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Originally published In Press as doi:10.1074/jbc.M400283200 on January 30, 2004

J. Biol. Chem., Vol. 279, Issue 17, 17047-17053, April 23, 2004
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Physiological and Biochemical Defects in Functional Interactions of Mitochondrial DNA Polymerase and DNA-binding Mutants of Single-stranded DNA-binding Protein*

Carol L. Farr, Yuichi Matsushima, Anthony T. Lagina, III, Ningguang Luo, and Laurie S. Kaguni{ddagger}

From the Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824-1319

Received for publication, January 12, 2004 , and in revised form, January 30, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Functional interactions between mitochondrial DNA polymerase (pol {gamma}) and mitochondrial single-stranded DNA-binding protein (mtSSB) from Drosophila embryos greatly enhance the overall activity of pol {gamma} by increasing primer recognition and binding and stimulating the rate of initiation of DNA strands (Farr, C. L., Wang, Y., and Kaguni, L. S. (1999) J. Biol. Chem. 274, 14779–14785). We show here that DNA-binding mutants of mtSSB are defective in stimulation of DNA synthesis by pol {gamma}. RNAi knock-down of mtSSB reduces expression to <5% of its normal level in Schneider cells, resulting in growth defects and in the depletion of mitochondrial DNA (mtDNA). Overexpression of mtSSB restores cell growth rate and the copy number of mtDNA, whereas overexpression of a DNA-binding and functionally impaired form of mtSSB neither rescues the cell growth defect nor the mtDNA depletion phenotype. Further development of Drosophila animal models, in which induced mtDNA depletion is manipulated by controlling exogenous expression of wild-type or mutant forms, will offer new insight into the mechanism and progression of human mtDNA depletion syndromes and possible intervention schemes.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Mitochondrial biogenesis is a key process in animal cell proliferation, and mtDNA replication is an essential component of that process. Whereas a large number of proteins participate in multi-component replication machines in bacterial and nuclear systems, a minimal set of essential proteins are likely involved in the mtDNA replication process (1). We have demonstrated functional interactions between the two-subunit Drosophila mitochondrial DNA polymerase (pol {gamma})1 and the homotetrameric mitochondrial single-stranded DNA-binding protein, mtSSB, at the levels of template-primer binding and initiation and elongation of DNA strand synthesis (2). These studies have documented roles for mtSSB in enhancing primer recognition and binding and in stimulation by 20- to 30-fold of the rate of initiation of DNA strands by pol {gamma}. Moreover, mtSSB increases severalfold the processivity of pol {gamma} in DNA strand elongation.

To probe further the biochemical and physiological importance of these functional interactions, we have evaluated the effects of altered forms of mtSSB on DNA binding per se and upon the coordinated reactions involving mtSSB and pol {gamma}. We find that even modest DNA-binding defects in mtSSB substantially affect DNA synthesis by pol {gamma}. Furthermore, under conditions that reduce cellular levels of endogenous wild-type mtSSB, a mtDNA depletion phenotype develops that is rescued by production of exogenous wild-type but not mutant mtSSB.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Nucleotides and Nucleic Acids—Unlabeled deoxy- and ribonucleotides were purchased from Amersham Biosciences. [3H]dTTP, [{alpha}-32P]dATP, and [{gamma}-32P]ATP were purchased from ICN Biochemicals. Wild-type (6,407 nucleotides (nt)) and recombinant (10,650 nt) M13 DNAs were prepared by standard laboratory methods. Oligodeoxynucleotides complementary to the M13 viral DNAs were synthesized in an Applied Biosystems oligonucleotide synthesizer. The primer for the recombinant M13 DNA that was used in the pol {gamma} stimulation assay was 17 nt in length and the primer for the M13 wild-type DNA used in the processivity analysis was 15 nt in length. To prepare template primers for DNA synthesis, M13 DNAs were added to the 15- and 17-mer oligonucleotide primers to a concentration of ~70 mM (as nt, in 4-fold molar excess over homologous oligonucleotide), and the DNA mixtures were precipitated with ethanol. The pellets were resuspended in a buffer (0.1 ml) containing 10 mM Tris-HCl, pH 8.0, 0.3 M NaCl, and 0.03 M sodium citrate and were incubated at 65 ° C for 1 h, followed by incubation at 37 ° C for an additional hour to anneal the primer to the template. The sequence of the 38-mer oligonucleotide used in the DNA binding and gel mobility shift assays (GMSA) is complementary to positions 6291–6329 in M13mp7 DNA.

