©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Stimulation of Drosophila Mitochondrial DNA Polymerase by Single-stranded DNA-binding Protein (*)

(Received for publication, September 22, 1994)

Andrea J. Williams Laurie S. Kaguni (§)

From the Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Mitochondrial DNA polymerase from Drosophila embryos has been characterized with regard to its mechanism of DNA synthesis in the presence of single-stranded DNA-binding protein from Escherichia coli. The rate of DNA synthesis by DNA polymerase was increased nearly 40-fold upon addition of single-stranded DNA-binding protein. Processivity of mitochondrial DNA polymerase was increased 2-fold, while its intrinsic rate of nucleotide polymerization was unaffected. Primer extension analysis showed that the rate of initiation of DNA strand synthesis by DNA polymerase was increased 25-fold in the presence of single-stranded DNA-binding protein. Our results indicate that the stimulation of Drosophila DNA polymerase by single-stranded DNA-binding protein results primarily from an increased rate of primer recognition and binding. Concurrent achievement of maximal activity and processivity by mitochondrial DNA polymerase in the presence of binding protein suggests that DNA polymerase , like other replicative DNA polymerases, associates with accessory factors in vivo to catalyze efficient and processive DNA synthesis.


INTRODUCTION

Single-stranded DNA-binding protein (SSB) (^1)from Escherichia coli has numerous roles in the replication of the bacterial DNA genome. It enhances helix destabilization by DNA helicase by preventing renaturation of unwound duplex (1) and protects single-stranded DNA from nuclease digestion(2) . SSB increases the fidelity of DNA polymerase, likely by increasing the rigidity of the DNA template, promoting increased steric hindrance between template and incorrect nucleotides(3, 4) . Furthermore, SSB increases the processivity of DNA polymerase by destabilizing the DNA secondary structure that causes enzyme pausing and dissociation(5, 6) . The presence of E. coli SSB in sufficient amounts to saturate the single-stranded DNA (ssDNA) template results in the stimulation of E. coli DNA polymerases II and III holoenzyme(7, 8) , bacteriophage T7 DNA polymerase(9) , herpes simplex virus-1 DNA polymerase(10) , and human DNA polymerase holoenzyme(11) . In contrast, SSB has no effect on E. coli DNA polymerase I (12) or on bacteriophage T4 DNA polymerase(13) .

The mitochondrial counterpart of E. coli SSB, mitochondrial SSB (mtSSB), has been purified from rat liver(14) , Xenopus laevis(15) , and Saccharomyces cerevisiae(16) . The gene encoding mtSSB from S. cerevisiae(16) and the corresponding cDNAs from X. laevis(17) and rat and human tissues (18) have been cloned and sequenced. Mitochondrial SSB comprises a 13-15-kDa polypeptide with an estimated native molecular mass of 56 kDa, suggesting that it is a tetramer in solution(14, 16, 18, 19) . It binds cooperatively to DNA with a binding site size of 8-9 nucleotides and has an affinity for DNA that is similar to that displayed by E. coli SSB(15, 20) , to which it is 25% identical and 50% similar by deduced amino acid sequence(16, 18, 19) .

Mitochondrial SSB is required for mtDNA maintenance in yeast, in which it was identified as a suppressor of mtDNA thermosensitivity associated with disruption of a mtDNA helicase gene, PIF1(16) . mtSSB has been shown to coat the displaced ssDNA that is the template for lagging DNA strand synthesis in mtDNA replication(21) , and its apparent role in preventing the renaturation of displacement loops likely enhances DNA helicase activity(22) . Furthermore, mtSSB is proposed to aid in the prevention of nonspecific lagging DNA strand initiation by mtDNA primase(23, 24) . However, the effect of mtSSB on the function of mitochondrial DNA polymerase (pol ) is unclear. Rat mtSSB stimulates the activity of partially purified rat pol 10-fold on the synthetic homopolymeric substrate poly(dT)bulletoligo(dA), but the activity of rat pol was unaffected on poly(dA)bulletoligo(dT), for which rat mtSSB exhibits a low DNA binding affinity(20) . X. laevis mtSSB was shown to stimulate severalfold a partially purified form of X. laevis pol on poly(dA)bulletoligo(dT) and on poly(rA)bulletoligo(dT)(25) . In contrast, X. laevis mtSSB had no effect on DNA synthesis in a mitochondrial lysate and was completely inhibitory to mitochondrial DNA polymerase activity on singly primed single-stranded viral DNA(25) .

