Reduced Stimulation of Recombinant DNA Polymerase γ and Mitochondrial DNA (mtDNA) Helicase by Variants of Mitochondrial Single-stranded DNA-binding Protein (mtSSB) Correlates with Defects in mtDNA Replication in Animal Cells*

Background: Mitochondrial single-stranded DNA-binding protein (mtSSB) coordinates the functions of the mitochondrial DNA (mtDNA) polymerase and helicase. Results: mtSSB variants are defective in stimulating mtDNA polymerase and helicase. Conclusion: mtSSB uses distinct structural elements to interact with mtDNA polymerase and helicase. Significance: Novel insights are presented into the mechanism of mtDNA replication and the role of mtSSB in human diseases involving mtDNA depletion. The mitochondrial single-stranded DNA-binding protein (mtSSB) is believed to coordinate the functions of DNA polymerase γ (pol γ) and the mitochondrial DNA (mtDNA) helicase at the mtDNA replication fork. We generated five variants of the human mtSSB bearing mutations in amino acid residues specific to metazoans that map on the protein surface, removed from the single-stranded DNA (ssDNA) binding groove. Although the mtSSB variants bound ssDNA with only slightly different affinities, they exhibited distinct capacities to stimulate the DNA polymerase activity of human pol γ and the DNA unwinding activity of human mtDNA helicase in vitro. Interestingly, we observed that the variants with defects in stimulating pol γ had unaltered capacities to stimulate the mtDNA helicase; at the same time, variants showing reduced stimulation of the mtDNA helicase activity promoted DNA synthesis by pol γ similarly to the wild-type mtSSB. The overexpression of the equivalent variants of Drosophila melanogaster mtSSB in S2 cells in culture caused mtDNA depletion under conditions of mitochondrial homeostasis. Furthermore, we observed more severe reduction of mtDNA copy number upon expression of these proteins during recovery from treatment with ethidium bromide, when mtDNA replication is stimulated in vivo. Our findings suggest that mtSSB uses distinct structural elements to interact functionally with its mtDNA replisome partners and to promote proper mtDNA replication in animal cells.

The mitochondrial single-stranded DNA-binding protein (mtSSB) is believed to coordinate the functions of DNA polymerase ␥ (pol ␥) and the mitochondrial DNA (mtDNA) helicase at the mtDNA replication fork. We generated five variants of the human mtSSB bearing mutations in amino acid residues specific to metazoans that map on the protein surface, removed from the single-stranded DNA (ssDNA) binding groove. Although the mtSSB variants bound ssDNA with only slightly different affinities, they exhibited distinct capacities to stimulate the DNA polymerase activity of human pol ␥ and the DNA unwinding activity of human mtDNA helicase in vitro. Interestingly, we observed that the variants with defects in stimulating pol ␥ had unaltered capacities to stimulate the mtDNA helicase; at the same time, variants showing reduced stimulation of the mtDNA helicase activity promoted DNA synthesis by pol ␥ similarly to the wild-type mtSSB. The overexpression of the equivalent variants of Drosophila melanogaster mtSSB in S2 cells in culture caused mtDNA depletion under conditions of mitochondrial homeostasis. Furthermore, we observed more severe reduction of mtDNA copy number upon expression of these proteins during recovery from treatment with ethidium bromide, when mtDNA replication is stimulated in vivo. Our findings suggest that mtSSB uses distinct structural elements to interact functionally with its mtDNA replisome partners and to promote proper mtDNA replication in animal cells.
