©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Differences in the Mechanism of Stimulation of T7 DNA Polymerase by Two Binding Modes of Escherichia coli Single-stranded DNA-binding Protein (*)

Mary N. Rigler (§) , Louis J. Romano (¶)

From the (1) Department of Chemistry, Wayne State University, Detroit, Michigan 48202

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Escherichia coli single-stranded DNA-binding protein ( Eco SSB) has been shown previously to display several DNA binding modes depending on the ionic conditions. To determine what effect these various binding modes have on DNA replication, we have studied DNA synthesis by the T7 DNA polymerase under ionic conditions where Eco SSB interacts with either 72 or 91 nucleotides of M13 DNA. These forms presumably correspond to the previously described (SSB)and (SSB)(Lohman and Ferrari, 1994) that were determined using the binding of SSB to homopolymers. Here we report the stimulation induced by (SSB)to be 4-fold greater than that produced by (SSB)under conditions where the template is in large excess. Surprisingly, when the polymerase level is raised so that it is in molecular excess, (SSB)no longer stimulates synthesis while (SSB)affords a 4-fold stimulation, which is the same level of stimulation as when the template was in excess. Both SSB forms increase the rate of DNA synthesis and were found to stimulate synthesis by relieving template secondary structures. However, (SSB)specifically increases strand displacement synthesis, while (SSB)stimulates synthesis by increasing the affinity of the polymerase for the template.


INTRODUCTION

Escherichia coli single-stranded DNA-binding protein ( Eco SSB)()binds tightly and cooperatively to single-stranded DNA to produce a complex that has been shown to participate in DNA replication, repair, and recombination (Chase and Williams, 1986; Meyer and Laine, 1990). This protein is essential for the replication of the E. coli chromosome as well as several other coliphages, including bacteriophage T7 (Meyer and Laine, 1990). Eco SSB exists as a stable homotetramer over a wide range of protein concentrations and solution conditions, and it is this tetrameric form of Eco SSB that binds to single-stranded DNA (Lohman and Ferrari, 1994). Electron microscopy, nuclease digestion (Chrysogelos and Griffith, 1982; Griffith et al., 1984), nuclear magnetic resonance experiments (Greipel et al., 1987), and the stoichiometry of oligonucleotide binding (Krauss et al., 1981; Bujalowski and Lohman, 1989) support the idea that the ssDNA is wound around an Eco SSB core in a structure reminiscent of a eukaryotic nucleosome and that, depending on the solution conditions, several complexes can be formed, which produce different percent decreases in the contour length of ssDNA.

The Eco SSB tetramer has been shown to interact with ssDNA in a number of different binding modes (Lohman and Ferrari, 1994), which differ in the number of nucleotides occluded by a single Eco SSB tetramer. These binding modes are referred to as the binding size (SSB), where n indicates the average number of nucleotides bound by the Eco SSB tetramer in that mode. Variation in early estimates of the binding site sizes (Krauss et al., 1981; Weiner et al., 1975) was explained by a series of experiments showing that the different complexes of Eco SSB and ssDNA which form depend on the salt concentrations, cation and anion valence or type, temperature, pH (Lohman and Overman, 1985; Bujalowski et al., 1988), and protein binding density (Bujalowski et al., 1988; Griffith et al., 1984; Lohman et al., 1986b). Lohman and co-workers showed that by increasing the ionic strength of the medium, the Eco SSB tetramer can form three distinct binding modes in complexes with poly(dT) at 25 °C, pH 8.1, where n = 35, 56, or 65 (Lohman and Overman, 1985), and one additional binding mode of n = 42 at 37 °C (Bujalowski and Lohman, 1986). Although complexes with native ssDNA appear to undergo the same salt-dependent size transitions, reported values of n have been larger than those for poly(dT) (Lohman and Overman, 1985; Morrical and Cox, 1990). These larger values may be due to stabilization of secondary structures in the native ssDNA as salt levels are raised, excluding protein from these regions.