The nearly homogeneous mitochondrial DNA polymerase from Drosophila melanogaster embryos is a heterodimeric enzyme (26) whose mechanism of DNA strand synthesis has been well characterized biochemically(27, 28, 29) . It is capable of the highly processive DNA strand synthesis characteristic of bacterial, bacteriophage, and nuclear replicative DNA polymerases, but only under conditions that are suboptimal for DNA synthetic rate(29) . Although mtDNA replication in vivo is slow (4.5 nucleotides/s/DNA strand)(30) , Drosophila pol catalyzes DNA synthesis at an even lower rate when carrying out highly processive DNA synthesis in vitro (0.5 nucleotides/s/DNA strand) (27, 29) . Considering the effects of E. coli SSB on bacterial DNA replication and because mitochondrial SSB both is critical for mtDNA replication in vivo and affects the activity of partially purified forms of pol , we have investigated the effect of E. coli SSB on Drosophila pol . The high amino acid sequence conservation between mtSSBs and E. coli SSB and their physical and biochemical similarities suggest that theeffects of E. coli SSB on mitochondrial DNA polymerase will reflect those of mtSSB as well.


EXPERIMENTAL PROCEDURES

Materials

Nucleotides and Nucleic Acids

Unlabeled deoxy- and ribonucleoside triphosphates were purchased from P-L Biochemicals. [^3H]dTTP was purchased from ICN Biochemicals; [alpha-P]dTTP and [-P]ATP were purchased from DuPont NEN.

Recombinant and wild-type M13 viral DNAs (10,650 and 6407 nucleotides, respectively) were prepared by standard laboratory methods. Synthetic oligodeoxynucleotides (15 nucleotides) complementary to the M13 viral DNAs were synthesized in an Applied Biosystems Model 477 oligonucleotide synthesizer.

Enzymes

Drosophila DNA polymerase (Fraction VI) was prepared as described by Wernette and Kaguni(26) . Drosophila SSB was generously provided by Dr. I. R. Lehman (Stanford University). E. coli SSB and bacteriophage T4 gp32 were from United States Biochemical Corp. E. coli DNA polymerase III subunits were generously provided by Dr. Charles McHenry (University of Colorado Health Sciences Center) and Dr. Mike O'Donnell (Cornell University Medical College).

Methods

DNA Polymerase Assay

Reaction mixtures (0.05 ml) contained 50 mM TrisbulletHCl (pH 8.5), 4 mM MgCl(2), 10 mM dithiothreitol, 30 or 120 mM KCl, 400 µg/ml bovine serum albumin, 20 µM dATP, 20 µM dCTP, 20 µM dGTP, 20 µM [^3H]dTTP (1000 cpm/pmol), 10 µM (as nucleotides) singly primed recombinant M13 DNA, and 0.1 unit of Fraction VI enzyme (7-fold excess of primer ends over pol molecules). Incubation was at 30 °C for 30 min. Specific modifications are described in the figure legends. 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. Here, we define standard activity as that activity exhibited by pol in the presence of 120 mM KCl on singly primed M13 DNA.