Enzymes and Proteins—Drosophila DNA polymerase {gamma} (Fraction VI, >90% homogeneous) was prepared from embryonic mitochondria, as described by Wernette and Kaguni (3). Recombinant Drosophila mtSSB (>90% homogeneous) was prepared as described by Farr et al. (2). T4 polynucleotide kinase and restriction endonucleases were purchased from Roche Applied Science. T4 DNA ligase and Taq DNA polymerase were purchased from Invitrogen. Pfu DNA polymerase was purchased from Stratagene.

Mutagenesis, Overexpression, and Purification of Recombinant mtSSBs—The 372-bp coding sequence of the mature Drosophila mtSSB was cloned previously into the bacteriophage T7 promoter-based expression vector pET-11a (Novagen) at its unique NdeI site (2). The mtSSB mutant variants were constructed by overlap extension PCR using Pfu DNA polymerase and standard laboratory methods. The Escherichia coli strain BL21 ({lambda}DE3) (Novagen) was used for transformation, and plasmids extracted from ampicillin-resistant colonies were selected for DNA sequence analysis to confirm their structure and sequence integrity. Wild-type and mutant mtSSBs were produced by overexpression in E. coli BL21 ({lambda}DE3) and purified to homogeneity, as described by Farr et al. (2).

DNA Polymerase {gamma} Stimulation Assay—Reaction mixtures (0.05 ml) contained 50 mM Tris-HCl, pH 8.5, 4 mM MgCl2, 10 mM dithiothreitol, 0–185 mM KCl as indicated, 400 µg/ml bovine serum albumin, 20 µM each of dGTP, dATP, dCTP, and [3H]dTTP (1000 cpm/pmol), 10 µM (as nt) singly primed recombinant M13 DNA, and 0.1 unit of Fraction VI enzyme (6-fold excess of primer ends over pol {gamma} molecules). Wild-type and mutant mtSSBs (0.8 µg, 2-fold excess of mtSSB over DNA, assuming a binding site size of 17 nt per protomer) were added as indicated in the legend to Fig. 4. Incubation was at 30° C for 30 min. One unit of activity is that amount that catalyzes the incorporation of 1 nmol of deoxyribonucleoside triphosphate into acid insoluble material in 60 min at 30° C using DNase I-activated calf thymus DNA as the substrate.



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  		FIG. 4.
Mutant forms of Drosophila mtSSB are defective in stimulation of DNA synthesis by pol {gamma}. A, DNA synthesis by Drosophila pol {gamma} was measured on singly primed M13 DNA, as described under "Experimental Procedures," in the presence of the indicated KCl concentrations and in the presence ({circ}) or absence () of wild-type or DNA-binding mutant mtSSBs (F85A, {triangleup}; W79Y, {blacktriangleup}; W79T, {square}; W79A, {blacksquare}; W79T/F85A, {diamondsuit}). B, the data from A were replotted to show the ratio of nucleotide incorporation by Drosophila pol {gamma} in the presence versus absence of mtSSB at each KCl concentration.

 
Analysis of pol {gamma} Processivity—Reactions were as described for the pol {gamma} stimulation assay, except that the reaction mixtures contained 30 or 120 mM KCl, 30 µM each of dGTP, dATP, dCTP, and 7 µM [{alpha}-32P]dTTP (2 x 104 cpm/pmol), and 20 µM (as nt) singly primed wild-type M13 DNA. Wild-type and mutant mtSSBs (2.4 µg, 3-fold excess of mtSSB over DNA, assuming a binding site size of 17 nt per protomer) were added as indicated in the legend to Fig. 5, and the mixtures were preincubated for 5 min at 30° C. pol {gamma} Fraction VI (0.02 unit) was then added (100-fold excess of primer ends over pol {gamma} molecules), followed by incubation for 8 min at 30° C. Reaction products were isolated and analyzed by gel electrophoresis, as described by Williams and Kaguni (4).



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  		FIG. 5.
Mutant forms of Drosophila mtSSB are defective in increasing the processivity of DNA synthesis by pol {gamma}. DNA synthesis by Drosophila pol {gamma} was measured on singly primed M13 DNA, as described under "Experimental Procedures." DNA product strands were isolated, denatured, and electrophoresed in denaturing 1.5% agarose (upper panel) and 6% polyacrylamide (lower panel) gels, and the gels were autoradiographed. Reactions were performed at 30 mM KCl (lanes 1-5) or 120 mM KCl (lanes 6-10) in the absence (lanes 1 and 6; apu = 2190 and 146 nt, respectively) or presence of wild-type (lanes 2 and 7; apu = 2998 and 264 nt, respectively) or DNA-binding mutant mtSSBs F85A (lanes 3 and 8; apu = 3152 and 261 nt, respectively), W79A (lanes 4 and 9; apu = 1674 and 151 nt, respectively), and W79T/F85A (lanes 5 and 10; apu = 1507 and 144 nt, respectively).

