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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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Terminal deletion variants of HsmtSSB bind to ssDNA with similar affinities.A, ssDNA-binding affinity was evaluated by GMSA. HsmtSSBwt and its deletion variants were pre-incubated with a radiolabeled 48-mer oligonucleotide at 50 mM NaCl in the presence of increasing mtSSB concentrations: 1.25, 2.5, 3.75, 5, 7.5, 10, 15, 20, 30, 40 and 50 nM (as tetramer), as described under “Materials and Methods”. “−” denotes no added protein. The fraction of unbound (U) and bound (B) oligomer were quantitated, and the data were plotted in B as the percent of substrate utilized. The data represent the average of three experiments.
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pone-0015379-g004: Terminal deletion variants of HsmtSSB bind to ssDNA with similar affinities.A, ssDNA-binding affinity was evaluated by GMSA. HsmtSSBwt and its deletion variants were pre-incubated with a radiolabeled 48-mer oligonucleotide at 50 mM NaCl in the presence of increasing mtSSB concentrations: 1.25, 2.5, 3.75, 5, 7.5, 10, 15, 20, 30, 40 and 50 nM (as tetramer), as described under “Materials and Methods”. “−” denotes no added protein. The fraction of unbound (U) and bound (B) oligomer were quantitated, and the data were plotted in B as the percent of substrate utilized. The data represent the average of three experiments.

Mentions: We proceeded to evaluate the HsmtSSB deletion variants by examining their biochemical activities as compared to the wild-type protein. Using a gel mobility shift assay (GMSA), we examined the apparent DNA-binding affinities of HsmtSSBwt and the terminal deletion variants using a 48-mer ssDNA oligonucleotide (Fig. 4), whose size is close to the binding-site size determined previously for HsmtSSB [16]. A titration of the proteins at 50 mM NaCl revealed no significant differences in ssDNA-binding affinities between HsmtSSBwt and the deletion variants, with apparent Kds of ∼5 nM. Interestingly, the lack of the C-terminal region does not appear to interfere with the ssDNA-binding efficacy of HsmtSSB, in contrast to EcSSB and T7 gp2.5. A recent report by Kozlov et al. [17] shows that an increased ssDNA binding of EcSSB lacking the C-terminus is observed only at 100 and 200 mM NaCl, and not at low salt concentration (20 mM NaCl). We also performed the GMSA assays with the HsmtSSB proteins at 20 and 100 mM NaCl, but failed to observe any differences in binding affinities (data not shown). At the 20–100 mM range, salt does not seem to affect the ssDNA-binding affinity of either the wild-type or variant forms of HsmtSSB.


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)

Terminal deletion variants of HsmtSSB bind to ssDNA with similar affinities.A, ssDNA-binding affinity was evaluated by GMSA. HsmtSSBwt and its deletion variants were pre-incubated with a radiolabeled 48-mer oligonucleotide at 50 mM NaCl in the presence of increasing mtSSB concentrations: 1.25, 2.5, 3.75, 5, 7.5, 10, 15, 20, 30, 40 and 50 nM (as tetramer), as described under “Materials and Methods”. “−” denotes no added protein. The fraction of unbound (U) and bound (B) oligomer were quantitated, and the data were plotted in B as the percent of substrate utilized. The data represent the average of three experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2965674&req=5

pone-0015379-g004: Terminal deletion variants of HsmtSSB bind to ssDNA with similar affinities.A, ssDNA-binding affinity was evaluated by GMSA. HsmtSSBwt and its deletion variants were pre-incubated with a radiolabeled 48-mer oligonucleotide at 50 mM NaCl in the presence of increasing mtSSB concentrations: 1.25, 2.5, 3.75, 5, 7.5, 10, 15, 20, 30, 40 and 50 nM (as tetramer), as described under “Materials and Methods”. “−” denotes no added protein. The fraction of unbound (U) and bound (B) oligomer were quantitated, and the data were plotted in B as the percent of substrate utilized. The data represent the average of three experiments.
Mentions: We proceeded to evaluate the HsmtSSB deletion variants by examining their biochemical activities as compared to the wild-type protein. Using a gel mobility shift assay (GMSA), we examined the apparent DNA-binding affinities of HsmtSSBwt and the terminal deletion variants using a 48-mer ssDNA oligonucleotide (Fig. 4), whose size is close to the binding-site size determined previously for HsmtSSB [16]. A titration of the proteins at 50 mM NaCl revealed no significant differences in ssDNA-binding affinities between HsmtSSBwt and the deletion variants, with apparent Kds of ∼5 nM. Interestingly, the lack of the C-terminal region does not appear to interfere with the ssDNA-binding efficacy of HsmtSSB, in contrast to EcSSB and T7 gp2.5. A recent report by Kozlov et al. [17] shows that an increased ssDNA binding of EcSSB lacking the C-terminus is observed only at 100 and 200 mM NaCl, and not at low salt concentration (20 mM NaCl). We also performed the GMSA assays with the HsmtSSB proteins at 20 and 100 mM NaCl, but failed to observe any differences in binding affinities (data not shown). At the 20–100 mM range, salt does not seem to affect the ssDNA-binding affinity of either the wild-type or variant forms of HsmtSSB.

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