Comparison of the physical properties of the three Eco SSB binding modes shows substantial differences. Eco SSB can exhibit a very high cooperativity when mixed with DNA at the low concentration of NaCl required to form the (SSB)binding mode. In this low salt mode, ssDNA interacts with only two subunits of the Eco SSB and seems to form unlimited clusters (Lohman and Ferrari, 1994). This may be the binding mode of smooth contoured complexes visualized in the electron microscope, where approximately 60% compaction of M13 occurs (Griffith et al., 1984; Bujalowski and Lohman, 1986). However, (SSB)exhibits a transient or ``metastable'' unlimited cooperativity, which is not a property of the binding mode at equilibrium (Lohman et al., 1986b). In both the (SSB)and (SSB)modes, all four subunits of the Eco SSB tetramer interact with ssDNA (Lohman and Ferrari, 1994), and it is thought that these forms produce the beaded SSB-ssDNA morphology observed in the electron microscope. Conditions that favor formation of both the (SSB)and the (SSB)binding modes are associated with approximately 80% compaction of M13 DNA (Griffith et al., 1984; Lohman and Bujalowski, 1990).

Because of the multiple roles that Eco SSB plays in the life cycle of E. coli, it has been suggested that the different properties of the SSB binding modes might allow it to selectively participate in this wide range of processes (Lohman et al., 1986b, 1988; Lohman and Bujalowski, 1990). Studies to support this suggestion have been limited to the effect of Eco SSB binding modes on the activities of E. coli RecA protein and these have shown that the (SSB)mode selectively inhibits reactions of this protein (Griffith et al., 1984; Morrical and Cox, 1990; Muniyappa et al., 1990). The fact that (SSB)displays unlimited cooperative binding has been used as evidence to support the suggestion that this mode might be present at the replication fork since this form can completely coat the ssDNA (Lohman and Bujalowski, 1990; Lohman et al., 1986b). There are no experimental data that address this possibility.

This paper is a continuation of our systematic study of the mechanisms by which SSB stimulates DNA synthesis. The goal of the present study is to determine if the different binding modes of Eco SSB differentially effect DNA synthesis and the mechanisms by which any differences occur. To address these questions we have used a chloride-free potassium glutamate buffer (KGB) system (McClelland et al., 1988) that did not inhibit DNA synthesis at the high concentrations required to induce the highest binding site modes. We incorporated these buffer conditions into an in vitro system, comprising a single-stranded viral DNA template hybridized to an oligonucleotide pentadecamer primer, which serves as a model for lagging strand DNA synthesis. In this study we show that the magnitude of stimulation of the T7 DNA polymerase and molecular mechanism which induces this stimulation is dependent on the binding site size with which Eco SSB interacts with the single-stranded template.


EXPERIMENTAL PROCEDURES

Materials

Bacterial Strains and Bacteriophages

E. coli JM103 and bacteriophage M13mp9 and M13mp7 have been described (Messing and Vieira, 1982). E. coli M72SmlacZambio-uvrBtrpEA2 ( Nam7-Nam53cI857H1/pTL119A-5) was obtained from Dr. Timothy M. Lohman (Texas A & M University) and has been described previously (Lohman et al., 1986a).

DNA

ssM13 DNA was prepared as described by Hayes and LeClerc (1983).

Nucleotides

Unlabeled nucleotides and (dT)were purchased from Pharmacia Biotech Inc. The (dT)and (dA)were purchased from the Midland Certified Reagent Co. The 15-mer and 17-mer primers (5`-TCCCAGTCACGACGT-3` and 5`-GTTTTCCCAGTCACGAC-3`) were purchased from New England Biolabs. [H]TTP (80 Ci/mmol) was from DuPont NEN, [-S]dATP (1000 Ci/mmol) was from Amersham Corp., and [-P]dATP (25 Ci/mmol) and [-P]dATP (7000 Ci/mmol) were from ICN Corp.

Proteins

T7 DNA polymerase Form II (95% pure) was either prepared by Dr. William Clay Brown (Brown and Romano, 1989) or purchased from U. S. Biochemical Corp. The specific activity of these preparations ranged 3000 to 7000 units/mg. E. col SSB (99% pure) was prepared from E. coli M72SmlacZambio-uvrBtrpEA2 ( Nam7-Nam53cI857H1/pTL119A-5) (Lohman et al., 1986a). The concentration of Eco SSB was determined spectrophotometrically by using the extinction coefficient of = 1.5 ml mgcm(1.13 10 Mcmtetramer) in a standard buffer (pH 8.1) of 10 m M Tris, 0.1 m M NaEDTA, and 0.20 M NaCl (Lohman and Overman, 1985). The purity of the above proteins was determined by polyacrylamide gel electrophoresis in the presence of SDS. T4 polynucleotide kinase and proteinase K were obtained from U. S. Biochemical Corp.