 
Preparation of 5'-32P-Labeled DNA Substrate for GMSA—The 38-mer oligodeoxyribonucleotide described earlier was 5'-end-labeled. The kinase reaction (0.08 ml) contained 50 mM Tris-HCl, pH 8.3, 10 mM MgCl2, 0.1 mM EDTA, 5 mM dithiothreitol, 0.1 mM spermidine, [{gamma}-32P]ATP (0.3 µM, 4500 Ci/mmol), 170 pmol (as nt) of oligonucleotide, and 20 units of T4 polynucleotide kinase. Incubation was for 30 min at 37° C, and the 5'-end-labeled 38-mer oligonucleotide was precipitated with ethanol, resuspended in water, and stored at –20 °C.

DNA Binding and GMSA—Reaction mixtures (0.025 ml) contained 20 mM Tris-HCl, pH 7.5, 8 mM dithiothreitol, 0–840 mM KCl, 80 µg/ml bovine serum albumin, 4% sucrose, 200 fmol 5'-end-labeled 38-mer oligonucleotide, and wild-type or mutant mtSSBs (400 pg), as indicated in the legend to Fig. 3. Incubation was at 20° C for 30 min. Samples were processed and electrophoresed, and the amounts of shifted and unshifted oligonucleotide were quantitated as described by Thommes et al. (5).



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  		FIG. 3.
Mutant forms of Drosophila mtSSB are defective in DNA binding. DNA-binding affinity and stability were evaluated in a GMSA using a radiolabeled 38-mer oligonucleotide, as described under "Experimental Procedures." The amounts of shifted and unshifted oligonucleotide were quantitated, and the data are presented as the percent of substrate utilized. A, DNA binding was measured in the presence of the indicated KCl concentrations and in the presence of wild-type ({circ}) or mutant mtSSBs (F85A, {triangleup}; W79Y, {blacktriangleup}; W79T, {square}; W79A, {blacksquare}; W79T/F85A, {diamondsuit}). B, the stability of mtSSB·DNA complexes was measured by saltback titration. Complexes formed at 30 mM KCl as in A were incubated further in the presence of increasing levels of KCl, as indicated, prior to processing and electrophoresis.

 
In the saltback titration analysis, reactions were as above, except that 30 mM KCl was used, and incubation was at 20° C for 20 min. Aliquots (15 µl) were removed and adjusted to 30–800 mM KCl in a final volume of 0.025 ml, and the reactions were incubated further for 10 min at 20° C prior to processing and electrophoresis.

Protein Gel Electrophoresis, Transfer, and Immunoblotting—Proteins were separated in 17% SDS-polyacrylamide gels according to Laemmli (6). Proteins were transferred to nitrocellulose membranes (BA85, Schleicher & Schuell) and probed by immunoblotting. Filters were preincubated for 1 h with 5% skim milk in phosphate-buffered saline, followed by incubation for 1 h with Drosophila mtSSB antibody (1:500 dilution in phosphate-buffered saline containing 0.1% Tween 20). Filters were washed four times with phosphate-buffered saline containing 0.1% Tween 20, incubated for 1 h with horseradish peroxidase-conjugated anti-rabbit IgG (Bio-Rad), and washed with phosphate-buffered saline containing 0.1% Tween 20. Protein bands were visualized using ECL Western blotting reagents (Amersham Biosciences).

Southern Blotting—Genomic DNA was purified from Drosophila Schneider S2 cells by standard methods. DNA (10 µg per lane) was cleaved with XhoI, fractionated in an 0.8% agarose gel/TBE buffer (90 mM Tris, 64.6 mM boric acid, 2.5 mM EDTA, pH 8.3) and transferred to nylon membrane (Amersham Biosciences). Hybridization was carried out for 16 h at 42 °C in 5x SSPE (150 mM NaCl, 10 mM sodium phosphate, pH 7.4, 1 mM EDTA), 0.5% SDS, 5x Denhardt's solution, and 50% formamide. Filters were washed three times for 10 min at room temperature with 2x SSC containing 0.1% SDS, once for 30 min at 65 °C with 0.2x SSC containing 0.1% SDS, and then analyzed with a PhosphorImager (Molecular Dynamics). Blots were probed with radiolabeled DNAs for the mitochondrial gene ATPase6 and the nuclear histone gene cluster. The ratio of the signals for these two genes was used to estimate the relative copy number of mtDNA.

