Mechanism of Stimulation of T7 DNA Polymerase by Escherichia coli Single-stranded DNA Binding Protein (SSB)*

Single-stranded DNA binding protein is a key com-ponent in growth of bacteriophage T7. In addition, DNA synthesis by the purified in vitro replication system is markedly stimulated when the DNA template is coated with Escherichia coli single-stranded DNA binding protein (SSB). In an attempt to understand the mechanism for this stimulation, we have studied the effect of E. coli SSB on DNA synthesis by the T7 DNA polymerase using a primed single-stranded M13 DNA template which serves as a model for T7 lagging strand DNA synthesis. Polyacrylamide gel analysis of the DNA product synthesized on this template in the absence of SSB indicated that the T7 DNA polymerase pauses at many specific sites, some stronger than others. By comparing the position of pausing with the DNA sequence of this region and by using a DNA template that contains an extremely stable hairpin structure, it was found that many, but not all, of these pause positions correspond to regions of potential sec- ondary structure. The presence of SSB during synthesis resulted in a large reduction in the frequency of hesitations at many sites that correspond to these sec- ondary structures. However, the facts that a large percentage of the pause sites remain unaffected even at saturating levels of SSB and that SSB stimulates synthesis on a singly primed poly(dA) template suggested that other mechanisms also contribute to the stimulation of DNA synthesis caused by SSB. Using a sucrose gradient analysis, we found that SSB increases the affinity of the polymerase for single-stranded DNA that this increased binding is only noticed when the polymerase concentration is limiting. The effect of this difference in polymerase affinity


Mechanism of Stimulation of T7 DNA Polymerase by Escherichia coli
Single-stranded DNA Binding Protein (SSB)* (Received for publication, August 25, 1987) Thomas W. Myerst  Single-stranded DNA binding protein is a key component in growth of bacteriophage T7. In addition, DNA synthesis by the purified in vitro replication system is markedly stimulated when the DNA template is coated with Escherichia coli single-stranded DNA binding protein (SSB). In an attempt to understand the mechanism for this stimulation, we have studied the effect of E. coli SSB on DNA synthesis by the T7 DNA polymerase using a primed single-stranded M13 DNA template which serves as a model for T7 lagging strand DNA synthesis. Polyacrylamide gel analysis of the DNA product synthesized on this template in the absence of SSB indicated that the T7 DNA polymerase pauses at many specific sites, some stronger than others. By comparing the position of pausing with the DNA sequence of this region and by using a DNA template that contains an extremely stable hairpin structure, it was found that many, but not all, of these pause positions correspond to regions of potential secondary structure. The presence of SSB during synthesis resulted in a large reduction in the frequency of hesitations at many sites that correspond to these secondary structures. However, the facts that a large percentage of the pause sites remain unaffected even at saturating levels of SSB and that SSB stimulates synthesis on a singly primed poly(dA) template suggested that other mechanisms also contribute to the stimulation of DNA synthesis caused by SSB. Using a sucrose gradient analysis, we found that SSB increases the affinity of the polymerase for single-stranded DNA that this increased binding is only noticed when the polymerase concentration is limiting. The effect of this difference in polymerase affinity was clearly observed by a polyacrylamide gel analysis of the product DNA synthesized during a limited DNA synthesis reaction using conditions where only two nucleotides are added to the primer. Under these circumstances, where the presence of hairpin structures should not contribute to the stimulatory effect of SSB, we found that the extension of the primer is stimulated 4-fold if the DNA template is coated with SSB. Furthermore, SSB had no effect on this synthesis at large polymerase to template ratios.
Escherichia coli single-stranded DNA binding protein (SSB)' plays an essential role in the replication of the E. coli chromosome (1)(2)(3) as well as in the life cycle of several DNA phages which infect E. coli (4). SSB is also involved in several other cellular processes including DNA recombination (2,5) and repair (2,3,(6)(7)(8)(9). Although the involvement of SSB in these cellular events has been studied extensively, the molecular mechanism by which SSB acts in these processes is not understood. SSB has been shown to have differential effects on DNA synthesis by purified DNA polymerases; T7 DNA polymerase, E. coli DNA polymerase 11, and DNA polymerase I11 holoenzyme are stimulated by SSB, while little stimulation is observed with either E. coli DNA polymerase I or T4 DNA polymerase (10)(11)(12)(13). SSB has also been shown to increase the fidelity of DNA synthesis by a wide range of DNA polymerases (14,15). Recently, SSB was found to have contrasting effects on translesion DNA synthesis of either aminofluorene-modified or UV-irradiated single-stranded templates by various DNA polymerases (16,17). For the most part, these diverse effects have been accounted for by the ability of SSB to alter the secondary structure of single-stranded DNA thus preventing loop-back or hairpin structures and also by providing a more rigid DNA template that presumably alters the interaction between the enzymes and the DNA (18).