Other Materials

DE81 filter discs were obtained from Whatman. Methods

Buffers

Potassium glutamate buffers were prepared essentially as described by McClelland et al. (1988), where 2.0 KGB denotes a buffer (KGlutamateBuffer) that contains 200 m M potassium glutamate, 50 m M Tris acetate (pH 7.5), 20 m M magnesium acetate. 1.5 , 1.25 , 1.0 , and 0.8 are dilutions of 2 KGB made with distilled water, except that in 1.0 and 0.8 the concentration of Tris acetate was raised to 30 m M in each.

Fluorescence Measurements

Reverse titrations of E. col SSB protein with ssM13mp9 were monitored by the quenching of intrinsic tryptophan fluorescence as described by Lohman and Overman (1985) using a Spex Spectramate 1680. The excitation wavelength of 300 nm and excitation band pass = 0.9 nm (0.5-mm slit width) were used, while the emission at 347 nm was monitored; emission bandpass = 4.5 nm (2.5-mm slit width). All titrations were carried out in a cell compartment maintained at 37.0 ± 0.2 °C using a thermostated cuvette holder and a circulating water bath. A 3.0-ml solution of SSB in a 4.0-ml quartz fluorescence cell (Starna, 10 10 4.5 cm) was stirred constantly with a Teflon-coated magnetic stir bar (8 8 mm) while concentrated ssM13mp9 DNA was titrated in 2-5-µl aliquots at 2-min intervals, with a 5-s acquisition time.

The fluorescence measurements were corrected for dilution, photobleaching, and inner filter effects using the method of Lohman and Mascotti (1992). The fluorescence signal from a blank containing the standard KGB buffer solution was subtracted from all data. Photobleaching of SSB was determined under each set of buffer conditions that were used in the experimental titrations and was found to be less than 2%. The SSB concentration used in these experiments was 1.11 10 M (tetramer) (8.47 µg/ml). At a given salt concentration, the SSB binding sizes are constant over a 5-fold range of SSB concentration (6.3-29.7 µg/ml). Using the method of Lohman and Overman (1985), no precipitation of SSB was detected at the salt and SSB concentrations used in these studies. The relationship between the corrected fluorescence intensity and concentration of SSB (Lohman and Mascotti, 1992) was determined in total reaction buffer over the range of 1.0 to 2.0 KGB at 37.0 ± 0.2 °C. A linear response was observed over a range of [SSB] from 0.41 to 20.5 µg/ml, which corresponds to 5.0% to greater than 200% of the concentration of SSB (8.47 µg/ml) used in binding site size determinations.

DNA Synthesis Assays

M13 single-stranded DNA (either 95 or 150 µg/ml) and 7-fold molar excess 15-mer primer (5`-TCCCAGTCACGACGT-3`) or (dA)(65 µg/ml) and a 1:1 molar ratio of the hexadecamer (dT)were annealed in 100 m M potassium glutamate by heating to 65 °C for 3 min, then allowing the solutions to cool for 1 h. The indicated amounts of primed DNA were added to a reaction containing the indicated level of KGB, 5 m M 2-mercaptoethanol, 200 µ M dATP, dCTP, dGTP, and [H]dTTP (approximately 100 cpm/pmol). Reactions using poly(dA) as a template contained only 300 µ M [H]dTTP. T7 DNA polymerase and Eco SSB were added to the reaction mix as indicated. The reaction mixtures (100 µl) were incubated at 37 °C for 30 min and stopped by the addition of 3 ml of ice-cold 100 m M NaPOin 1 M HCl. DNA synthesis was determined by measuring the amount of acid-insoluble radioactivity by a modification of the procedure described by Richardson (1971). The precipitate was collected on Whatman GF/C filter discs, then rinsed three times with 3 ml of the acid solution and once with 95% ethanol. Acid-precipitable counts were measured in 5 ml of a nonaqueous scintillation mixture on a Beckman LS 7500 liquid scintillation counter.

Formation of Linear Single-stranded M13mp7L1 DNA

Linear single-stranded M13mp7L1 DNA was prepared as described (Banerjee et al., 1990).