Preparation of Inducible Plasmids Expressing Drosophila mtSSB and mtSSB-targeted RNAi—Plasmids in which wild-type or mutant Drosophila mtSSB cDNA is regulated by the metallothionein promoter were constructed as follows: a fragment of Drosophila mtSSB cDNA was amplified by PCR using as the 5'-primer, 5'-GCGCCTCGAGTGCAACCACAAGGCGCATGCTGAATCCTCTGTTGACC-3', and as 3'-primer, 5'-GCGCACTAGTTTAGTTGTTGGCATCACGG-3'. The PCR fragment was cleaved by XhoI and SpeI and subcloned. The plasmid pMt/Hy/INV carried an inverted repeat of a nucleotide sequence from the 3'-UTR of the Drosophila mtSSB cDNA that was transcribed from the metallothionein promoter. The insert in pMt/Hy/INV was generated from two PCR-amplified fragments of the Drosophila mtSSB cDNA. One fragment had terminal XhoI and EcoRI sites and was prepared using the following pair of primers: 5'-GCGCCTCGAGACTAGTAATTTAAGCCCAGATCAC-3' (forward) and 5'-GCGCGAATTCGGGATCGATGGAGTACGACTACGCATG-3'(reverse). A second fragment had terminal SpeI and EcoRI sites and was prepared using the primers 5'-GCGCCTCGAGACTAGTAATTTAAGCCCAGATCAC-3' (forward) and 5'-GCGCGAATTCAAAAAGCTTGGAGTACGACTACGCATG-3' (reverse). The two PCR products were ligated and cloned into the pMt/Hy vector cleaved with XhoI and SpeI.

Generation and Induction of Stable Cell Lines—Drosophila Schneider S2 cells were cultured at 25 °C in Drosophila Schneider medium (Invitrogen) supplemented with 10% fetal bovine serum and were subcultured to 3 x 106 cells/ml every third day. Cells were transfected using Effectene (Qiagen). Hygromycin-resistant cells were selected with 200 µg/ml hygromycin. Cells were passaged at least five times in hygromycin-containing medium and then cultured in standard medium. The cell lines were grown to a density of 3 x 106/ml and then treated with 0.4 mM CuSO4 to induce expression from the metallothionein promoter (7).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Biochemical Defects in Mutant Drosophila mtSSBs—Our previous studies documented a functional interaction between pol {gamma} and mtSSB in replicating single-stranded DNAs that mimic lagging DNA strand synthesis during mtDNA replication (2, 5). We sought to explore the possible consequences of perturbing that interaction, both in biochemical assays and in vivo. To do so, we designed several site-directed mutants of mtSSB, targeting its DNA-binding domain. Several key amino acids have been identified in E. coli SSB and mammalian mtSSBs that affect DNA binding without affecting assembly of the tetramer (8–12). We made three substitutions of Trp79 in Drosophila mtSSB, with changes to Tyr (W79Y), Thr (W79T), and Ala (W79A), converted Phe85 to Ala (F85A), and constructed a double mutant containing the Trp79 -> Thr and the Phe85 -> Ala substitutions (W79T/F85A, Fig. 1, and "Experimental Procedures"). The recombinant proteins were produced by overexpression from pET-11a non-fusion constructs and purified to homogeneity as described by Farr et al. (2) (Fig. 2). Velocity gradient sedimentation analysis showed that each of the variants exists as a homotetramer in solution (data not shown).



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  		FIG. 1.
Structure and mutagenesis of Drosophila mtSSB. A schematic representation of the structure of Drosophila mtSSB is shown. The mature protein comprises 124 amino acids that are preceded in the precursor protein by a 16-amino acid leader peptide. Alignment of the central DNA-binding domain among SSB proteins shows a high degree of sequence similarity. Two conserved amino acids, W79 and F85, were mutagenized to generate the five mutant mtSSBs used in this study: Dm, Drosophila melanogaster; Hs, Homo sapiens; Rr, Rattus ratti; Xl, Xenopus laevis; Sc, Saccharomyces cerevisiae; Ec, Escherichia coli.

