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Functional roles of the N- and C-terminal regions of the human mitochondrial single-stranded DNA-binding protein.

Oliveira MT, Kaguni LS - PLoS ONE (2010)

Bottom Line: Notably, we find that unlike the high level of stimulation that we observed previously in the Drosophila system, stimulation of DNA synthesis catalyzed by human mtDNA polymerase is only moderate, and occurs over a narrow range of salt concentrations.Interestingly, each of the deletion variants of human mtSSB stimulates DNA synthesis at a higher level than the wild-type protein, indicating that the termini modulate negatively functional interactions with the mitochondrial replicase.We discuss our findings in the context of species-specific components of the mtDNA replisome, and in comparison with various prokaryotic DNA replication machineries.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Center for Mitochondrial Science and Medicine, and Graduate Program in Genetics, Michigan State University, East Lansing, Michigan, United States of America.

ABSTRACT
Biochemical studies of the mitochondrial DNA (mtDNA) replisome demonstrate that the mtDNA polymerase and the mtDNA helicase are stimulated by the mitochondrial single-stranded DNA-binding protein (mtSSB). Unlike Escherichia coli SSB, bacteriophage T7 gp2.5 and bacteriophage T4 gp32, mtSSBs lack a long, negatively charged C-terminal tail. Furthermore, additional residues at the N-terminus (notwithstanding the mitochondrial presequence) are present in the sequence of species across the animal kingdom. We sought to analyze the functional importance of the N- and C-terminal regions of the human mtSSB in the context of mtDNA replication. We produced the mature wild-type human mtSSB and three terminal deletion variants, and examined their physical and biochemical properties. We demonstrate that the recombinant proteins adopt a tetrameric form, and bind single-stranded DNA with similar affinities. They also stimulate similarly the DNA unwinding activity of the human mtDNA helicase (up to 8-fold). Notably, we find that unlike the high level of stimulation that we observed previously in the Drosophila system, stimulation of DNA synthesis catalyzed by human mtDNA polymerase is only moderate, and occurs over a narrow range of salt concentrations. Interestingly, each of the deletion variants of human mtSSB stimulates DNA synthesis at a higher level than the wild-type protein, indicating that the termini modulate negatively functional interactions with the mitochondrial replicase. We discuss our findings in the context of species-specific components of the mtDNA replisome, and in comparison with various prokaryotic DNA replication machineries.

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Related in: MedlinePlus

Stimulation of HsmtDNA helicase by terminal deletion variants of HsmtSSB.DNA unwinding assays were performed as described under “Materials and Methods”. A, a constant concentration of HsmtDNA helicase (3.5 nM as hexamer) and KCl (50 mM) were used in each assay. The concentration of HsmtSSBs (as tetramer) that was used is indicated above each lane. “−” and “+ boil” lanes represent the intact and denatured substrate (heated to 100°C for 2 min prior to loading), respectively. B, analysis of the data shown in A together with the data from two other independent experiments. The data represent the average of unwound substrate as percent.
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pone-0015379-g008: Stimulation of HsmtDNA helicase by terminal deletion variants of HsmtSSB.DNA unwinding assays were performed as described under “Materials and Methods”. A, a constant concentration of HsmtDNA helicase (3.5 nM as hexamer) and KCl (50 mM) were used in each assay. The concentration of HsmtSSBs (as tetramer) that was used is indicated above each lane. “−” and “+ boil” lanes represent the intact and denatured substrate (heated to 100°C for 2 min prior to loading), respectively. B, analysis of the data shown in A together with the data from two other independent experiments. The data represent the average of unwound substrate as percent.

Mentions: We extended our analysis by titrating HsmtSSBwt, ΔN, ΔC, and ΔNΔC in DNA unwinding assays conducted at 50 mM KCl (Fig. 8). None of the concentrations of the HsmtSSBs used were sufficient to cause any dsDNA destabilization in the absence of helicase (Fig. 8A). HsmtDNA helicase shows maximal DNA unwinding activity in the presence of 100 nM HsmtSSB, a concentration corresponding to coating of ∼80% of the ssDNA substrate (according to our GMSA data). No significant differences in stimulation were observed between HsmtSSBwt and deletion variants; stimulation of the HsmtDNA helicase was ∼8 fold at the highest HsmtSSB concentrations.