Replication of animal mitochondrial DNA (mtDNA) 2 is mediated by three protein players that function directly at the replication fork: DNA polymerase ␥ (pol ␥), which catalyzes DNA synthesis per se; mtDNA helicase (also known as Twinkle), which unwinds double-stranded DNA to provide a singlestranded DNA (ssDNA) substrate for pol ␥; and mitochondrial single-stranded DNA-binding protein (mtSSB), which binds ssDNA to protect it against damage and to coordinate the functions of pol ␥ and mtDNA helicase (1,2). mtSSB has been shown to stimulate the DNA polymerase activity of pol ␥ both in the Drosophila and human systems (3,4) and the unwinding activity of the human mtDNA helicase (HsmtDNA helicase) (4,5). In addition, human mtSSB (HsmtSSB) stimulates stranddisplacement DNA synthesis promoted by the combined action of the human pol ␥ (Hspol ␥) and HsmtDNA helicase (2). In vivo, absence or depletion of mtSSB protein causes reduction of mtDNA copy number in Drosophila and human cells in culture (6,7), and lethality at the third larval stage in developing D. melanogaster (8). Although mtSSB mutations have not thus far been documented in association with human diseases, as is the case for the genes encoding pol ␥ and mtDNA helicase, a recent report shows a correlation between the levels of mtSSB protein, mtDNA copy number and the aggressiveness of human osteosarcoma cells, suggesting a link between mtDNA replication and cancer progression (9).
To date, mutagenesis studies of mtSSB have focused largely on biochemical and physiological properties of amino acid residues involved in tetramerization and/or ssDNA binding affin-ities. Li and Williams (10) demonstrated the importance of highly conserved residues among tetrameric SSBs for protomer-protomer interactions in the mouse mtSSB, including H69 (H69, HsmtSSB; H64, DmmtSSB (fly)), and for ssDNA binding, including W49, W68, and F74 (W49, W68 and F74, HsmtSSB; Y50, W63, and F69, DmmtSSB). DmmtSSB variants bearing W63A and/or F69A substitutions were defective in DNA binding in vitro as predicted, and the corresponding mutant genes promoted mtDNA depletion when expressed in Drosophila S2 cells in culture (6). Interestingly, these proteins were also defective in stimulating the DNA polymerase activity of Drosophila pol ␥ (Dmpol ␥). The homologous residues in the prototypical tetrameric SSB from Escherichia coli (EcSSB) have been implicated in similar functions (11)(12)(13), in keeping with the high degree of conservation between bacterial and mitochondrial SSBs.
We sought to investigate properties that are specific to animal mtSSBs in the context of mtDNA replication by targeting amino acid residues that are well-conserved across animal species, but differ from those in EcSSB. Unlike mtSSBs, the catalytic subunit of pol ␥ and the mtDNA helicase share a common ancestry with the bacteriophage T7 DNA polymerase and primase-helicase enzymes, respectively (14). This raises the interesting question of how the bacterial-like mtSSB has evolved to function in concert with the T7-like pol ␥ and mtDNA helicase in the mtDNA replisome. In an earlier study, we explored biochemically the capacity of terminal deletion variants of HsmtSSB to bind ssDNA and stimulate Hspol ␥ and HsmtDNA helicase (4). We determined that the absence of the termini was not deleterious and in fact, the stimulation of Hspol ␥ was higher in the presence of the variants than with wild-type HsmtSSB. In the present report, we describe biochemical and physiological studies of additional mtSSB variants carrying amino acid substitutions or deletions in residues that map outside of the DNA binding groove, mostly in loop regions on the surface of the protein (Fig. 1). Our findings provide insight into how mtSSB coordinates the functions of pol ␥ and mtDNA helicase and participates in the formation of the mtDNA replisome, a multi-protein complex in which defects caused by genetic mutations can culminate in aging, and in a variety of human diseases (15)(16)(17)(18).