 



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  		FIG. 2.
SDS-polyacrylamide gel electrophoresis of wild-type and mutant forms of Drosophila mtSSB. The glycerol gradient fractions of recombinant wild-type (lane 1) and DNA-binding mutant (F85A, lane 2; W79T, lane 3; W79Y, lane 4; W79A, lane 5; W79T/F85A, lane 6) mtSSBs (0.4 µg) were denatured and electrophoresed in an SDS-17% polyacrylamide gel, as described under "Experimental Procedures." Proteins were detected by silver staining.

 
To initiate our functional analysis of the mutant mtSSBs, we performed two assays to evaluate DNA-binding efficacy as compared with the wild-type protein. Using a GMSA, we examined relative DNA-binding affinity and stability of the mtSSB·DNA complexes in salt titrations using a 38-mer single-stranded DNA oligonucleotide (Fig. 3). First, a titration was performed to determine that amount of the wild-type mtSSB needed to bind and shift the electrophoretic position of the radiolabeled oligomer. Equivalent amounts of mutant homotetramers were then compared over a broad range of KCl concentrations.

The standard assay for stimulation of pol {gamma} by mtSSB on single-stranded DNA substrates is performed at 30 mM KCl, where maximal DNA synthesis combined with the highest enzyme processivity is observed (4, 5). Under this ionic condition, all of the mtSSB variants with single amino acid substitutions displayed DNA-binding activities >= 90% of wild type, whereas the double mutant W79T/F85A showed 50% of wild-type DNA binding (Fig. 3A). There was no change in the binding ability of the wild-type mtSSB up to 840 mM KCl, the highest salt concentration tested. The same profile was observed for the F85A mutant. The most conservative substitution of Trp79, Trp -> Tyr, showed binding at a level of ~90% of wild-type mtSSB, whereas the Trp -> Thr and Ala variants showed steadily reduced binding activity with increasing salt, with values dropping to ~50% at 840 mM KCl. The apparently synergistic effect of the W79T/F85A double substitution was exacerbated with increasing salt: a 10-fold drop in binding was observed at 840 mM KCl, as compared with binding at 30 mM KCl. Further discrimination was observed among the variants when the stability of mtSSB·DNA complexes was analyzed by saltback titration of complexes formed at 30 mM KCl (Fig. 3B). Again, the mtSSB variants with single amino acid substitutions displayed DNA-binding stabilities at 30 mM KCl that were similar to wild type, whereas the W79T/F85A mutant showed only 30% of the wild-type level. At 100 mM KCl, the W79Y complexes remained stable, but the Thr and Ala substitutions showed reduced stability, which became more apparent with increasing salt. There was no change in the fraction of bound complexes of wild-type mtSSB with increasing salt to 400 mM KCl, and at 800 mM KCl binding was ~80% of that observed at 30 mM. The same profile was observed for the F85A mutant, whereas each of the Trp79 substitutions showed more pronounced decreases with increasing salt concentrations. The stability of the W79T/F85A complexes was less than 10% of that observed with the wild-type mtSSB. Taken together, the DNA binding and stability analyses of the mtSSB mutants would predict modest defects under ionic conditions favorable for functional interaction with pol {gamma} during DNA synthesis, with increasingly more apparent defects with increasing ionic strength.

At 120 mM KCl in the absence of mtSSB, pol {gamma} shows maximal activity on singly primed M13 DNA (13). Addition of mtSSB to the reaction lowers the KCl optimum ~8-fold to 15–30 mM, while stimulating activity at low salt up to 18-fold (2, 5). Surprisingly, although the DNA-binding activities of the mutants with single amino acid substitutions were all within 10% of that of the wild-type mtSSB at 30 mM KCl, defects in their abilities to stimulate DNA synthesis by pol {gamma} were apparent in the range of 0–30 mM KCl (Fig. 4). Stimulation by the F85A mutant was reduced 1.5-fold, whereas that for the Trp79 mutants was reduced 2.0- to 3.7-fold, indicating a clear reduction in the functional interaction between pol {gamma} and mtSSB. This was amplified in the W79T/F85A double mutant, in which binding activity was 50% of the wild-type level, yet its stimulation of pol {gamma} activity dropped 8-fold, to less than twice the activity of pol {gamma} alone. At higher KCl concentrations, where the activity of pol {gamma} alone is highest, the effects of the mutant mtSSBs were less apparent, although it seems likely from their DNA-binding defects at elevated ionic strength that functional interactions with pol {gamma} are in fact further reduced.