The observation that T7 mutants in gene 2.5 (singlestranded DNA binding protein) will not grow on an E. coli strain (ssb) that lacks SSB (19) indicates that the presence of a viable single-stranded DNA binding protein is required for the growth of this virus. The precise role it plays in the T 7 life cycle is not known, although it has been assumed to be directly involved in DNA replication based on the effect of these proteins on replication by the purified T7 in uitro system. Both E. coli and T7 SSB provide a large stimulation to both the purified T7 DNA polymerase (20,21) and the T7 in vitro replication system (22-25) on single-stranded DNA templates. The mechanism by which SSB causes this stimulation has not been determined, but the fact that the stimulation observed seems to be specific for RNA-primed DNA synthesis (22) suggested that the involvement of SSB may not be solely as a helix-destabilizer.
In this paper, we present the first findings of a systematic study which we hope will lead to a better understanding of the essential role of SSB in cellular growth. The goal of the present study is aimed specifically at determining the mechanism by which SSB stimulates DNA synthesis. To accomplish this, we have utilized an in vitro system, comprised of a single-stranded viral DNA template hybridized to an oligonucleotide pentadecamer primer, as a model for lagging strand DNA synthesis. We find that the T7 DNA polymerase hesi-' The abbreviation used is: SSB, E. coli single-stranded DNA binding protein.
tates in regions of potential secondary structure in the template DNA and that the presence of SSB decreases the extent of hesitation in these regions. Experiments using a uniquely primed poly(dA) template, which lacks any potential stable secondary structures, indicates that the SSB stimulation of DNA synthesis also occurs by a mechanism separate from its ability to destabilize hairpin structures in the template DNA. In this regard, we have examined the influence of SSB on the interaction between the T7 DNA polymerase and the DNA template and find that the presence of SSB greatly increases their affinity.

Materials
Bacterial Strains and Bacteriophages-E. coli DllO Suthy end polyAl has been described previously (26). E. coli 011' Su' thy, T7 wild type phage and T7 amber mutants were obtained from Dr. F. W. Studier (Brookhaven National Laboratory). The low thymidine-requiring mutant of E. coli 71.18 has been described previously (27) and was obtained from Dr. C. Richardson (Harvard Medical School). T7 amber mutants are designated by subscript notation indicating the mutant gene only. The amber mutations used are: gene 3, am29; gene 4, am20; gene 6, am147. T7 phages were grown on E. coli 011' Su' thy as described by Studier (2&30). E. coli JM103, M13mp7, and M13mp9 bacteriophage have been described (31) and were obtained from Dr. J. E. LeClerc (University of Rochester).
[3H]M13mp9 single-stranded DNA was prepared as follows: the low thymidine-requiring mutant of E. coli 71.18 was grown in Frasier's media containing 5 pg/ml thymine to an A,, of 0.5, and 250 pg/ml 2'-deoxyadenosine was added. Aeration was continued at 37 'C for 15 min, and 2.5 pCi/ml of ['HI thymidine (ICN Radiochemicals) was added. Aeration was continued at 37 "C to an A590 of 1.5 and M13mp9 phage were added at a multiplicity of infection of 0.5. Aeration was continued for 4.5 h at 37 "C. The phage were harvested by precipitation with 6% polyethylene glycol and the phage DNA isolated as described (33). The specific activity of the DNA obtained was 9.2 cpm/pmol. The poly(dA) template (dA)Bm(dC),s was prepared as described (34) and the amount of dCMP added to the poly(&) was monitored by duplicate reactions utilizing [3H]dCTP.
Nucleotides-Unlabeled nucleotides were purchased from Pharmacia LKB Biotechnology Inc. The (dA), was purchased from the Midland Certified Reagent Company. The oligonucleotide pentadecamer primers were synthesized on a Beckman System 1 DNA synthesizer and purified by high performance liquid chromatography on either a Beckman Ultrasphere ODS column or on a Du Pont Zorbax Oligo column. Radioactive nucleotides were obtained from ICN Radiochemicals.
Enzymes-T7 DNA polymerase Form I1 (95% pure) was prepared from Tlls4,-infected E. coli DllO (35). Conversion of T7 DNA polymerase from Form I1 to Form I was performed as described (36). The specific activity of the preparation used in these experiments was 7,800 units/mg. E. coli DNA binding protein (95% pure) was purified by the method of Weiner et al. (11). The purity of the above proteins was determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. E. coli DNA polymerase I large fragment (Klenow fragment) having a specific activity of 17,000 units/mg was purchased from New England Biolabs. T4 polynucleotide kinase was obtained from United States Biochemical Corporation. stated. T7 DNA polymerase and SSB were added to the reaction mix as indicated. The reaction mixtures (100 p l ) were incubated at 37 "C for 30 min and stopped by the addition of 10 p l of 0.5 M EDTA (pH 8). Incorporation of nucleotides was determined by measuring the amount of acid-insoluble radioactivity by a modification of the procedure described by Richardson (37). In the experiments where high specific activity radionucleotide (<3000 cpm/pmol) was used, the DNA was first pelleted by the addition of 10 p1 of calf thymus DNA (2.5 mg/ml) as a carrier and 1 ml of ice-cold 100 mM N&PZ0, in 1 M HC1 and centrifugation in an Eppendorf microcentrifuge for 5 min. The pellet was washed with 1 ml of the acid solution and centrifuged for 5 min. The supernatant was again decanted and the pellet redissolved in 500 p l of 0.1 N NaOH. The solution was allowed to set at room temperature for 5 min and then the DNA was precipitated with 500 p1 of the acid solution. The precipitate was collected on a Whatman GF/C filter and rinsed three times with 3 ml of the acid solution and once with 5 ml of 95% ethanol. Acid-precipitable counts were measured on Beckman LS 7500 liquid scintillation counter.
