binding is still weak.

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