EXPERIMENTAL PROCEDURES
Nucleotides and Nucleic Acids-Unlabeled deoxy-and ribonucleotides were purchased from Amersham Biosciences. [␣-32 P]dATP and [␥-32 P]ATP were purchased from MP Biomedicals. Wild-type M13 (6,407 nt) and pBSKSϩ (2,958 nt) DNAs were prepared by standard laboratory methods. Oligodeoxynucleotides complementary to these DNAs were synthesized in an Applied Biosystems oligonucleotide synthesizer. The singly primed M13 DNA used in DNA polymerase assays was prepared as described previously (19). The substrate for the DNA unwinding assays and the 48-mer oligodeoxynucleotide used in gel mobility shift assays (GMSA) were prepared as described in Oliveira and Kaguni (4). The dsRNA used for knockdown of endogenous DmmtSSB (DmmtSSB endo ) in S2 cells was produced in vitro using MEGAscript T7 kit (Ambion), according to manufacturer's specifications. The DNA used as template in the reaction to produce the dsRNA was generated via PCR with the following primers: 5Ј-TAA-TACGACTCACTATAGGGAGAAATTTAAGCCCAGAT-CAC-3Ј and 5Ј-TAATACGACTCACTATAGGGAGATG-GAGTACGACTACGCATG-3Ј, where the underlined sequences represent the minimal promoter needed for T7 RNA polymerase transcription initiation. The amplified fragment (ϳ120 bp) contained the entire sequence of the 3Ј-UTR of the mRNA of DmmtSSB endo .
ssDNA Binding and Gel Mobility Shift Assays-Reaction mixtures (20 l) contained 20 mM Tris-HCl, pH 7.5, 1 mM DTT, 4 mM MgCl 2 , 50 mM NaCl, 36 fmol 5Ј-end-labeled 48-mer, and the indicated amounts of the HsmtSSB proteins. Incubation was at 20°C for 10 min. Samples were processed and electrophoresed in 6% native polyacrylamide gels. The amounts of shifted and free oligonucleotide were quantitated as follows: % ssDNA bound ϭ (V S /(V S ϩ V F )) x 100, where V S represents the volume of the shifted and V F the volume of unshifted oligonucleotide in the sample lane of interest.
dsDNA Unwinding Assays-Reaction mixtures (50 l) contained 20 mM Tris-HCl, pH 7.5, 10% glycerol, 500 g/ml bovine serum albumin, 10 mM DTT, 4 mM MgCl 2 , 3 mM ATP, 50 mM KCl, 0.4 nM of DNA unwinding substrate, 3.5 nM of mtDNA helicase (as hexamer), and the indicated concentrations of HsmtSSB proteins. The reactions were pre-incubated at 37°C for 5 min prior to the addition of the helicase. Once the helicase was added, the reactions were incubated further at 37°C for 30 min and then stopped by the addition of 5 l of 10ϫ stop solution (6% SDS, 100 mM EDTA, pH 8.0), followed by 5 l of 10ϫ loading buffer (50% glycerol, 0.25% bromphenol blue). DNA products were fractionated from substrate by electrophoresis in a 22% polyacrylamide gel (59:1 acrylamide/bisacrylamide) using 1ϫ TBE (90 mM Tris-HCl-borate, 2 mM EDTA) at 600 V for ϳ30 min. After electrophoresis, the gel was processed and the unwinding activity was quantitated as described in Oliveira and Kaguni (4).
Generation of Stable Cell Lines, Knockdown of DmmtSSB endo , and Induction of DmmtSSB Variants-Drosophila Schneider S2 cells were cultured at 25°C in Drosophila Schneider medium supplemented with 10% fetal bovine serum, 50 units/ml penicillin G and 50 g/ml streptomycin sulfate (Invitrogen). For establishment of stable lines, ϳ1.2 ϫ 10 7 cells were transfected with 1 g of pMt/Hy vector carrying DmmtSSBwt or variant genes using the Effectene kit (Qiagen), according to manufacturer's specifications. Hygromycin-resistant cells were selected with 0.2 mg/ml hygromycin for at least 5 passages, before transfer to standard growth medium. For knockdown of endogenous DmmtSSB protein, 3 g of dsRNA (representing the 3Ј-UTR of DmmtSSB endo mRNA) were delivered to 3 ϫ 10 6 cells using the Effectene kit. Cells were cultured for 3 days before reduction in levels of DmmtSSB endo protein was observed. In the experiments with EtBr, cells were treated with 0.2 g of EtBr per ml of growth medium 3 days prior to treatment with dsRNA. For induction of expression of DmmtSSB variants, cells treated with dsRNA for 3 days were subjected to the indicated concen-trations of CuSO 4 to promote appropriate expression from the metallothionein promoter (22).