Polyacrylumide Gel Electrophoresis-DNA synthesis reactions (50 p l ) were carried out as described above except that the dTTP was unlabeled and the pentadecamer primer was labeled at the 5' end with 32P (1-4 X 10, cpm/pmol). The reactions were 50 p~ in dNTP concentration. The labeled primer was prepared as described (16,38). Synthesis reactions were ended by the addition of 50 pl of phenol. The aqueous phase was recovered and extracted twice with ether. The sample volume was reduced in uacuo. The reaction was loaded onto a denaturing (7 M urea) polyacrylamide gel and electrophoresed as indicated. Sequencing reactions were performed by the dideoxy sequencing method of Sanger et al. (39). Gels were autoradiographed at -70 "C using Kodak XAR5 film and a Du Pont Cronex Lighting Plus intensifying screen. The experiments with limited DNA synthesis were carried out as described above except that the 25 p~ (a-32P] dTTP (25 Ci/mmol) and ddGTP were the only nucleotides present. SSB was present as indicated. Synthesis reactions were ended by the addition of 2.5 p1 of 0.5 M EDTA (pH 8). To remove the possibility that the presence of different levels of SSB affected the electrophoresis or sample loading, the concentration of SSB was adjusted so that each sample contained 7.5 pg of SSB prior to loading on the gel. To determine the amount of nucleotide added, the gel slice containing the 32P-labeled product was excised, and the radioactivity contained in it was determined by Cerenkov counting.
Computer Analysis-The computer program of Queen et al. (40) was utilized to search for potential hairpin structures in the M13 DNA template.

Effect of SSB on DNA Synthesis-Previous studies have
shown that SSB stimulates DNA synthesis by the T7 DNA polymerase on denatured T7 DNA templates (22,24). Furthermore, DNA synthesis on a duplex template by the polymerase and T7 gene 4 protein is stimulated by SSB, and this stimulation may be specific for RNA-primed lagging strand DNA synthesis (22)(23)(24).
In this study, we have used several oligonucleotide-primed single-stranded DNA templates to specifically determine the effect of SSB on lagging strand synthesis. It is well established that the T7 DNA polymerase is capable of using short RNA primers synthesized by the T7 gene 4 protein to initiate DNA synthesis on single-stranded DNA templates in uitro (22)(23)(24)(25)41). These uniquely primed single-stranded DNA templates thus serve as models for T7 gene 4 protein-primed lagging

Effect of
SSB on DNA Synthesis strand DNA synthesis. Addition of SSB to this in vitro replication system resulted in a substantial stimulation of DNA synthesis on primed M13 DNA by the T7 DNA polymerase, consistent with observations reported using other in vitro T7 DNA replication systems (22)(23)(24)(25)41). Maximum stimulation was observed at a level of SSB that presumably saturates the DNA template, that is, at levels where the SSB completely coats the single-stranded DNA. Shown in Fig. 1 is a time course comparing DNA synthesis by the T7 DNA polymerase in the presence and absence of SSB. Interestingly, the extent of stimulation by SSB is not consistent through the reaction; in the initial stages of synthesis, SSB stimulates over 11-fold, whereas later, the stimulation drops to about 5fold. Correlation of Hesitation Sites to Template Secondary Structure-Although the mechanism by which SSB stimulates the T7 DNA polymerase is not known, many have suggested that this stimulation could be explained by the ability of SSB to alter the secondary structure of the template DNA (20,41,42). We have obtained evidence in support of this proposal for replication by the T7 system. Analysis of products from synthesis on a primed M13mp9 DNA template using polyacrylamide gel electrophoresis determined the relative effect of SSB on the size distribution of the products of DNA synthesis. In the absence of SSB, the T7 DNA polymerase was shown to hesitate or stop at specific sites on the DNA template. This was demonstrated by the production of discrete bands visible on the polyacrylamide gel following autoradiography (Fig. 2, -SSB). The pentadecamer primer was radiolabeled at the 5' terminus in these experiments so that the abundance of a particular end-labeled DNA fragment was directly proportional to the intensity of the resulting band in the autoradiograph and thus independent of chain length. Others (43-48) have shown that sequence-dependent hesitation by various DNA polymerases occurs during in vitro synthesis and have suggested that this pausing results from a combination of both the particular nucleotide sequence that the polymerase is replicating (primary structure) and the existence of sequences capable of forming stable hairpins (secondary structure).