Analysis of Products of Processive DNA Synthesis by Gel Electrophoresis

Reactions were as described above, except that reaction mixtures contained 30 µM dATP, 30 µM dCTP, 30 µM dGTP, 10 µM [alpha-P]dTTP (2 times 10^4 cpm/pmol), 20 µM (as nucleotides) singly primed wild-type M13 DNA, and 0.02 units of Fraction VI enzyme (100-fold excess of primer ends over pol molecules). Incubation was at 30 °C for 4 or 8 min. Reaction mixtures were made 1% in SDS and 10 mM in EDTA, heated for 4 min at 80 °C, phenol/chloroform-extracted, and precipitated with ethanol in the presence of 0.5 µg of tRNA as carrier. The ethanol precipitates were resuspended in 30 mM NaOH and 20 mM EDTA and electrophoresed on a 1.5% agarose slab gel (13 times 18 times 0.7 cm) containing 30 mM NaCl and 2 mM EDTA in 30 mM NaOH and 2 mM EDTA. Approximately equal amounts of radioactivity (1000 cpm) were loaded in each lane. After electrophoresis, the gel was washed in distilled water for 20 min, dried under vacuum, and exposed at -80 °C to Kodak X-Omat AR x-ray film using a DuPont NEN Quanta III intensifying screen. Quantitation of the data was performed by scanning the autoradiographs using a BioImage Visage 110 digital imager. The area under the peaks was determined by computer integration analysis and was normalized to the nucleotide level to correct for the uniform labeling of the DNA products. In the determination of processivity values, the length of the primer (15 nucleotides) was subtracted from the DNA product strand lengths.

Time Course of pol DNA Synthesis

Reaction mixtures containing 30 mM KCl were as described for the DNA polymerase assay, except that they were 450 µl, contained 0.9 units of Fraction VI enzyme (7-fold excess of primer ends over pol molecules), and lacked dNTPs. After incubation at 30 °C for 5 min, dNTPs were added, and the incubation was continued. At the time points specified in the figure legends, aliquots (0.05 ml) were removed, and the reaction was stopped by the addition of 10% trichloroacetic acid containing 10 mM sodium pyrophosphate. For analysis of products by gel electrophoresis, [P]dTTP (3000 cpm/pmol) replaced [^3H]dTTP (1000 cpm/pmol), aliquots were processed and electrophoresed, and the DNA strand products were analyzed as described for the processivity assay.

Primer Extension Assay

Reaction mixtures (0.05 ml) contained 50 mM TrisbulletHCl (pH 8.5), 4 mM MgCl(2), 10 mM dithiothreitol, 30 mM KCl, 400 µg/ml bovine serum albumin, 30 µM dATP, 30 µM dGTP, 30 µM dTTP, 20 µM (as nucleotides) 5`-end labeled singly primed recombinant M13 DNA, and 0.4 units of Fraction VI enzyme (3-fold excess of primer ends over pol molecules). Incubation was at 30 °C for 5 min. Samples were made 1% in SDS and 10 mM in EDTA, heated for 10 min at 65 °C, phenol/chloroform-extracted, and precipitated with ethanol in the presence of 0.5 µg of tRNA as carrier. The ethanol precipitates were resuspended in 80% formamide and 90 mM Tris borate. Aliquots were denatured for 2 min at 100 °C and electrophoresed on an 18% polyacrylamide slab gel (13 times 24 times 0.075 cm) containing 7 M urea in 90 mM Tris borate (pH 8.3) and 25 mM EDTA. After electrophoresis, the gel was washed in distilled water for 20 min and exposed at -80 °C to Kodak X-Omat AR x-ray film using a DuPont NEN Quanta III intensifying screen. Quantitation was as described above.


RESULTS

E. coli SSB Stimulates DNA Synthesis by Drosophila DNA Polymerase

E. coli SSB was shown previously to have no effect on the activity of partially purified X. laevis mitochondrial DNA polymerase(25) . However, when nearly homogeneous D. melanogaster DNA polymerase was assayed on singly primed M13 DNA in the presence of SSB and 120 mM KCl, we found that polymerase activity was stimulated 2.5-fold (250% of standard activity) (Fig. 1). Furthermore, when Drosophila pol was assayed in the presence of SSB and 30 mM KCl, activity was stimulated 25-fold (625% of standard activity). Maximal stimulation was observed when the amount of SSB present was sufficient to coat all of the single-stranded DNA in the reaction: the presence of salt alters the binding characteristics of E. coli SSB, resulting in saturation of the M13 DNA by binding protein at 0.5 and 1.0 µg of SSB in the presence of 120 and 30 mM KCl, respectively(31) . This result suggests that SSB stimulation of pol is caused by its DNA coating capacity and not by a stoichiometric interaction with DNA polymerase .