The processivity of pol {gamma} on singly primed M13 DNA decreases with increasing ionic strength, such that at moderate KCl concentrations (120 mM), where DNA polymerase activity is highest, processivity is reduced severalfold, as compared with that at low ionic strength (30 mM) (13). mtSSB increases pol {gamma} processivity over a broad salt range, though in a disproportional manner relative to stimulation of DNA polymerase activity at the same salt concentrations (5). Given the variable effects of the mutations generated in mtSSB upon DNA binding and pol {gamma} stimulation, we examined the effects of several of the mutants on pol {gamma} processivity at both low and moderate salt. At 30 mM KCl, the processivity of pol {gamma} alone is high (~2100 nt) and is increased modestly by the addition of wild-type mtSSB to ~3100 nt (Fig. 5), whereas a more pronounced effect is observed at 120 mM KCl, with an increase of 2.8-fold, from ~70 to 200 nt. A similar effect is observed with mtSSB F85A, which showed wild-type DNA-binding activity and slightly reduced stimulatory activity. Although mtSSB W79A showed substantially greater DNA binding and stimulatory activity as compared with mtSSB W79T/F85A, neither mutant was able to enhance pol {gamma} processivity.

Drosophila mtSSB Models of Mitochondrial Dysfunction—We developed several approaches to assess the impact on mitochondrial DNA metabolism of reduced DNA binding by mtSSB and loss of functional interactions with pol {gamma}, including both animal and cell culture models. First, we constructed transgenic Drosophila strains that express either the wild-type or mutant forms of mtSSB under the control of the Drosophila hsp70 promoter. In a previous study (14), we showed that overexpression of the catalytic subunit of pol {gamma} using such a strategy resulted in both developmental and morphological defects, depending on the level and temporal pattern of overexpression. Despite our ability to demonstrate expression of the transgenes in both the wild-type and mutant mtSSB lines, both in the presence or absence of heat shock over six generations, we observed no aberrant phenotypes in terms of developmental timing, eclosion frequency, adult body weight, or locomotion, nor did we find a reduction in mtDNA copy number (data not shown). Although there are a number of possible explanations for this finding, one that seemed likely is the substantially higher level of endogenous mtSSB as compared with pol {gamma}, which is apparent both in protein and mRNA levels at various stages in fly development (Refs. 14–16, and data not shown).

To circumvent this putative problem and issues related to cell type and tissue specificity of expression during development, we evaluated the effects of high-level overexpression of mutant mtSSB in cultured cells. To do so, we developed stably transfected Drosophila Schneider S2 lines, in which the wild-type and mutant mtSSB genes are expressed under the control of the Drosophila metallothionein promoter in the vector pMt/Hy (see "Experimental Procedures"). Following induction with 0.4 mM CuSO4, immunoblot analysis of whole-cell extracts using antiserum directed against Drosophila mtSSB showed protein overexpressed at levels 3- to 7-fold above the endogenous level (data not shown). Although there was no effect of overexpression of wild-type mtSSB upon either cell growth rate or mtDNA content, overexpression of the W79T/F85A mtSSB produced an mtDNA-depletion phenotype. After culturing the cells in 0.4 mM CuSO4 for a period of 21 days, there was no apparent effect on growth rate, but Southern analysis demonstrated a 40% reduction in mtDNA copy number in the double mutant as compared with both control cells and those overexpressing wild-type mtSSB (data not shown).

These data are consistent with our hypothesis that mutant mtSSB expression would result in defects of mtDNA replication that should lead to mtDNA depletion such as that found in human mitochondrial diseases. We explored this possibility further using the cell culture system, first by examining the effects of lowering endogenous mtSSB levels, and second, by introducing exogenously expressed wild-type and mutant forms. To inhibit expression of endogenous mtSSB, we developed a strategy to produce double-stranded-interfering RNAs targeted to the 3'-UTR of the mtSSB gene (Fig. 6). Two inverted repeat fragments were generated by PCR and inserted into the pMt/Hy vector for construction of stably transfected cell lines. After induction with 0.4 mM CuSO4 for 10 days, immunoblot analysis of whole-cell extracts showed a dramatic reduction in the level of endogenous mtSSB (Fig. 7). This was accompanied by a substantial reduction in cell growth (Fig. 8). Southern blot analysis demonstrated the generation of an mtDNA depletion phenotype: mtDNA levels were reduced 4-fold after 10 days of constitutive expression of the dsRNA (Fig. 9).



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  		FIG. 6.
Scheme for selective suppression of endogenous Drosophila mtSSB expression using RNAi in Schneider S2 cells. A 3'-UTR fragment of mtSSB was cloned as an inverted repeat into the inducible vector pMt/Hy. In the presence of 0.4 mM CuSO4, dsRNA and exogenous mtSSB mRNA were induced. Induction of dsRNA triggers degradation of endogenous mtSSB mRNA. However, the dsRNA does not affect the induced exogenous mtSSB mRNA (either wild-type or mutant), which lacks its native 3'-UTR.