FIG. 2. Polyacrylamide gel of synthesis by T7
DNA polymerase in regions of potential secondary structure in the presenceor absence of SSB. DNA synthesis by the T7 DNA polymerase (0.1 unit) in the absence or presence of 3.8 pg of SSB was carried out on an M13mp9 DNA template (0.18 pg) as described under "Experimental Procedures." Synthesis was primed with a 32P-labeled oligonucleotide and the labeled DNA products were run on an 8% denaturing polyacrylamide gel. Potential secondary structures in this region of the mp9 template are depicted to the left, with lines connecting the base of each potential hairpin stem with the corresponding position in the autoradiogram as determined by the "dideoxy" sequencing lanes on the right.
The addition of SSB to the reaction resulted in a change in the banding pattern on the autoradiograph (Fig. 2, +SSB) such that the intensity of many of the bands presumably caused by the polymerase pausing at particular DNA sequences in the DNA template was greatly reduced by the addition of SSB. Other investigators have utilized this same type of approach and have shown that SSB has similar effects with E. coli DNA polymerase I1 (43) and E. coli DNA polymerase I11 (46). Although we observed that many of the sites of hesitation were either reduced or eliminated by the addition of SSB, some of the pause sites remained unaffected even at saturating levels of SSB (Fig. 2). Similar results have also been observed with the T4 DNA polymerase using the T4 gene 32 protein (single-stranded DNA binding protein) (49-51).
In order to determine whether these pause sites were resulting from primary or secondary structural effects, the precise nucleotide sequence near these pause sites was determined from the sequencing reactions run in adjacent lanes.
Computer analysis of this region identified numerous locations of potential hairpin structures, and three of the most energetically favorable structures are depicted (Fig. 2). The strong pause sites near position 6130 corresponded precisely with the base of the one of the more energetically favored hairpin structures. A series of numerous bands occurred in this region centered around the position where the hairpin starts, but starting five nucleotides before the hairpin and extending several nucleotides into the stem. Surprisingly, only very minor pause sites were seen at position 6090 where the predicted hairpin structure shown is actually more favorable energetically than the structure at position 6130. The weak pause sites observed at position 6064 could not be correlated with any region of potential secondary structure. Finally, the strong pause site at position 6008 is located seven nucleotides past the 15 base loop of the hairpin a t position 6041. This hesitation site could not be simply accounted for since the expected hesitations sites on the primer proximal side of the hairpin were not observed.
The addition of SSB (3.8 pg) to the synthesis reaction almost completely eliminates the hesitation that occurs through the first potential hairpin structure. However, numerous weak pausing sites remained further downstream from the proposed hairpin. Additionally, the strong pause site at position 6008 was only able to be partially removed by SSB.
The variations observed in the particular sites of hesitation at each potential hairpin could be accounted for by several possible explanations. For example, some of the hesitation sites may be a response to the primary structure of the template rather than the secondary structure. Alternatively, these sequences may represent structures which are not removed in the presence of SSB. In any event, the fact that it is not possible to precisely map the positions of these hairpins makes the interpretations of these results difficult and prompted us to use a more well defined template. Characterization of the SSB-mediated Helix Destabilization-In order to visualize the effect of SSB on DNA synthesis by the T7 DNA polymerase through a hairpin region in a well characterized situation, synthesis was carried out on a M13mp7 DNA template (Fig. 3). This template has a 22-base pair perfect homology hairpin structure containing a 4-base loop (31) located 19 bases downstream from the pentadecamer primer used in these studies. The use of this template allowed the visualization of synthesis through a single hairpin known to be very stable (-57 kcal mol") under in vitro synthesis conditions.
In the absence of SSB (Fig. 3, lane a), the majority of the pause sites are at the base of the primer proximal side of the hairpin, although some hesitation is observed continuing up the stem of the hairpin. The addition of SSB (Fig. 3, lane b ) results in a dramatic decrease in the number of hesitation sites. These data strongly suggest that SSB is aiding the T7 DNA polymerase by allowing it to synthesize through this extremely stable secondary structure. It should be noted, however, that although the hairpin structure in Fig. 3 appears to be a strong block to replication, even in the absence of SSB, a large percentage of polymerase molecules must be able to synthesize through the hairpin since a substantial amount of full-length products can be observed near the top of the gel.
In order to exclude the possibility that SSB was simply stimulating synthesis on all possible sequences with no particular preference for stimulating synthesis through hairpins, this experiment was carried out using decreasing levels of polymerase (Fig. 3, lanes c-h). For each level, the major product produced when SSB was present was long-length  (lanes a, c, e, and g) or presence (lanes b, d, f, and h ) of SSB (9.5 pg) was carried out on an M13mp7 template (0.45 pg) as described under "Experimental Procedures." Four sets of reactions were run each set +SSB and with either 0 .36 (lanes a and b), 0.036  (lanes c and d), 0.0036 (lanes e and f ) , or 0.00036 units (lanes g and h ) of T7 DNA polymerase. Synthesis was primed with a 32P-labeled oligonucleotide, and the labeled DNA products were run on a 10% denaturingpolyacrylamide gel. The nucleotide sequence of the hairpin structure is illustrated to the kft, with the position of the base of the hairpin indicated on the autoradiogram to the right.