Figure 1: E. coli SSB stimulates the rate of DNA synthesis by Drosophila pol . DNA synthesis was measured on singly primed M13 DNA as described under ``Methods'' in the presence of the indicated amounts of E. coli SSB and 30 (closedcircles) or 120 (opencircles) mM KCl. STD, standard.



We found previously that the mechanism of DNA synthesis by Drosophila pol is dependent on the KCl concentration at which it was assayed(29) : at 120 mM KCl, pol is most active (standard activity), yet only moderately processive (average processive unit of 45 nucleotides); at 30 mM KCl, pol is less active (25% of standard activity), but highly processive (average processive unit of 2500 nucleotides). Therefore, we investigated the effect of KCl concentration on the stimulation of Drosophila pol by E. coli SSB. The stimulation of mitochondrial DNA polymerase by SSB was maximal from 20-90 mM KCl (Fig. 2), thus lowering the KCl concentration required to achieve optimal DNA polymerase activity as much as 6-fold. Furthermore, SSB inhibited completely pol when the concentration of KCl was >150 mM. This shift in KCl optimum may reflect a change in DNA binding mechanism with varying salt concentration both by SSB (31) and by pol in the presence of SSB and/or a change in protein-protein interactions.


Figure 2: E. coli SSB lowers the KCl optimum of Drosophila pol . DNA synthesis was measured on singly primed M13 DNA as described under ``Methods'' in the presence of the indicated amounts of KCl and in the absence (closedcircles) or presence (opencircles) of E. coli SSB (1 µg). STD, standard.



SSB Stimulation of DNA Synthesis by Drosophila DNA Polymerase Exhibits a Lag Time

E. coli SSB stimulates bacteriophage T7 DNA polymerase 11-fold following 2 min of DNA synthesis, with stimulation decreasing over time to 5-fold after 15 min of catalysis(32) . To determine the maximal stimulation of Drosophila pol by SSB, we examined the extent of DNA synthesis by pol in the absence and presence of SSB and 30 mM KCl over a time course of incubation. Nucleotide incorporation by pol alone was linear to 60 min of incubation at 30 °C (Fig. 3A)(28) , while DNA synthesis in the presence of SSB was linear only to 8 min of incubation. The stimulation of mitochondrial DNA polymerase was 7-fold initially, then >30-fold from 8 to 16 min, decreasing to 22-fold following 60 min of incubation at 30 °C (Fig. 3B). Thus, a lag time is required to achieve maximal stimulation of Drosophila pol by E. coli SSB.


Figure 3: Time course of stimulation of Drosophila pol by E. coli SSB. DNA synthesis was measured on singly primed M13 DNA over a time course of incubation as described under ``Methods'' in the presence of 30 mM KCl and in the absence (closedcircles) or presence (opencircles) of E. coli SSB (1 µg). A, nucleotide incorporation by pol was determined after incubation at 30 °C for 0, 1, 2, 4, 8, 16, 30, 45, and 60 min. In the absence of SSB, 1% of the input DNA template was copied after 60 min of incubation versus 17% in its presence. B, the data from A were replotted to show the ratio of nucleotide incorporation by Drosophila pol in the presence versus absence of E. coli SSB at each time point.



Surprisingly, only 17% of the template DNA was copied after 60 min of DNA synthesis in the presence of SSB, corresponding to approximately the amount expected if each DNA polymerase molecule copied only a single DNA molecule and did not cycle to an unused primer terminus. That at least 70% of the input DNA could serve as template for DNA replication was shown by readdition of pol after 60 min of incubation (data not shown). Furthermore, the extent of DNA synthesis was proportional to the amount of DNA polymerase added: in enzyme titrations, the percentage of DNA copied, ranging from 4 to 70%, reflected that expected for a single binding and polymerization cycle (data not shown).

SSB Increases the Processivity of Drosophila DNA Polymerase

The stimulation of Drosophila mitochondrial DNA polymerase by E. coli SSB may result from an increase in the initiation or elongation rate or in the rate of polymerase cycling or from some combination of these parameters. To determine the mechanism by which SSB stimulates Drosophila pol , we first examined its effect on enzyme processivity. The addition of SSB at 30 mM KCl resulted in a more uniform distribution of DNA synthetic products, suggesting the elimination of some DNA polymerase pause sites, and a substantial increase in the amount of full-length DNA product strands (Fig. 4). However, the average processivity of mitochondrial DNA polymerase was increased only 2-fold, even though the stimulation of DNA polymerase activity by SSB was 37-fold after 8 min of incubation.