Quantitative Real Time-PCR-Total DNA was extracted from S2 cells using phenol/chloroform protocol and standard laboratory procedures. mtDNA copy number was determined by quantification of relative amounts of mtDNA to nuclear DNA via real time amplification of a fragment of the mitochondrial 16 S gene (primers: 5Ј-AAAAAGATTGCGACCTC-GAT-3Ј and 5Ј-AAACCAACCTGGCTTACACC-3Ј) and the nuclear RpL32 gene (primers: 5Ј-AGGCCCAAGATCGTGAA-GAA-3Ј and 5Ј-TGTGCACCAGGAACTTCTTGAA-3Ј). Reactions were performed using SYBR Green JumpStart TM Taq ReadyMix TM (Sigma-Aldrich) on a 7500 Real Time PCR System instrument (Applied Biosystems), according to manufacturer's specifications.
Molecular Modeling-To construct the model of HsmtSSB bound to ssDNA, the crystal structure of HsmtSSB (PDB code 3ULL (23)) was aligned to the structure of EcSSB bound to ssDNA (PDB code 1EYG (13)) using PyMol (The PyMOL Molecular Graphics System, Version 1.3, Schrödinger, LLC). The atomic coordinates of EcSSB were then removed, allowing placement of the ssDNA in the DNA binding groove of HsmtSSB without major atomic collisions.
To construct the model of the Hspol ␥-HsmtSSB interaction, the regions of HsmtSSB that are disordered in the crystal structure (E1-V9, N terminus; S54-L59, loop 2,3; and D126-E132, C terminus; PDB code 3ULL (23)) were first modeled using MODELLER (24) at default parameters. The tetrameric HsmtSSB structure containing the modeled termini and loop 2,3 was submitted to the software ClusPro 2.0 (25) for docking onto the crystal structure of the Hspol ␥ holoenzyme (PDB code 3IKM (26)) at default parameters. All of the figures were generated and the model was analyzed using the software PyMol.

RESULTS
We selected candidate regions of the mtSSB protein for mutagenesis based on the assumption that residues important for functional and/or physical interactions with pol ␥ and mtDNA helicase would be well conserved among animal mtSSB sequences, and also locate on the surface of the protein structure. We avoided residues conserved between mtSSBs and EcSSB, because of their essential roles in the stabilization of the tertiary/quaternary structure of the protein and/or in contacting DNA directly; disrupting such properties would be expected to cause indirect effects on the ability of the protein to stimulate its mtDNA replisome partners (6). The selected residues and an evaluation of their locations in a molecular model of HsmtSSB bound to ssDNA that indicates none of them are likely to interact directly with ssDNA are shown in Fig. 1.
We then examined the ability of the HsmtSSB variants and non-cognate SSBs to stimulate the dsDNA unwinding activity of HsmtDNA helicase. Interestingly, helicase stimulation by HsmtSSBloop12 and HsmtSSBloop23 reflects their increased ssDNA binding, as maximal stimulation is achieved at lower concentrations of these variants (37 nM) as compared with HsmtSSBwt (50 nM). Nevertheless, maximal stimulation by HsmtSSBloop12 is reduced ϳ40% as compared with that by HsmtSSBwt, whereas the reduction caused by HsmtSSBloop23 is only ϳ15% (Fig. 2B). In addition, HsmtSSBloop45-2 also showed a ϳ40% reduced capacity to stimulate HsmtDNA helicase in vitro, suggesting that residues in loop 1,2 and 4,5-2 are responsible for the functional interactions that we observed between HsmtSSB and HsmtDNA helicase. In comparison, we found that the maximal stimulation of the HsmtDNA helicase by HsmtSSBwt is similar to that contributed by the non-cognate DmmtSSBwt and EcSSB (Fig. 2C), in contrast to the results reported by Korhonen et al. (5).