DNA shown at the top of the gel (Fig. 3) indicating that even at extremely low polymerase concentration there is little blockage of synthesis at the hairpin in the presence of SSB.
However, it must be noted that at lower polymerase concentrations additional bands also appear which correspond to synthesis stopping in the middle of the hairpin. These products probably result from hesitation by the polymerase as it proceeds through the hairpin. However, in no case does synthesis "pile up" prior to the hairpin as occurs in the absence of SSB.
Evidence for a Dual Role for SSB in DNA Synthesis-There is a good deal of evidence that suggest that the mechanism of stimulation by SSB may not be solely to eliminate base-paired secondary structures. First, prior studies show that the effect of SSB is greatest under conditions where synthesis is initiated with short RNA or DNA primers (22)(23)(24). Second, the time course shown in Fig. 1 indicates that SSB has a greater effect during the initial stages of synthesis. Third, SSB had very little effect on many of the sites of hesitation by the T7 DNA polymerase, and even in its absence, much of the synthesis is full length. Fourth, preliminary experiments established a correlation between the extent of stimulation and the polymerase-template ratio.
In order to test the possibility that stimulation might result from a second role of the SSB in addition to destabilizing hairpin blocks to synthesis, we constructed a singly primed poly(&) template that lacks the ability to form hairpin structures. This poly(dA) template consisted of a long poly(dA) homopolymer (dA)aoo which had an 18-nucleotide stretch of dC residues added to its 3' terminus by terminal deoxynucleotidyl transferase. The use of a pentadecamer primer with the sequence (dG)ll (dT), allowed synthesis to initiate from a unique site at the 5'-end of the poly(&) template. Since this template contained a long single-stranded region and was primed at a unique site, this template provided a direct comparison between the synthetic template and the natural M13 templates. In the absence of SSB, the level of DNA synthesis was approximately 2-fold greater for the poly(&) template than for the M13 mp9 (Table I). This difference may be due to the secondary structures present on the M13 template which would decrease the total amount of DNA synthesis. However, we cannot rule out the possibility that the T7 DNA polymerase simply has a higher affinity for the poly(&) template versus the M13 template which results in an increased level of DNA synthesis. In the presence of SSB, the extent of DNA synthesis was nearly the same for all three templates (Table I).
On the poly(&) template, the stimulation by SSB was approximately 4-fold suggesting that the mechanism for stimulation may involve properties other than its role in destabilizing potential hairpin structures. It must be noted that approximately %fold more SSB was required for full stimulation on the poly(&) template as compared to the amount of SSB required for maximum stimulation on M13 DNA. However, the need for additional protein might be accounted for by the lower affinity of SSB for the poly(dA) (52). Stimulation by SSB Is Dependent on the Polymerase to DNA Ratio-To more fully characterize the role polymerase concentration plays in the stimulation by SSB, we have determined the relative stimulation caused by the presence of SSB under conditions where the level of M13mp9 template DNA and SSB concentration were held constant, and the amount of polymerase was increased over a wide range (Fig.   4A). As expected, the absolute amount of DNA synthesis increases with increasing levels of polymerase in both the presence and absence of SSB. However, the stimulatory effect of SSB decreases from 12-to 1.4-fold as the polymerase concentration is increased from 0.06 to 2 units/ml. Thus, when there is approximately 1 polymerase molecule/template molecule, the effect of SSB is negligible, but when the template is present in large excess, the stimulation is increased 10-fold. Furthermore, most of this increased stimulation is a result of a large decrease in DNA synthesis by the polymerase in the absence of SSB (195 pmol to 10 pmol). Over this same range of polymerase levels, synthesis in the presence of SSB only drops from 275 to 120 pmol.
In order to discount the possibility that we were approaching the maximum level of synthesis possible at the upper polymerase concentrations, we carried out a parallel study where the level of polymerase was held constant and the amount of SSB-coated template was varied (Fig. 4B). In this experiment, the T7 DNA polymerase concentration was held at 0.15 units/ml, which is near the midpoint of the range used in Fig. 4 A , and the template concentration was increased from 0.01 pmol of DNA molecules to 0.32 pmol(O.02 pg to 0.76 pg). Under these conditions and in the absence of SSB, the amount of DNA synthesis produced is essentially independent of the amount of DNA template present, suggesting that even at the lower DNA concentrations the template is in excess. The presence of SSB resulted in a stimulation of synthesis which was dependent on the template concentration and increased from 2-to over 6-fold. In this experiment, the SSB to template ratio was held constant at a level sufficient to completely coat the DNA template and the polymerase to template ratio decreased from near stoichiometric to a 16-fold excess of template. The data presented in Fig. 4 clearly show that the extent of stimulation by SSB is greatest when there are fewer polymerase molecules relative to template molecules, Similar results were obtained when the experiments shown in Fig. 4 were conducted using a poly(&) template DNA (data not shown).