Figure 4: E. coli SSB increases the processivity of Drosophila pol . DNA synthesis was performed on singly primed M13 DNA (6407 nucleotides) at 30 mM KCl as described under ``Methods,'' and the DNA product strands were isolated, denatured, and electrophoresed on a 1.5% denaturing agarose gel. Reactions were performed in the absence (lanes1 and 2) or presence (lanes3 and 4) of SSB (2 µg) and incubated at 30 °C for 4 (lanes1 and 3) or 8 (lanes2 and 4) min. Numbers at left indicate the positions and sizes (in nucleotides) of HindIII restriction fragments of -DNA that were electrophoresed in an adjacent lane.



SSB Increases Dramatically the Rate of Initiation of DNA Synthesis by DNA Polymerase

Next, we investigated the effect of SSB on the rate of initiation of DNA synthesis by Drosophila pol by primer extension analysis. Limited DNA synthesis on M13 DNA containing a 5`-end-labeled 15-nucleotide primer was carried out in the presence of dATP, dGTP, and TTP, and the DNA product strands were examined by denaturing gel electrophoresis (Fig. 5). In the absence of dCTP, termination of DNA synthesis occurs after the incorporation of 8 or 11 nucleotides, the first and second positions where dCTP is the required substrate. Hence, the primer extension assay allows an evaluation of the initial stages of DNA synthesis: primer recognition and binding by pol and limited primer extension.


Figure 5: E. coli SSB increases the rate of initiation of DNA synthesis by Drosophila pol . Primer extension was performed at 30 mM KCl in the absence of dCTP as described under ``Methods.'' DNA product strands were isolated, denatured, and electrophoresed on an 18% denaturing polyacrylamide gel. Reactions were performed in the absence (lane2) or presence (lane3) of SSB (2 µg). Lane1 represents a control reaction lacking both pol and SSB.



Quantitation of the DNA product strands in the presence and absence of SSB revealed that SSB stimulates primer extension 25-fold at 30 mM KCl following 5 min of incubation at 30 °C (Fig. 5). This corresponds closely to the 30-fold stimulation of polymerase activity at this time of incubation (Fig. 3B), indicating that 80% of pol stimulation by SSB is due to an increase in the rate of initiation of DNA synthesis by mitochondrial DNA polymerase. The stimulation may result from SSB-facilitated primer recognition accomplished by elimination of nonproductive binding of DNA polymerase to excess single-stranded DNA or by increasing the affinity of unbound polymerase molecules for DNA. In support of this hypothesis is the observation that SSB stimulates to a similar extent the 3` 5`-exonuclease activity of DNA polymerase on M13 DNA containing mispaired primers. (^2)

SSB Has No Effect on the Intrinsic Rate of DNA Synthesis by DNA Polymerase

To explore further the mechanism of stimulation of mitochondrial DNA polymerase by SSB, we examined the products of DNA synthesis by pol in the absence and presence of SSB over the same time course of incubation shown in Fig. 3A (Fig. 6). Based on the average DNA product strand lengths observed after 1 and 2 min of DNA synthesis, we can estimate the intrinsic rate of nucleotide polymerization by Drosophila pol to be 15-20 nucleotides/s, both in the absence and presence of SSB. Likewise, full-length DNA product strands were observed after 8 min of DNA synthesis in either case, corresponding to a polymerization rate of 20 nucleotides incorporated per s. Although E. coli SSB apparently has no effect on the intrinsic rate of DNA synthesis by pol , it eliminates a strong pause site at the position of DNA product strands of 2600 nucleotides in length, resulting in a greater accumulation of full-length DNA products. As in the analysis shown in Fig. 4, the processivity of pol was increased 2-fold in the presence of SSB. Based on the distribution of DNA product strand lengths after various times of DNA synthesis (Fig. 6), Drosophila pol synthesizes a processive product of 2600 nucleotides in the absence of SSB and then cycles to an unused primer terminus. In the presence of SSB, the average processive unit increases to 5500 nucleotides.