Polyacrylamide Gel Electrophoresis

Primer-template annealing reactions were carried out as described except that the pentadecamer primer was labeled at the 5` end with P. The reaction mixture (80 µl) contained 50 m M Tris-Cl, pH 7.6, 10 m M MgCl, 10 m M 2-mercaptoethanol, 0.48 µg (96 pmol) of primer, 5 µ M [-P]dATP(7000 Ci/mmol), and 20 units of T4 polynucleotide kinase. Incubation was for 90 min at 37 °C, followed by 10 min at 75 °C to inactivate the kinase. The DNA synthesis reactions were carried out as described above except the dTTP was unlabeled. The reactions were then incubated with proteinase K (80 µg) at 65 °C for 20 min. This step was repeated twice, to insure maximum removal of Eco SSB from DNA synthesis products. To remove the possibility that different levels of Eco SSB affected the electrophoresis or sample loading, either the Eco SSB concentration was adjusted so that all reactions had the same concentration of Eco SSB prior to loading the gel, or it was verified that reactions were identical except for their Eco SSB level appeared the same on the gel. Reactions were diluted 1:1 with a mixture of 100% formamide, 0.1% bromphenol blue, and 0.1% xylene cyanol, then boiled for 5 min and quick cooled in ice water and immediately loaded onto a 10% wedge denaturing polyacrylamide gel. Gels were autoradiographed at -70 °C using Kodak XAR-5 film and a DuPont Cronex Lighting Plus intensifying screen. Gels were also scanned using a Molecular Dynamics PhosphorImager SF and radioactivity quantitated using ImageQuant software.

Alkaline Agarose Gel Electrophoresis

DNA synthesis reactions (50 µl) were carried out as described above except that dTTP was unlabeled and 300 µ M [-S]dATP (1000 Ci/mmol) was used. Eco SSB was present as indicated. To determine the extent of strand displacement DNA synthesis, DNA synthesis reactions (50 µl) were carried out as described above except that 300 µ M dATP was present and the pentadecamer primer labeled at the 5` end with P as described above. The reactions were stopped by addition of 5 µl of 0.2 M EDTA (pH 8). Eleven µl of 300 m M NaOH, 6 m M EDTA (pH 8), 18% Ficoll type 400 (in water), 0.15% bromcresol green, and 0.25% xylene cyanol was added to each reaction and the samples were loaded onto a 0.7% alkaline agarose gel prepared as described (Sambrook et al., 1989). Gels were soaked in 7% trichloroacetic acid for 15 min, then dried and autoradiographed at room temperature using Kodak XAR-5 film. To determine the extent of strand displacement DNA synthesis, radioactivity in the dried gels was quantitated using a Molecular Dynamics PhosphorImager SF.

Single Round of DNA Synthesis on M13mp9

M13 single-stranded DNA (270 µg/ml) and 7-fold molar excess of the 15-mer primer (5`-(TCCCAGTCACGACGT-3`) or (dA)(average size 1400 bases) (260 µg/ml) and a 3-fold molar excess of the hexadecamer (dT)were annealed in 100 m M potassium glutamate by heating to 65 °C for 3 min, and then allowing the solutions to cool for 1 h. All incubations were carried out at 25 °C. The preincubation mixture (120 µl) contained indicated amounts of primed DNA, the indicated level of KGB, and 5 m M 2-mercaptoethanol. Eco SSB was present or not, as indicated. Preincubation from 2 to 15 min gave identical results. A standard preincubation time of 2 min was chosen. The reaction was started by the addition of a 80-µl start mix containing dCTP, dGTP, dTTP, [-P]dATP (3000 Ci/mmol), and the indicated amount of primed (dA)in the indicated level of KGB to give final concentrations of 50 µ M dCTP, dGTP, [-P]dATP (3000 Ci/mmol), and 150 µ M dTTP. Eco SSB (13.2 µg) was present as indicated. Reactions were stopped by addition of 60 µl of 0.5 M EDTA (pH 8) at the indicated times. The reactions containing Eco SSB were ethanol-precipitated as described (Sambrook et al., 1989). [-P]dAMP incorporation was measured on DE81 filter discs as described (Bryant et al., 1983).