Effect of SSB on Polymerase Binding-Although several mechanisms might explain why the stimulation of the T7 DNA polymerase by SSB is greatest at low polymerase concentrations, a likely possibility, which has some precedent in the literature (53,54), is that SSB is preventing nonproductive binding by the polymerase to the single-stranded template. This mechanism predicts that the random binding by the polymerase to single-stranded DNA is inhibited by SSB thus promoting productive binding to the primer template site. In order to test this potential explanation, we measured the binding of the T7 DNA polymerase in the presence and absence of SSB at both high and low polymerase to template ratios.
The extent of binding of the T7 DNA polymerase to a single-stranded M13mp9 template was determined by band sedimentation through neutral sucrose gradients followed by T7 DNA polymerase assays to locate the position of the polymerase in the gradient. Fig. 5 shows the results obtained when 12 units of T7 DNA polymerase were incubated with 0.6 c(g of M13mp9 DNA at 4 "C for 20 min. At this level, approximately 60 polymerase molecules were present for each template molecule. Under these conditions, essentially all of the T7 polymerase present in the gradient co-sediments with the DNA template (Fig. 5 A ) . This band position is well separated from the position to which unbound polymerase was found to sediment in parallel gradients. It is also faster than the sedimentation of unbound M13mp9 DNA because of the increased mass the bound polymerase affords to the

DNA.
When SSB was present during the polymerase-template incubation (Fig. 5 B ) , there was no change observed in the sedimentation profile, other than the fact that the DNA sediments somewhat faster due to the presence of SSB on the template. However, the most important feature of this profile is that no polymerase is observed sedimenting free of the template, suggesting that under these conditions SSB is not blocking nonproductive binding of the polymerase to the single-stranded DNA. This experiment was carried out with sufficient SSB to coat all of the DNA present in the incubation.
We have shown that the stimulation by SSB increases as the polymerase to template ratio is reduced (Fig. 4) seems reasonable that this effect might be more pronounced when the concentration of polymerase is lowered. In order to test this possibility, a band sedimentation analysis was carried out analogous to that shown in Fig. 5 but under conditions where the polymerase concentration was 20-fold lower and the template concentration was 12-fold higher (at these levels approximately four template molecules were present for each polymerase molecule). Under these conditions and in the absence of SSB, we found, surprisingly, that the polymerase no longer binds efficiently to the DNA template (Fig. 6A).
Instead, approximately 60% of the T7 DNA polymerase activity which was recovered from the gradient sediments at the position of unbound polymerase, with the remainder associated with the template. However, the presence of SSB (Fig.  6 B ) resulted in a large reduction in the polymerase not bound to the template; under these conditions, less than 5% of the activity is recovered at the position where the free polymerase sediments and greater than 95% of the activity co-sediments with the DNA template. These results are most surprising and discount the hypothesis that SSB is inhibiting of nonproductive binding. Instead, these data suggest that when the polymerase is present at concentrations more similar to in uiuo levels, its binding affinity for a single-stranded template is reduced and, further, that the presence of SSB increases the binding affinity of the polymerase to the template.
Nucleotide Addition to a Primer-If this model for stimulation of the T7 DNA polymerase by SSB is correct, then it predicts that SSB should be able to stimulate the incorporation of a very small number of nucleotides to the 3' terminus of a primer. Furthermore, the stimulation should be dependent upon the polymerase-template ratio and thus be largest when the relative polymerase level is low. Moreover, if stimulation is observed, it should be independent of any effect SSB might have on destabilizing hairpin structures or on the processivity of the polymerase.
To test this model, we carried out synthesis on uniquely primed M13mp9 single-stranded DNA template in the presence of only [ L U -~* P ]~T T P and the chain terminator ddGTP over a range of polymerase-template ratios. Synthesis carried out using the pentadecamer primer (Fig. 7, top) in the presence of only these nucleotides will stop after the addition of 2 bases and result in the conversion of the pentadecamer into a heptadecamer. The dideoxynucleotide was added as a chain terminator to inhibit artifactual extension of the primer by the misincorporation of dTTP and to prevent the incorporation of nucleotides which may contaminate the polymerase, SSB, or nucleotide preparations at low levels.
DNA synthesis in this in vitro replication system was visualized by autoradiography following electrophoresis of the product on a 23% polyacrylamide gel (Fig. 7). It is evident from this experiment that at a high polymerase concentration (Fig. 7, lunes a and b ) there is little stimulation observed upon the addition of SSB. However, as the polymerase level is reduced from approximately equal stoichiometry (Fig. 7, lunes  a and b ) to a 75-fold excess of DNA, the stimulation produced by the presence of SSB increases until, at the lowest polymerase concentration, a substantial level of stimulation is  ; lanes e and f, 0.005 units; lanes g and h, 0.001 units. Lanes b, d, f, h, and i,  reached (Fig. 7, lanes g and h). Had the effect of SSB been to alter the secondary structure of the DNA, then this stimulation should have been independent of the polymerase concentration. Instead, we find that the maximal stimulation by SSB is observed at low polymerase levels.