Figure 6: Gel analysis of the effect of E. coli SSB on a time course of DNA synthesis by Drosophila pol . A time course of DNA synthesis was performed on singly primed recombinant M13 DNA (10,650 nucleotides) at 30 mM KCl as described under ``Methods.'' DNA product strands were isolated, denatured, and electrophoresed on a 1.5% denaturing agarose gel. Reactions were performed in the absence (lanes 1-8) or presence (lanes 9-16) of SSB (1 µg) and were incubated at 30 °C for 1 (lanes1 and 9), 2 (lanes2 and 10), 4 (lanes3 and 11), 8 (lanes4 and 12), 16 (lanes5 and 13), 30 (lanes6 and 14), 45 (lanes7 and 15), and 60 (lanes8 and 16) min. The arrow indicates the position of full-length products. Numbers at left indicate the positions and sizes (in nucleotides) of HindIII restriction fragments of -DNA that were electrophoresed in an adjacent lane.



While SSB has no effect on the intrinsic rate of DNA synthesis by pol and only a modest effect on pol processivity, the extents of DNA synthesis by pol varied 10-20-fold over the time course of incubation in the absence and presence of SSB: in the absence of SSB, 0.06% of the DNA template was copied after 1 min and 1% after 60 min of incubation, as compared with the presence of SSB, where the extents of DNA synthesis were 0.6 and 17% after 1 and 60 min, respectively. Interestingly, the 10-20-fold variation in the extent of DNA synthesis by pol in the absence versus presence of SSB correlates with a 5-10-fold variation in the fraction of pol molecules actively engaged in DNA synthesis, which can be estimated using the average DNA strand length to calculate an average rate of nucleotide polymerization at each time point. That the extent of stimulation of pol by SSB is generally proportional to the number of active pol molecules supports the hypothesis that the mechanism of stimulation of mitochondrial DNA polymerase by E. coli SSB is via an increase in the rate of initiation.

Drosophila SSB and Bacteriophage T4 gp32 Stimulate DNA Synthesis by Drosophila DNA Polymerase

The stimulation of Drosophila mitochondrial DNA polymerase by E. coli SSB is dramatic and appears to be due primarily to an increase in primer recognition and binding. Single-stranded DNA-binding proteins from sources as diverse as bacteria and humans appear to function via specific protein-protein interactions in addition to their ability to coat single-stranded DNA. To examine the specificity of stimulation of mitochondrial DNA polymerase by E. coli SSB, the effects of Drosophila SSB (DSSB) and bacteriophage T4 gp32 on pol activity were examined (Fig. 7). Both DSSB and gp32 stimulated Drosophila pol , although to a lesser extent than E. coli SSB (Fig. 7). Maximal stimulation was 7.5-fold for DSSB and 20-fold for gp32, as compared with 37-fold for E. coli SSB. As with E. coli SSB, DNA synthesis by mitochondrial DNA polymerase in the presence of DSSB and gp32 was linear only to 16 min of incubation at 30 °C. Likewise, maximal stimulation by all three single-stranded DNA-binding proteins was achieved after 8 min, although the extent of stimulation of pol by DSSB and gp32 was relatively constant, varying <2-fold over the time course of incubation at 30 °C.


Figure 7: Drosophila SSB and bacteriophage T4 gp32 stimulate DNA synthesis by Drosophila pol . The rate of DNA synthesis was determined at 30 mM KCl over a time course of incubation at 30 °C as described under ``Methods'' in the absence (closedcircles) or presence of saturating Drosophila SSB (1 µg; closedtriangles) or gp32 (1 µg; opencircles).