RESULTS

Identification of Eco SSB-ssDNA Binding Modes in KGB

Reverse titrations, where a lattice of ssM13mp9 DNA was added to the Eco SSB ligand, were used to monitor the quenching of the intrinsic tryptophan fluorescence induced upon binding (Lohman and Overman, 1985; Lohman and Mascotti, 1992) (data not shown). This procedure was performed over a range of KGB concentrations, with at least four replicate determinations at each salt concentration (Table I). Two binding site sizes were obtained for which n = 72 ± 4 in 0.8-1.0 KGB, corresponding to 80-100 m M potassium glutamate and 8-10 m M magnesium acetate, and n = 91 ± 4 in 2-2.5 KGB, corresponding to 200-250 m M potassium glutamate and 20-25 m M magnesium acetate. We believe that these binding modes correspond to the two largest Eco SSB binding modes determined for Eco SSB-poly(dT) complexes (Bujalowski and Lohman, 1986) of n = 56 and 65 nucleotides/ Eco SSB tetramer (see Discussion). The relationship between fluorescence and concentration of Eco SSB was verified to be linear in both 1 and 2 KGB over a range of Eco SSB concentrations from 5% to greater than 200% of the concentration used in fluorescence determinations of Eco SSB binding mode. At a given salt level, the Eco SSB binding sizes are constant over a 5-fold range of Eco SSB concentration (6.3-29.7 µg/ml) (data not shown), suggesting that our measurements of binding site size are not influenced by Eco SSB affinity (Lohman and Mascotti, 1992). We also found that the fluorescence of Eco SSB by ssM13mp9 was dependent on salt concentration, and the fluorescence of free Eco SSB was independent of salt concentration (not shown).

Stimulation of DNA Synthesis by Eco SSB Is Dependent on the Binding Site Size

Prior studies have shown that Eco SSB can stimulate DNA synthesis by the T7 DNA polymerase and gene 4 protein on a duplex template (Scherzinger et al., 1977; Romano and Richardson, 1979a), and this stimulation is greatest for RNA-primed lagging strand synthesis (Romano and Richardson, 1979a, 1979b). It has also been shown that on single-stranded templates the T7 DNA polymerase can use short RNA primers synthesized by the T7 gene 4 protein to initiate DNA synthesis (Romano and Richardson, 1979a, 1979b). For this study, we used oligonucleotide-primed single-stranded DNA templates as a model for T7 gene 4 protein-primed lagging strand synthesis. Addition of Eco SSB to this system (Myers and Romano, 1988) and to many in vitro replication systems (Meyer and Laine, 1990) results in a substantial stimulation of DNA synthesis.

To determine if the binding site size affected the stimulation induced by Eco SSB, we have measured DNA synthesis by the T7 DNA polymerase on a primed single-stranded M13 template under conditions that span the transition from (SSB)to (SSB)in both the absence and presence of Eco SSB (Fig. 1). In the absence of Eco SSB, synthesis levels remained in the 10-20 pmol range, showing that this change in salt concentration has little or no effect on synthesis when Eco SSB is not present (Fig. 1). However, in the presence of Eco SSB, we obtained a dramatic increase in the level of synthesis when the binding site size was increased from 72 to 91 (Fig. 1). These increased levels of synthesis correspond to a 4-fold stimulation of DNA synthesis by (SSB), while (SSB)provided a 14-fold stimulation.


Figure 1: Stimulation of T7 DNA polymerase by Eco SSB depends on SSB binding mode. DNA synthesis by the T7 DNA polymerase (0.005 units) was performed on a primed M13mp9 DNA template (0.60 µg) in the absence or presence of Eco SSB (15.8 µg) using the indicated concentration of KGB as described under ``Experimental Procedures.'' DNA synthesis levels were measured by the incorporation of [H]dTTP into acid-precipitable DNA.



The data presented in Fig. 1 used conditions where the template was in large molar excess, conditions that provide the largest effect by Eco SSB. Prior studies (Myers and Romano, 1988) have shown that under these conditions Eco SSB stimulates synthesis by increasing the affinity of the polymerase for the template. It was also found that when the polymerase is present at high concentrations, it binds stably to the template in the absence of Eco SSB and the stimulation in this situation was the result of other Eco SSB properties. Therefore, it is important to explore the effect Eco SSB binding site size has on synthesis under conditions where the template is limiting. We carried out a similar set of experiments to that shown in Fig. 1, but used a 40-fold higher level of polymerase. Under these conditions, the binding site size had an effect but, surprisingly, it was the reverse of that observed when the template was in excess (Fig. 2). Thus, at low binding site size ( n = 72), we observed a 3-4-fold stimulation by Eco SSB, while at high binding site size ( n = 91), we obtained no stimulation by Eco SSB. Note also that even at the high polymerase level the KGB concentrations had no effect on synthesis in the absence of Eco SSB (Fig. 2).