Next, we measured the stimulation of the DNA polymerase activity of Hspol ␥ on a singly primed ssDNA template. As we reported previously (4), we observed that the DNA polymerase activity of Hspol ␥ in the absence of HsmtSSB was ϳ2-fold higher at 100 mM than at 20 mM KCl. In contrast, in the presence of the variant HsmtSSBs, Hspol ␥ was stimulated at 20 mM KCl, but inhibited completely at 100 mM KCl (Fig. 3). Furthermore, the variants HsmtSSBloop23, HsmtSSB␣1, and HsmtSSBloop45-1 show a reduced capacity to stimulate DNA synthesis by Hspol ␥ (ϳ60% of that of HsmtSSBwt), which indicates that the altered amino acid residues in these variants are important for the functional interactions between HsmtSSB and Hspol ␥. Notably, the stimulation of Hspol ␥ by these defective variants is similar to that observed with the non-cognate EcSSB, and the HsmtSSB residues identified as important for Hspol ␥ stimulation differ from those shown to be important for stimulation of HsmtDNA helicase (Fig. 2B).

mtDNA Depletion in Cells Overexpressing Variants of DmmtSSB under Conditions of Mitochondrial Homeostasis-
We developed a Drosophila S2 cell system in which we knocked down the DmmtSSB protein (DmmtSSB endo ) by treatment with dsRNA homologous to the 3Ј-UTR of the endogenous gene and expressed an exogenous DmmtSSB variant, to evaluate mtDNA maintenance and replication under various physiological conditions. dsRNA treatment depleted DmmtSSB endo and mtDNA copy number to ϳ10 and ϳ40% of the levels in the control cells, respectively. In agreement with previous data from our laboratory (6), expression of an exogenous DmmtSSBwt gene promoted an increase in mtDNA copy number proportional to the level of protein induction (Fig. 4A).
Defects in mtDNA Repletion upon Expression of DmmtSSB Variants-We then investigated mtDNA repletion in S2 cells expressing the DmmtSSB variants after mtDNA depletion with ethidium bromide (EtBr). Application of low concentrations of EtBr in the cell culture (0.2 g/ml growth media) depleted FIGURE 2. ssDNA binding affinities and stimulation of HsmtDNA helicase by HsmtSSB variants. A, ssDNA-binding affinity was evaluated by GMSA, as described under "Experimental Procedures." The fraction of unbound and bound oligomer was quantitated, and the data were plotted as the average percent of substrate utilized from three independent experiments. B and C, DNA unwinding assays were performed as described under "Experimental Procedures," using 3.5 nM of HsmtDNA helicase (as hexamer). "Ϫ" and "ϩ boil" lanes represent the intact and denatured (heated to 100°C for 2 min prior to loading) substrate, respectively. The data in B represent the average of unwound substrate as percent from three independent experiments. mtDNA to ϳ30% of that of control cells after 3 days of treatment, without affecting DmmtSSB endo levels (Fig. 5A). After removal of EtBr from the culture, control cells recovered completely from mtDNA depletion in ϳ9 days, whereas mtDNA levels in DmmtSSB endo -knockdown cells remained substantially lower (55% of that of control cells) for as long as 15 days. Cells carrying the exogenous DmmtSSBwt gene were treated with dsRNA to knock down DmmtSSB endo , and with CuSO 4 to induce the expression of DmmtSSBwt after EtBr treatment. When DmmtSSBwt was expressed at a level comparable to DmmtSSB endo in control cells (in the presence of 0.04 mM CuSO 4 ), recovery from mtDNA depletion also occurred in ϳ9 days, after a 3-day lag time. This delay may result from our experimental design, which requires CuSO 4 addition to the cells 3 days after the dsRNA treatment (see "Experimental Procedures" for details). In contrast to endogenous-level DmmtSSBwt expression, the 3-day lag time was not observed upon DmmtSSBwt overexpression, and mtDNA copy number was restored to normal levels at a faster rate (Fig. 5A, right panel).