E. coli DNA Polymerase III Subunits Have No Effect on pol Activity or DNA Binding

The strong homology of mtSSB to E. coli SSB and the stimulation of pol by the latter suggested that E. coli processivity and primer recognition factors may substitute for putative mitochondrial DNA polymerase accessory factors as well. The subunits of DNA polymerase III responsible for processivity and primer recognition were examined with regard to their effects on mitochondrial DNA polymerase activity and DNA binding. Neither the beta-subunit (processivity) nor the -complex (primer recognition), either alone or in combination, affected pol activity at 30 or 120 mM KCl in the absence or presence of E. coli SSB (data not shown). Furthermore, pol binding to template-primer DNA, examined in a gel mobility shift assay, was unaffected by either the beta-subunit or the -complex, either alone or in combination (data not shown).


DISCUSSION

Replication of procaryotic, eucaryotic, and viral DNA genomes requires DNA polymerase in conjunction with its accessory factors and single-stranded DNA-binding protein. Mitochondrial single-stranded DNA-binding proteins homologous to E. coli SSB have been identified in X. laevis, S. cerevisiae, and rat and human tissues(14, 15, 18, 19) . The substantial homology between mtSSBs and E. coli SSB in both sequence and proposed subunit structure and the requirement for mtSSB in yeast mtDNA replication led us to examine the effects of E. coli SSB on Drosophila mitochondrial DNA polymerase.

E. coli SSB stimulates Drosophila pol at both 30 and 120 mM KCl. KCl affects greatly the mode and cooperativity of SSB binding even though it has little effect on the affinity of SSB for DNA: below 10 and above 200 mM NaCl, E. coli SSB binds to ssDNA in two modes that have site sizes of 33 and 65 nucleotides/tetramer, respectively (31) . In the range between these salt concentrations, SSB tetramers bind in both modes, resulting in apparent binding site sizes between the two limits(31, 33, 34) . Consistent with the known binding properties of E. coli SSB, we found that maximal stimulation of mitochondrial DNA polymerase at both 30 and 120 mM KCl occurs at the SSB concentrations required to saturate the ssDNA template.

The ability of E. coli SSB to increase the processivity of E. coli DNA polymerases II and III and bacteriophage T7 DNA polymerase (5, 6, 9) suggested that it might also increase the processivity of mitochondrial DNA polymerase. We found that the presence of SSB increases the processivity of Drosophila pol 2-fold at 30 mM KCl (this report) and 3-fold at 120 mM KCl, (^3)reducing or eliminating pausing of DNA polymerase at specific sites and increasing the fraction of completed DNA product strands. Interestingly, the increase in processivity corresponds closely with the 2-3-fold stimulation of pol at 120 mM KCl, but cannot account for the 37-fold stimulation observed at 30 mM KCl.

The stimulation of several DNA polymerases by E. coli SSB has been documented and, in some cases, appears to be due to the ability of SSB to increase primer recognition by either preventing nonproductive binding of DNA polymerase to or enhancing the affinity of DNA polymerase for single-stranded DNA. SSB stimulation of herpes simplex virus-1 DNA polymerase occurs by the former mechanism(10) , while that of bacteriophage T7 DNA polymerase is proposed to occur by the latter(32) . The elimination of nonproductive binding by DNA polymerase may result from a decreased affinity of DNA polymerase for SSB-coated ssDNA, causing multiple binding and dissociation events that continue until the primer terminus is recognized and bound. Alternatively, the presence of SSB may maintain or enhance DNA binding and facilitate a sliding or looping mechanism by which DNA polymerase locates efficiently the primer terminus. Our primer extension data show that the stimulation of mitochondrial DNA polymerase by E. coli SSB is due primarily to an increased rate of primer recognition and binding, consistent with either mechanism.

By determining the extents of DNA synthesis and the average lengths of DNA product strands over a time course of incubation, we could estimate the number of active DNA polymerase molecules at each time point. In doing so, we conclude that in the presence of SSB, nearly all of the pol molecules were polymerizing DNA after 16 min of incubation. In contrast, in the absence of SSB, only 12% of the pol molecules were active after 16 min and 25% after 60 min of incubation. In fact, the stimulation of pol by SSB at each time point was generally proportional to the increase in the estimated number of pol molecules actively synthesizing DNA in the presence and absence of SSB, providing further support for the hypothesis that SSB stimulates pol by increasing the rate of primer recognition.