Figure 2: Stimulation by Eco SSB binding mode depends on the T7 polymerase:template ratio. DNA synthesis by the T7 DNA polymerase (0.2 units) was performed on a primed M13mp9 DNA template (0.38 µg) in the absence or presence of Eco SSB (10.1 µg) using the indicated concentration of KGB as described under ``Experimental Procedures.'' DNA synthesis levels were measured by the incorporation of [H]dTTP into acid-precipitable DNA.



What mechanisms might account for these intriguing differences? Prior studies have suggested that Eco SSB stimulates by at least three mechanisms. One of these involves the relief of secondary structures that inhibit the progress of the polymerase (Myers and Romano, 1988; Kowalczykowski et al., 1981; Tabor et al., 1987). A second mechanism suggests that Eco SSB specifically increases strand displacement synthesis (Nakai and Richardson, 1988b). Finally, Eco SSB has been shown to increase the affinity of the polymerase for the DNA template (Myers and Romano, 1988). To more fully characterize the effect of Eco SSB binding site size on polymerase stimulation, we attempted to determine how each binding mode affected each mechanism of stimulation.

Eco SSB Stimulation by Removal of Template Secondary Structures

As a first step in determining the mechanism by which Eco SSB binding site size is involved in the stimulation of T7 DNA polymerase, we determined the effect on synthesis through a stable 22-base perfect homology hairpin. In the absence of Eco SSB, the majority of the pause sites are on the proximal side of the hairpin over the range of KGB concentrations in which the (SSB)and (SSB)binding modes have been identified (Fig. 3, lanes a-e). The presence of Eco SSB (Fig. 3, lanes f-j) results in a dramatic decrease in the number of hesitation sites and a substantial increase of full-length product over the entire range of KGB concentrations. The extent of polymerase pausing was quantified by calculating the percentage of product formed that extended beyond the hairpin. In the absence of Eco SSB, between 15 and 20% of the synthesis was observed to synthesize past the hairpin no matter which KGB level was present. Similarly, between 70 and 76% of the synthesis bypassed the hairpin if Eco SSB was present in either binding site mode. These data strongly suggest that both (SSB)and (SSB)aid the T7 DNA polymerase by allowing it to synthesize through this extremely stable secondary structure. Moreover, any differences in stabilization of secondary structure over the range of KGB concentrations needed to study the Eco SSB binding modes are not sufficiently large to inhibit the ability of Eco SSB to melt secondary structures.


Figure 3: Both (SSB) and (SSB) binding modes increase synthesis by T7 DNA polymerase through the hairpin structure of M13mp7. DNA synthesis by the T7 DNA polymerase (0.20 units) in the absence ( lanes a-e) or presence ( lanes f-h) of Eco SSB (10.1 µg) was carried out on an M13mp7 template (0.38 µg) in the indicated concentration of KGB. DNA synthesis reaction conditions are described under ``Experimental Procedures.'' The primer-template was the P-labeled 15-mer primer annealed to M13mp7 as described under ``Experimental Procedures.'' The labeled DNA products were run on a 10% denaturing polyacrylamide wedge gel and were visualized by autoradiography.



Stimulation of T7 DNA Polymerase by Eco SSB on a Poly(dA) Template

Because no differences were observed in relieving secondary structure inhibition by the two Eco SSB binding modes, other mechanisms must be involved, which lead to the observed differences in synthesis levels. This was confirmed by carrying out synthesis reactions on a poly(dA) template that lacks the ability to form secondary structures. When the template is present in large excess, we obtained a result using this template similar to the result obtained using M13. Thus (SSB)afforded no stimulation of synthesis, while (SSB)produced a large stimulation (3-5-fold) (Fig. 4). Interestingly, when the template was not present in excess, Eco SSB did not stimulate under any conditions (data not shown). The implication of this latter result is that at high polymerase to template ratios, (SSB)might be stimulating on an M13 template by the relief of secondary structures but on a poly(dA) template (where no secondary structure can exist) no stimulation is obtained. Alternatively, other differences between the M13 and poly(dA) templates, such as the potential for strand displacement synthesis on the circular M13 template via a rolling circle model, may play a role.