Next, we induced the five DmmtSSB variant proteins at two expression levels after the combined EtBr and dsRNA treatments: expression levels equivalent to DmmtSSB endo under normal growth conditions (endogenous-level) and overexpression (Fig. 5B). We observed distinct phenotypes of mtDNA depletion, allowing us to categorize the variants in three groups. The first comprises DmmtSSBloop12, which is the only protein that caused strong reduction of mtDNA copy number under conditions of mitochondrial homeostasis (40% of that of control cells), but showed only a small-to-moderate effect under recovery from depletion, either upon endogenous-level expression or overexpression of the protein (60 -80% of that of cells expressing DmmtSSBwt). The second group comprises DmmtSSBloop23, which caused a clear mtDNA depletion (15-30% of that of control cells) only when high levels of the protein were achieved (overexpression conditions). The third group comprises DmmtSSB␣1, loop45-1 and loop45-2, which caused reduction in mtDNA copy number under all conditions tested (10 -50% of that of control cells), with more severe phe-

DISCUSSION
DNA replisomes are well-coordinated machines in which the activity of one protein component is regulated by that of the others. For example, replicative helicases from bacteria and viruses that unwind dsDNA at low catalytic rates become highly efficient when coupled to DNA polymerases and/or SSBs (reviewed in Refs. 27,28). At the animal mtDNA replication fork, little is known about how the three key components of the replisome function coordinately to copy the mitochondrial genome. Studies to date have focused largely on structure-function relationships in pol ␥ and mtDNA helicase, and their association with human diseases, such as progressive external ophthalmoplegia (PEO) and Alpers syndrome (1,17,18,29,30). Furthermore, deregulation of replisome function in vivo has been shown in human cell lines expressing active site mutants of pol ␥ and mtDNA helicase: dysfunctional pol ␥ mutants induced delayed lagging-DNA strand synthesis causing replica-tion stalling, whereas defective helicases caused an increased rate of initiation of lagging-DNA strand synthesis relative to the rate of mtDNA fork progression (31).
Here, we present a biochemical and physiological analysis of the role of mtSSB at the mtDNA replication fork. We generated five recombinant HsmtSSB variants designed not to exhibit defects in ssDNA binding, and found that two of them (HsmtSSBloop12 and loop23) actually bound ssDNA with slightly higher affinity than HsmtSSBwt, indicating that loops 1,2 and 2,3 in HsmtSSB are either closer to the DNA binding channel and/or participate negatively in an alternative binding mode. We speculate that loop 2,3 of vertebrate mtSSBs, which contain a 6 -7 amino acid insertion relative to this region in invertebrate mtSSBs and bacterial SSBs, can form a flexible domain that may disturb ssDNA binding in the absence of pol ␥. Most of the amino acid residues in loop 2,3 are disordered in the crystal structure of HsmtSSB (23), the loop carries a slight negative charge as analyzed by electrostatic surface potential, and it appears to be important for pol ␥ stimulation in vitro and in vivo, as discussed below.