 



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  		FIG. 7.
Expression of mtSSB in Schneider S2 cells. Cellular proteins (10 µg) were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and immunoblotted with antiserum against Drosophila mtSSB, as described under "Experimental Procedures." Proteins were extracted from Schneider S2 cells (S2, lanes 1 and 2), S2 cells stably transfected with the pMt/Hy plasmid (Mt/Hy, lanes 3 and 4), S2 cells stably transfected with the inverted repeat containing pMt/Hy plasmid construct (INV, lanes 5 and 6), S2 cells stably transfected with both the inverted repeat and exogenous wild-type mtSSB containing pMt/Hy plasmid constructs (INV + wild type, lanes 7 and 8), and S2 cells stably transfected with both the inverted repeat-containing and exogenous W79T/F85A mtSSB-containing pMt/Hy plasmid constructs (INV + W79T/F85A, lanes 9 and 10) that were grown for 10 days in the absence (lanes 1, 3, 5, 7 and 9) or the presence (lanes 2, 4, 6, 8 and 10) of 0.4 mM CuSO4.

 



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  		FIG. 8.
An mtSSB RNAi-induced growth-retardation phenotype is rescued by exogenous expression of wild-type mtSSB but not by the DNA-binding mutant mtSSB W79T/F85A. Growth curves were generated for the Schneider S2 cell lines described in the legend to Fig. 7: S2 (A); Mt/Hy (B); INV (C); INV + wild-type mtSSB (D); and INV + W79T/F85A mtSSB (E). The cell lines were grown in medium in the presence (+) or absence (–) of 0.4 mM CuSO4, and cell concentrations were determined by hemacytometry. Cells were split to 3 x 106/ml every third day.

 



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  		FIG. 9.
mtSSB RNAi-induced depletion of mtDNA is rescued by exogenous expression of wild-type mtSSB but not by the DNA-binding mutant mtSSB W79AT/F85A. The mtDNA:nDNA ratio in the Schneider S2, Mt/Hy, INV, INV + wild type, and INV + W79T/F85A cell lines described in the legend to Fig. 7 was determined by Southern blot analysis. DNAs were isolated as described under "Experimental Procedures" after growth for 10 days in the presence or absence of 0.4 mM CuSO4. A fragment of the ATPase6 gene was used as the probe for determination of mtDNA content, and a DNA fragment from the nuclear histone gene cluster was used for normalization. The value for the S2 cell line grown in the absence of 0.4 mM CuSO4 was taken as 1.

 
To link directly the observed cell growth defect to the mtDNA depletion, we attempted to rescue the phenotype with the production of exogenous wild-type mtSSB. To do so, we developed doubly transfected cell lines using the plasmids carrying the 3'-UTR inverted repeats and an mtSSB cDNA that lacks the endogenous 3'-UTR. After induction by 0.4 mM CuSO4, both cell growth rate and mtDNA copy number were restored to near wild-type levels (Figs. 8 and 9). Finally, we examined the effects of co-expression of the W79T/F85A mtSSB using the same strategy. We found that under these conditions, in which wild-type mtSSB levels are low, this DNA-binding and functionally impaired form neither rescues the cell growth defect nor restores normal mtDNA copy number (Figs. 8 and 9).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
SSBs serve critical roles in DNA replication, repair, and recombination (17). Mitochondrial SSBs share similar physical and biochemical properties with E. coli SSB (5, 11, 18–21), with which they exhibit a high degree of amino acid sequence conservation (21–23). Although high affinity DNA binding by SSBs can occur independently of other proteins, both functional and physical interactions between SSBs and a variety of enzymes involved in the above processes have been documented and, in particular, interactions between SSBs and replicative DNA polymerases have been demonstrated in bacterial, nuclear, and viral systems (17). Our previous biochemical studies are consistent with an important role for mtSSB in initiation and elongation of DNA strands in mtDNA replication, which has been documented genetically by the fact that an insertion in the third intron of the Drosophila gene (lopo) results in developmental lethality, concomitant with the complete loss of mtDNA and respiratory capacity (24). In an extension of these studies, we document here the variable effects of DNA-binding mutants of Drosophila mtSSB upon the biochemical activity of pol {gamma} and, in a physiological context, upon cell growth and mtDNA maintenance.