Effect of Eco SSB Binding Mode on Strand Displacement DNA Synthesis

On a circular M13 template, once the leading strand has progressed once around the template, strand displacement will yield a (-) strand product that is larger than the 7.2 kb (+) M13 DNA. This process is referred to as a rolling circle model in which the leading strand synthesis continually displaces the newly synthesized (-) strand DNA so that very large products are produced. In the absence of other proteins, the native form of T7 DNA polymerase (Form II) lacks the ability to catalyze leading strand DNA synthesis by a rolling circle mechanism of strand displacement. The lack of strand displacement activity of native T7 DNA polymerase has been attributed to its exonuclease activity, which results in high nucleotide turnover as it stalls when synthesizing at a nick, or region of strong secondary structure (Engler et al., 1983; Tabor and Richardson, 1989). However, in the presence of Eco SSB, native T7 DNA polymerase can catalyze strand displacement synthesis on a primed ssM13 DNA template, and it has been suggested that this is the major mechanism by which Eco SSB stimulates DNA synthesis on a primed single-stranded circular template (Nakai and Richardson, 1988b).

To determine the effect of Eco SSB binding mode on strand displacement, DNA synthesis was carried out on a primed single-stranded M13mp9 template under conditions where either (SSB)or (SSB)formed and the products analyzed on an alkaline agarose gel (Fig. 5). In the absence of Eco SSB, no strand displacement synthesis was observed, regardless of the KGB concentration (Fig. 5). However, in the presence of Eco SSB, we found that the extent of strand displacement synthesis was very much dependent upon the Eco SSB binding site size. Under conditions where (SSB)was induced (2.25 KGB), we could not observe any strand displacement synthesis, even when polymerase was present in large excess (Fig. 5). But when the (SSB)binding mode formed, extensive strand displacement synthesis was observed, even when the polymerase concentration was limiting (Fig. 5, lanes a, e, and i). The extent of strand displacement synthesis was measured in a separate experiment using a 5` end labeled primer (not shown) so that the amount of radioactivity incorporated would be independent of the length of the product. This experiment indicated that approximately 3-fold more strand displacement synthesis was produced over full-length product when 3 polymerase/template were used. Finally, to verify that strand displacement synthesis was not being prohibited by the (SSB)mode due to an increase in exonuclease activity with increasing salt levels, we repeated this study using Sequenase version 2.0 T7 DNA polymerase, which lacks any 3` to 5` exonuclease activity (Tabor and Richardson, 1989). Using this polymerase, (SSB)was still the only binding mode able to stimulate strand displacement synthesis (data not shown).


Figure 5: Effect of Eco SSB binding mode on strand-displacement DNA synthesis by T7 DNA polymerase. DNA synthesis was performed in the indicated concentration of KGB with 0.474 units ( lanes a-d), 0.158 units ( lanes e-h), or 0.003 units ( lanes i-l) of T7 DNA polymerase on 0.19 µg ( lanes a-h) or 0.24 µg ( lanes i-l) of a primed M13mp9 template in the absence or presence of 5.05 µg ( lanes a-h) or 6.36 µg ( lanes i-l) of Eco SSB as described under ``Experimental Procedures.'' Synthesis was labeled with [-S]dATP with lanes i-l having 5 times the specific activity of lanes a-h. DNA products were denatured and resolved on a 0.7% alkaline agarose gel.



Interestingly, at high polymerase to template ratios, the amount of strand-displacement synthesis as a function of Eco SSB concentration correlated with the stimulation afforded by Eco SSB when it was in the (SSB)binding mode (Fig. 6). Fig. 6 A shows that strand displacement synthesis increases as the amount of Eco SSB present was increased, while the amount of full-length product (7.2 kb) remained constant. The radioactivity present in this gel was quantified, and these data are presented in Fig. 6 B. Total DNA synthesis as a function of Eco SSB concentration was then measured for both the (SSB)and (SSB)binding modes (Fig. 6 C). As expected, at a high polymerase to template ratio, the (SSB)binding mode produced no stimulation as the Eco SSB concentration was increased (Fig. 6 C). However, as the concentration of (SSB)was increased, a stimulation of total synthesis was observed that closely paralleled the stimulation of strand displacement synthesis that occurred (Fig. 6 B) with both reaching plateaus when 4 µg of Eco SSB was present. This is additional support for the idea that a significant mechanism by which (SSB)stimulates the T7 DNA polymerase is specifically through a stimulation of strand displacement synthesis.