Given the proposed mechanism of stimulation of Drosophila pol by E. coli SSB, it is not surprising that Drosophila SSB and bacteriophage T4 gp32 also stimulate mitochondrial DNA polymerase, albeit to different extents. While the three proteins bind to DNA with approximately equal affinity, they may impose different conformations onto single-stranded DNA(35, 36) . Thus, mitochondrial DNA polymerase may bind to DSSB- and gp32-coated DNA nonproductively with higher affinity than it binds to E. coli SSB-coated DNA. Alternatively, pol may displace E. coli SSB more effectively than DSSB or gp32 from the DNA template during polymerization. Because of its homology to the DNA-binding domain of E. coli SSB, mtSSB might be expected to impose a conformation onto ssDNA similar to that of E. coli SSB: mtSSBs are 50-67% identical to several regions of the amino-terminal two-thirds of E. coli SSB that is involved in DNA binding (16, 18, 19, 37) . In addition, two amino acids known to be involved in DNA binding by E. coli SSB are identical in all mtSSBs identified to date, and a third amino acid is identical in all mtSSBs except that in yeast(18, 38, 39, 40) . Hence, the effects of E. coli SSB on DNA polymerase may be expected to represent more closely those of mtSSB than those of bacteriophage SSBs or eucaryotic SSBs of nuclear origin.

Primer recognition and processivity factors from E. coli failed to alter the activity of mitochondrial DNA polymerase. This may be due to a lack of stable protein-protein associations. Stimulation of pol by E. coli SSB is likely mediated by the interaction of SSB with DNA and not through specific pol -SSB interactions. In contrast, primer recognition and processivity factors associate with DNA polymerase to tether it onto the DNA template(41) . Because the polymerase catalytic subunits of mitochondrial DNA polymerase and E. coli DNA polymerase III holoenzyme lack regions of significant homology(42) , specific association of the procaryotic accessory proteins with Drosophila pol is perhaps unlikely.

Animal mtDNA is replicated by a highly asymmetric mechanism(30, 43) . Leading DNA strand synthesis occurs on the double-stranded parental DNA template, while lagging DNA strand synthesis, which occurs on the displaced parental DNA strand, involves replication of a predominantly (or entirely) single-stranded DNA template. Thus, replication of the singly primed single-stranded viral DNA template used in this study provides an excellent model of lagging strand mtDNA synthesis. We have found previously that Drosophila mitochondrial DNA polymerase is capable of highly processive DNA synthesis in vitro only under reaction conditions that are suboptimal for DNA synthetic rate(29) . Here, we find that the addition of E. coli SSB enhances greatly the rate of DNA synthesis while maintaining high processivity, providing further support for an important role for SSB in mitochondrial DNA replication. That previous studies examining the effects of both E. coli SSB and homologous mtSSBs on pol did not yield a uniform conclusion (20, 25) may reflect both the purity of the pol preparation and the reaction conditions employed. We have found that both KCl concentration and enzyme purity have a substantial effect on the stimulation of Drosophila pol by E. coli SSB. For example, in Fractions I-V of our standard pol purification(26) , which vary from 0.04 to 20% in purity, SSB stimulation may vary from 0.5- to 20-fold. (^4)In any case, the SSB-induced ability of Drosophila pol to copy efficiently and processively long stretches of ssDNA is characteristic of a DNA polymerase that catalyzes continuous DNA strand synthesis, as proposed in current models of mtDNA replication(30, 43) .


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 by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

This paper is dedicated to I. Robert Lehman on the occasion of his 70th birthday.

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

(^1)
The abbreviations used are: SSB, single-stranded DNA-binding protein; ssDNA, single-stranded DNA; mtSSB, mitochondrial SSB; pol , DNA polymerase ; DSSB, Drosophila SSB.

(^2)
C. L. Farr and L. S. Kaguni, unpublished data.

(^3)
C. M. Wernette and L. S. Kaguni, unpublished data.

(^4)
A. J. Williams and L. S. Kaguni, unpublished data.


ACKNOWLEDGEMENTS

We thank Carol Farr for excellent technical assistance and Dr. Shelagh Ferguson-Miller for many helpful discussions.


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