Functional roles of the N- and C-terminal regions of the human mitochondrial single-stranded DNA-binding protein.

Oliveira MT, Kaguni LS - PLoS ONE (2010)

Stimulation of HsmtDNA helicase by terminal deletion variants of HsmtSSB.DNA unwinding assays were performed as described under “Materials and Methods”. A, a constant concentration of HsmtDNA helicase (3.5 nM as hexamer) and KCl (50 mM) were used in each assay. The concentration of HsmtSSBs (as tetramer) that was used is indicated above each lane. “−” and “+ boil” lanes represent the intact and denatured substrate (heated to 100°C for 2 min prior to loading), respectively. B, analysis of the data shown in A together with the data from two other independent experiments. The data represent the average of unwound substrate as percent.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2965674&req=5

pone-0015379-g008: Stimulation of HsmtDNA helicase by terminal deletion variants of HsmtSSB.DNA unwinding assays were performed as described under “Materials and Methods”. A, a constant concentration of HsmtDNA helicase (3.5 nM as hexamer) and KCl (50 mM) were used in each assay. The concentration of HsmtSSBs (as tetramer) that was used is indicated above each lane. “−” and “+ boil” lanes represent the intact and denatured substrate (heated to 100°C for 2 min prior to loading), respectively. B, analysis of the data shown in A together with the data from two other independent experiments. The data represent the average of unwound substrate as percent.
Mentions: We extended our analysis by titrating HsmtSSBwt, ΔN, ΔC, and ΔNΔC in DNA unwinding assays conducted at 50 mM KCl (Fig. 8). None of the concentrations of the HsmtSSBs used were sufficient to cause any dsDNA destabilization in the absence of helicase (Fig. 8A). HsmtDNA helicase shows maximal DNA unwinding activity in the presence of 100 nM HsmtSSB, a concentration corresponding to coating of ∼80% of the ssDNA substrate (according to our GMSA data). No significant differences in stimulation were observed between HsmtSSBwt and deletion variants; stimulation of the HsmtDNA helicase was ∼8 fold at the highest HsmtSSB concentrations.

Bottom Line: Notably, we find that unlike the high level of stimulation that we observed previously in the Drosophila system, stimulation of DNA synthesis catalyzed by human mtDNA polymerase is only moderate, and occurs over a narrow range of salt concentrations.Interestingly, each of the deletion variants of human mtSSB stimulates DNA synthesis at a higher level than the wild-type protein, indicating that the termini modulate negatively functional interactions with the mitochondrial replicase.We discuss our findings in the context of species-specific components of the mtDNA replisome, and in comparison with various prokaryotic DNA replication machineries.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Center for Mitochondrial Science and Medicine, and Graduate Program in Genetics, Michigan State University, East Lansing, Michigan, United States of America.

ABSTRACT
Biochemical studies of the mitochondrial DNA (mtDNA) replisome demonstrate that the mtDNA polymerase and the mtDNA helicase are stimulated by the mitochondrial single-stranded DNA-binding protein (mtSSB). Unlike Escherichia coli SSB, bacteriophage T7 gp2.5 and bacteriophage T4 gp32, mtSSBs lack a long, negatively charged C-terminal tail. Furthermore, additional residues at the N-terminus (notwithstanding the mitochondrial presequence) are present in the sequence of species across the animal kingdom. We sought to analyze the functional importance of the N- and C-terminal regions of the human mtSSB in the context of mtDNA replication. We produced the mature wild-type human mtSSB and three terminal deletion variants, and examined their physical and biochemical properties. We demonstrate that the recombinant proteins adopt a tetrameric form, and bind single-stranded DNA with similar affinities. They also stimulate similarly the DNA unwinding activity of the human mtDNA helicase (up to 8-fold). Notably, we find that unlike the high level of stimulation that we observed previously in the Drosophila system, stimulation of DNA synthesis catalyzed by human mtDNA polymerase is only moderate, and occurs over a narrow range of salt concentrations. Interestingly, each of the deletion variants of human mtSSB stimulates DNA synthesis at a higher level than the wild-type protein, indicating that the termini modulate negatively functional interactions with the mitochondrial replicase. We discuss our findings in the context of species-specific components of the mtDNA replisome, and in comparison with various prokaryotic DNA replication machineries.

Show MeSH
Related in: MedlinePlus