Several amino acid residues that are important for DNA binding were identified in E. coli SSB by a combination of biophysical methods, genetics, and site-directed mutagenesis (8). In particular, Trp54 and Phe60 function synergistically within an extended {alpha}-helical structure in which hydrophobic interactions with nucleotide bases contribute substantially to the stability of SSB·DNA complexes (25). These amino acids are conserved in mtSSBs, and mutagenesis studies of mouse and human mtSSB confirm their roles in DNA binding (11, 12). Furthermore, the crystal structure of human mtSSB shows that they occupy similar spatial positions (26). We show here that mutations of Trp79 and Phe85 in Drosophila mtSSB have variable effects on DNA binding depending on the specific amino acid substitution, as might be anticipated from the above studies. Notably, however, these DNA-binding defects are amplified when DNA synthesis is measured as the readout. Although all of the DNA-binding activities of single mutants were near wild-type levels under low salt conditions, their ability to stimulate DNA synthesis by pol {gamma} was reduced 1.5- to 3.7-fold, and the W79T/F85A mutant showed a dramatic reduction of 8-fold while retaining 50% of its DNA-binding activity. It seems likely that these effects would be translated into a correspondingly large effect in vivo, although earlier biochemical studies provide some evidence that elevated mtSSB concentrations might mitigate such an effect (12). In that regard, we have shown that both the temporal and spatial expression of mtSSB vary substantially during Drosophila development (15), and there may be a threshold that can not be met that would give rise to tissue- and/or stage-specific phenotypic defects.

Ionic conditions fluctuate substantially in the mitochondrial matrix (27), and we have shown that pol {gamma} exhibits broad salt optima for both its DNA polymerase and exonuclease activities (2, 28). Stimulation by mtSSB also occurs over a broad salt range (2, 5). The stimulatory effects of mtSSB are minimal at higher salt concentrations where activity is high and processivity is reduced. Although wild-type mtSSB enhances processivity ~3-fold at moderate salt, we find that neither the W79A mutant with only a modest defect in DNA binding nor the W79T/F85 mutant was able to do so.

What factors mediate the functional interactions of pol {gamma} with mtSSB? Functional interactions may occur upon DNA binding and/or by specific physical interactions. E. coli SSB is known to bind DNA in several modes depending on ionic conditions (29). Studies of mtSSBs also show salt-dependent effects on DNA binding and, in particular, upon binding-site size and cooperativity of DNA binding (5, 11, 18). Ionic conditions likely affect both DNA conformation and SSB structure and, hence, its interactions with pol {gamma}. It is perhaps not surprising then that mutant forms of mtSSB show variable defects in different assays and under different conditions, although taken together, our data suggest flexibility in presumptive protein-protein interactions and perhaps in the mode of mitochondrial DNA replication in vivo. In this regard, there is now substantial data supporting both strand-displacement and strand-coupled models of mtDNA replication (30–36), for which the versatility in pol {gamma}-mtSSB interactions seems particularly well suited.

mtDNA depletion syndromes are common in human mitochondrial diseases caused by defects in mitochondrial biogenesis that result from mutations in nuclear genes involved in mtDNA maintenance. To date, these genes include those encoding both mtDNA replication proteins and proteins involved in maintaining nucleotide pool balances (1, 37). Though no known human diseases have yet been mapped to mutations in the mtSSB gene, we found that a hypomorphic mutation in the Drosophila gene results in developmental lethality (24). In an extension of this finding, we show that, whereas overexpression of wild-type mtSSB affects neither growth rate nor mtDNA copy number in cultured cells, a modest reduction in mtDNA copy number is produced by simple overexpression of the W79T/F85A mutant mtSSB. We exploited our earlier finding to develop a strategy to demonstrate further that reduction of wild-type mtSSB protein levels by interfering dsRNA methodology results in a substantial mtDNA depletion phenotype that interferes with cell growth, and that these human disease-like features can be rescued by the expression of exogenous wild-type mtSSB but not the mutant form. The current studies open avenues for the development of new Drosophila animal models in which induced mtDNA depletion may be manipulated by controlling exogenous expression of either wild-type or mutant forms. Such investigations should offer substantial new insight into the mechanism and progression of human mtDNA depletion syndromes and possible intervention schemes.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant GM45295. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed. Tel.: 517-353-6703; Fax: 517-353-9334.

1 The abbreviations used are: pol, DNA polymerase; nt, nucleotide(s); mtSSB, mitochondrial single-stranded DNA-binding protein; mtDNA, mitochondrial DNA; GMSA, gel mobility shift assay; UTR, untranslated region. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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