Our group and others have shown that HsmtSSBwt interacts functionally with HsmtDNA helicase using a preformed repli- cation fork-like substrate to stimulate its dsDNA unwinding activity (4,5). On such a simple substrate one might predict that the helicase would be stimulated in vitro by any SSB capable of preventing reannealing of the unwound DNA. Indeed, we show here that both EcSSB and DmmtSSB can stimulate the activity of HsmtDNA helicase similarly to HsmtSSBwt. In contrast, Korhonen et al. (5) failed to show a stimulation by EcSSB and interpreted their findings as a specific interaction between HsmDNA helicase and HsmtSSBwt. Though we cannot document conclusively an explanation for the different observations discussed here, we note that the stimulation of HsmtDNA helicase by HsmtSSBwt reported by Korhonen et al. (5) was only moderate (ϳ2.5-fold), whereas our data show a ϳ7-fold effect ((4), Fig. 2B) that may allow a discrimination of specificity.
Our studies of the fly and human mtSSB variants suggest that residues in loop 1,2 and loop 4,5 serve important but distinct roles in interacting with the mtDNA helicase. Interestingly, loop 1,2 of DmmtSSB appears to be involved only in mtDNA homeostasis in S2 cells, whereas DmmtSSBloop45-2 induces moderate and severe mtDNA depletion under homeostasis and EtBr recovery conditions, respectively. During recovery from EtBr treatment, mtDNA replication is apparently stimulated and may occur by a different mechanism and/or more independently from the cell cycle than under homeostasis conditions. We speculate that loop 4,5-2 provides the primary point of interaction between mtSSB and mtDNA helicase that promotes efficient dsDNA unwinding during mtDNA replication. Loop 1,2 may also contact the helicase, perhaps facilitating enzyme function only to maintain mtDNA at normal levels under homeostasis conditions. This hypothesis correlates with our findings that HsmtSSBloop45-2 shows the lowest stimulation of HsmtDNA helicase in vitro, even at protein levels 3-fold higher than that required to saturate the ssDNA substrate (data not shown). Alternatively, disruption of loop 4,5 may cause a primary biochemical defect that we have not yet tested but that results in reduced DNA polymerase and/or helicase stimulation in vitro, and depletion of mtDNA in cultured cells. Though cooperativity in ssDNA binding is debated in mtSSBs (32,33), loss of this property would represent a plausible explanation, because loop 4,5 of EcSSB has been implicated in cooperative DNA binding in the bacterial protein (13).
Functional interactions between pol ␥ and mtSSB have been shown clearly with native and recombinant forms of the Drosophila proteins (3,6,8,33). Both the DNA polymerase and exonuclease activities of the Dmpol ␥ holoenzyme are stimulated 15-to 20-fold on singly primed ssDNA templates by DmmtSSB over a broad range of KCl concentrations. DmmtSSB increases primer recognition, DNA synthesis, processivity, and mispair hydrolysis during proofreading DNA synthesis by Dmpol ␥. In the human system, Hspol ␥ holoenzyme is stimulated by HsmtSSB only at very low ionic strength, and the overall stimulation is only ϳ2.5-fold (Ref. 4 Fig. 3). We showed here that EcSSB can also interact functionally with Hspol ␥ and stimulate its DNA polymerase activity ϳ1.5-fold at 20 mM KCl, establishing a baseline level of Hspol ␥ stimulation in vitro. The HsmtSSB variants in loop23, ␣1 and loop45-1 were able to stimulate the mitochondrial replicase similarly to EcSSB, suggesting possible defects in pol ␥-mtSSB interactions.