The effect of SSB on this synthesis reaction is more clearly seen in the experiment shown in Fig. 8 where the level of SSB was varied using the lowest polymerase to template ratio shown in Fig. 7. Following synthesis in the absence of SSB, an extremely faint band is observed which corresponds to the heptadecamer (Fig. &4, lane a). The presence of increasing amounts of SSB (Fig. 8A, lanes b-e) results in a substantial increase in the heptadecamer product as visualized on the gel. In order to quantitate the relative level of stimulation of DNA synthesis by SSB, the bands corresponding to the heptadecamer were excised from the gel, and the radioactivity present in each gel slice was determined by Cerenkov counting (Fig.  8B). At a level where SSB was present in sufficient quantity to fully coat the DNA (Fig. 8, lane d ) , the stimulation of synthesis is over 4-fold. When SSB was present in a 2-fold excess (Fig. 8, lane e) synthesis was somewhat reduced, presumably because of the ability of SSB to destabilize the primer from the template.

DISCUSSION
The discovery and isolation of the T4 gene 32 protein (55) and the demonstration that temperature-sensitive gene 32 mutants cease all replication within 2 min of shifting to a nonpermissive temperature (56,57) led to the conjecture that all cellular replication of single-stranded DNA required the presence of a single-stranded DNA binding protein. However, until recently, the involvement of E. coli single-stranded DNA binding protein in DNA replication was based on the requirement of this protein for various in vitro viral replication systems (58, 59). The identification and characterization of temperature-sensitive mutations in the structural gene for SSB (1) confirmed the requirement for this protein and further extended its role to both DNA repair and recombinational processes (2)(3)(4)(5)(6)(7).
The simplicity and detailed understanding of the T7 in vitro replication system (60) makes this an ideal paradigm to determine the role of SSB in cellular DNA replication. It has been shown that SSB is required for T7 DNA replication, but the precise role it plays is not known. Amber mutations in the T7 DNA binding protein show defects in DNA replication which are especially pronounced when infecting a host having a mutation in SSB (19). Either the T7 or E. coli SSB proteins stimulate DNA replication by the purified T7 replication system to similar extents, with the largest effects observed using a single-stranded DNA template under conditions where RNA primers are being produced (22). The T7 DNA polymerase, which is composed of a one-to-one complex of the T7 gene 5 protein and thioredoxin, and the T7 gene 4 protein (which has both helicase (61) and primase activities (23-25)) can together catalyze both the leading and lagging strand DNA synthesis during replication on a duplex DNA molecule (60). The interactions between the T7 DNA polymerase and gene 4 protein with each other and with the DNA template have recently been characterized, and models for the mechanism of synthesis by these two enzymes based on these properties have been proposed (62). Both the T7 DNA polymerase and gene 4 protein bind efficiently to single-stranded DNA in the absence of the other protein, but the gene 4 protein binding requires the presence of a nucleoside 5'triphosphate (63). However, gene 4 protein T7 DNA polymerase.DNA complexes can form in the presence or absence dNTP. The gene 4 protein has also been shown to form a tertiary complex with the template and primer presumably at the primer recognition site (62). These pieces of information have been fit into a model (62) which predicts that the gene 4 protein binds to the single-stranded template, translocates until it finds a primer recognition site, and synthesizes a primer at that site where it remains bound until a polymerase molecule joins the complex and utilizes the primer.
How might SSB participate in this process? The results presented in this paper suggest that SSB is involved in at least two stages of replication catalyzed by the T7 in vitro system. First, SSB overcomes the temporary blockages to elongation by the T7 DNA polymerase posed by hairpin loop structures which form in the single-stranded DNA templates. In the absence of SSB, there are numerous positions in the template at which the polymerase hesitates. Addition of SSB results in a reduction of the intensities of many of these bands as well as a concomitant increase in the total level of measured synthesis. These results suggest that SSB is stimulating synthesis by reducing the frequency of hesitation at these sites by removing these hairpin structures from the template. To have a more precise estimate for the position of the hairpin structure, we have used as a template M13mp7 DNA, which contains a single extremely stable, 22-base pair, perfect homology hairpin to remove the ambiguity for the presence or position of this structure. The T7 DNA polymerase displayed a strong tendency to hesitate at the base of the primer proximal side of the hairpin with some synthesis proceeding up the proximal stem. The addition of SSB to the reaction mix resulted in a large reduction in the extent of hesitation and a concomitant increase in the extent of DNA synthesis measured. Others have shown that various polymerases pause anywhere from 17 nucleotides on the primer proximal side of the base of the hairpin to approximately halfway up the stem of the hairpin (44-47). In addition, the proximity of polymerase pausing to sites adjacent to hairpin stems has been correlated with the size of the DNA polymerase (46). Furthermore, it was shown that the presence of E. coli SSB reduced the barriers to the progression of E. coli DNA polymerase I11 holoenzyme (46).
Although we did not directly measure the processivity of the T7 DNA polymerase in this study, it has been demonstrated that SSB can increase both the rate and processivity of the T7 DNA polymerase (64), presumably by preventing it from dissociating from the template at these secondary structures which tend to impede it. However, the importance of this role in the T7 replication mechanism is not clear. Since Ti' replicates as a duplex DNA molecule and produces singlestranded templates only transiently, the extrapolation of results obtained using primed single-stranded templates may not be entirely justified. Whether these secondary structures that block the polymerase form on the time scale that the single-stranded DNA is produced by the moving replication fork prior to being replicated is not known.
The second role of SSB in T7 DNA replication involves its effect on the interaction between the polymerase and the DNA template. A great deal is known about the physical interactions between SSB and a single-stranded DNA tem-plate (18). SSB binds cooperatively and without regard for sequence (65) to single-stranded DNA as a tetramer (18) with a binding affinity estimated to be on the order of 108-10'o M" (52,66). There appears to be at least two binding modes for SSB; at a low protein to DNA ratio, the DNA takes on a beaded appearance which may be the result of the DNA being wrapped around two SSB tetramers (67,68); at higher protein concentration, the DNA appears as a smooth nucleoprotein filament (68). The former conformation, which has the appearance of a nucleosome-like structure, is also favored at NaCl concentrations above 0.2 M and may also be in slow equilibrium with the smooth contoured structure at lower ionic strengths (66). Each bead has been shown to contain approximately 145 nucleotides with a stretch of about 30 bases free of bound SSB (67). These unbound regions have been proposed to be the site of interaction with other proteins (67,68), implying that the role of the SSB may be to provide a scaffold which holds the single-stranded DNA in a rigid conformation more able to interact with these proteins.
The results presented in this paper show that SSB can alter the affinity of the T7 DNA polymerase for the single-stranded DNA template. Prior studies have suggested that singlestranded DNA binding proteins stimulate synthesis by preventing nonproductive binding to the single-stranded template and thus directing it to the primer template site (53, 54). Our data do not support this model. Instead, we find that SSB increases the affinity of the polymerase for the template, an effect only observed when the template is present in large excess compared to the number of polymerase molecules. It is not clear why the affinity of the polymerase for the template is reduced at high template levels. One possible explanation is that the polymerase binds cooperatively to single-stranded DNA, and thus, when present at a high concentration, the association between it and the template is stronger. However, when the template is present in large excess, cooperative binding is less likely, causing binding to be weaker and resulting in the polymerase dissociating from the template during the sucrose gradient analysis (Fig. 6A). But in the presence of SSB, the polymerase remains associated with the template whether the polymerase to template ratio is high (Fig. 5B) or low (Fig. 6B).
The increased affinity of the T7 DNA polymerase in the presence of SSB explains why the relative stimulation afforded by the T7 DNA polymerase varies with the polymerase or DNA concentrations (Fig. 4). Also, others have noted that gene 4 protein-primed synthesis by the T7 DNA polymerase is extremely dependent on the relative polymerase-template concentrations (63); synthesis was shown to decrease by over 3-fold as the template level was increased. The explanation for this decrease could be accounted for by a decreased affinity of the polymerase for the single-stranded template possibly caused by a dilution of the enzymes bound together to a DNA molecule. Thus, the role of SSB in the replication process might be to increase the likelihood of forming a complex between the polymerase and gene 4 protein at the primer template site.
The results of this increased affinity by the T7 DNA polymerase for the template was directly visualized in the limited synthesis reaction where only two nucleotides were added to the 3' terminus of the primer. SSB stimulated this synthesis over 4-fold (Fig. 8), presumably by increasing the number of polymerase molecules that associated with the template DNA. When this experiment was carried out at a higher polymerase to template ratio, much less stimulation by SSB was observed (Fig. 7), suggesting that this stimulation was not the result of alteration in the secondary structure of the DNA caused by the presence of SSB.
We are unable to determine at this point whether SSB is providing a more favorable template conformation for the polymerase to bind to or if there is a specific interaction between the polymerase and the SSB. The fact that SSB cannot substitute for the gene 32 protein either in vivo or in vitro suggests that there might be a specific interaction between this class of DNA binding proteins and the enzymes involved in DNA metabolism (10,69,70). There is also limited physical evidence for the interaction between replication proteins and DNA binding proteins in vitro. It has been shown that a gene 32-bound affinity column specifically retains nine proteins which are known to be involved in either T4 DNA replication or recombination (71). SSB has also been proposed to have specific associations with the replication proteins (70, 41). Early reports described a specific interaction between the T7 DNA polymerase and SSB (72) but we, as well as others (43), have been unable to detect a complex between SSB and the T7 DNA polymerase (data not shown). Further physiochemical analysis will be required in order to determine the exact interactions between the T7 replication proteins and the SSB-coated template DNA. (73) recently reported that both E. coli and T7 DNA binding proteins stimulate the rate of initiation of gene 4 protein-primed lagging-strand DNA synthesis by the T7 DNA polymerase.