and
We found that DmmtSSB␣1 and loop45-1 caused mtDNA depletion in S2 cells under all conditions tested, whereas DmmtSSBloop23 only reduced mtDNA copy number when expressed Ͼ50-fold higher than DmmtSSB endo . Residual DmmtSSB endo , and mixed DmmtSSB endo -DmmtSSBloop23 tetramers in cells expressing DmmtSSBloop23 at levels equivalent to the endogenous level (resulting in a ratio of ϳ10 DmmtSSBloop23 to 1 DmmtSSB endo ) appear to be sufficient to maintain mtDNA at the same level as in DmmtSSB endo -knockdown cells (40 -50% of control S2 cells). Thus, at endogenouslevel expression, the increased ssDNA-binding ability of DmmtSSBloop23 may compensate for the loss of native pol ␥-mtSSB interactions. However, as indicated above, loop 2,3 is one of the most variable regions among animal mtSSBs, suggesting that the biochemical results observed with HsmtSS-Bloop23 and the physiological response observed with DmmtSSBloop23 are likely species-specific effects that represent distinct mechanisms of pol ␥ regulation. In fact, as we have speculated previously (4), the presence of a dimeric accessory subunit exclusive to vertebrate pol ␥s might increase DNA synthesis by the mitochondrial replicase to a level that makes the contribution of mtSSB to the overall rate of DNA synthesis only moderate. By comparison, the substantial stimulation of DmmtSSB on the DNA polymerase activity of Dmpol ␥ holoenzyme appears to correlate with the presence of a monomeric accessory subunit in insect pol ␥s, and to a short loop 2,3 in mtSSBs.
To provide structural support for our current results and hypotheses, we developed a computational model in which we docked the crystal structure of HsmtSSB onto the structure of Hspol ␥ (Fig. 6). The model offers insight into how loop 2,3 and ␣-helix 1 of HsmtSSB may interact with the intrinsic processivity (IP) and fingers subdomains of the catalytic subunit (Hspol ␥-␣) of the mitochondrial replicase (Fig. 6, A and B). We did not observe any evidence of protein-protein interactions involving loop 4,5-1 of HsmtSSB, presumably because the amino acid residues of additional sites of interaction with mtSSB are disordered in the crystal structure of Hspol ␥-␣, such as residues 319 -344 in the exonuclease domain and 674 -709 in the IP subdomain of the spacer domain (26). Notably, amino acid substitutions in Hspol ␥-␣ alleles that are associated with Alpers, Parkinson, Charcot-Marie Tooth, PEO, and ataxia-neuropathy diseases in human patients (17, 34 -38) locate in the vicinity of the region of potential interaction between loop 2,3 of HsmtSSB and Hspol ␥-␣ (Fig. 6C), suggesting that defects in Hspol ␥ stimulation by HsmtSSB can explain the phenotype of mtDNA depletion and/or deletions associated with such diseases. Interestingly, amino acid residues G619, W620, and Y622 of Hspol ␥-␣ are found in the same region (Fig. 6C), supporting our previous results that Dmpol ␥ variants bearing G575A/W576A/F578A substitutions had reduced stimulation by DmmtSSB in vitro (39). Additionally, the model suggests that either of the negatively charged termini of HsmtSSB could occupy partially the positively charged DNA-binding pocket of the mitochondrial replicase, disrupting the progression of DNA synthesis by dislodging transiently the primer-template DNA from the DNA polymerase active site of pol ␥ (Fig. 6D). HsmtSSB proteins lacking either or both termini would then render the holoenzyme readily available for DNA binding, thus promoting increased DNA synthesis, as shown in our previous report (4). Unfortunately, DmmtSSB variants lacking the N or C terminus are not stable when expressed in Drosophila S2 cells, which prevents us from exploring this phenomenon further at present.
Although physical interaction data are not presented here and similar structural analyses and modeling predictions cannot currently be performed reliably for mtSSB interactions with mtDNA helicase, our findings suggest that mtSSB uses a repertoire of structural elements in stimulating pol ␥ and mtDNA helicase to ensure proper mtDNA maintenance in animal cells. Its functional properties may result from multiple sites of physical interaction that may also be associated with different mtDNA replication modes in vivo. In any case, it is evident that the mechanisms by which mtSSBs function at the mtDNA rep-lication fork are distinct from those that bacterial, viral and nuclear eukaryotic SSBs employ in their respective replisomes, despite their shared features in ssDNA binding (23, 40 -43). Further biochemical, structural and physiological studies are clearly needed to test the relevant hypotheses and to help understand the mechanisms of mtDNA replication in healthy cells and in myriad disease states.