Figure 6: (SSB) stimulation of DNA synthesis by T7 DNA polymerase follows same pattern as (SSB) stimulation of strand displacement synthesis. DNA synthesis by the T7 DNA polymerase on a uniquely primed M13mp9 DNA template using approximately 3 polymerase molecules:1 template molecule in the presence of increasing levels of Eco SSB was carried out as described under ``Experimental Procedures.'' A, autoradiograph of the products of DNA synthesis in 0.8 KGB containing either 0, 0.86, 2.0, 4.1, 5.1, 6.3, or 8.0 µg of SSB ( lanes a-g, respectively) and labeled with [-S]dATP. The products were denatured, run on a 0.7% alkaline agarose gel, and the bands visualized by autoradiography. B, the gel in panel A was scanned with a Molecular Dynamics PhosphorImager using ImageQuant software. Full length indicates products having a length corresponding to one round of synthesis on the M13 template (7.2 kb). Strand displacement indicates products that are larger then the M13 template. C, DNA synthesis in either 0.8 or 2.25 KGB by 0.2 units of T7 DNA polymerase on 0.38 µg of M13mp9 containing the indicated levels of SSB. Synthesis levels were measured by incorporation of [H]dTTP into acid-precipitable DNA. All synthesis re-actions were carried out as described under ``Experimental Procedures.''



Effect of Eco SSB Binding Mode on the Binding of T7 DNA Polymerase to DNA

If (SSB)does not stimulate strand displacement, then what might be the basis for the differential stimulation observed by this binding mode at low polymerase to template ratios? Prior studies have shown that Eco SSB can increase the affinity of T7 DNA polymerase for the template (Myers and Romano, 1988), and therefore this might be a potential mechanism for this binding mode. To determine whether the (SSB)and (SSB)binding modes differ in their ability to alter the binding affinity of T7 DNA polymerase to the template, we measured the extent of disassociation of the T7 DNA polymerase in the presence and absence of both Eco SSB binding modes at both high and low polymerase to template ratios.

The extent of dissociation of the T7 DNA polymerase from performed polymerase-DNA complexes was determined by measuring the amount of DNA synthesis on a primed single-stranded DNA competitor (Fig. 7). The polymerase was allowed to associate with a pentadecamer-primed single-stranded M13mp9 template in the absence of dNTPs. After this preincubation, synthesis was initiated by addition of dNTPs. After 1 min EcoRI-linearized single-stranded M13mp7L1 primed with a 5`-end labeled primer 26 bases from the 3` terminus was added to the actively replicating complex to trap any dissociating polymerase. The extent of 42 base-long runoff synthesis product on the second template provides a measure of dissociation of the polymerase from the preformed polymerase-ssM13mp9 complexes after partially or completely copying it. At various time intervals after the addition of the second template, aliquots were withdrawn from the reaction mixture and the production of 42-mer was analyzed by polyacrylamide gel electrophoresis. Since the dissociation rate of preformed polymerase-template complexes is strongly dependent on competitor DNA concentration, probably due to direct transfer of the polymerase between DNA (Huber et al., 1987), we selected the concentration of competitor DNA to be used by increasing its concentration until a plateau run-off synthesis level was reached while unused challenger DNA remained (data not shown).

Autoradiographs of the gels were used to visualize the 42-mer synthesis products, and the amount of radioactivity in each band was quantified (Fig. 7). The average percent transfer of polymerase was determined from a standard curve of 42-mer production in the presence of only competitor DNA and amounts of T7 DNA polymerase used for the transfer experiment. At a low polymerase to template ratio, approximately 7% of the polymerase dissociates from the template when a 0.8 KGB buffer system is used (Fig. 7). However, in 2 KGB, approximately 70% of the polymerase dissociates from the template (Fig. 7). Repeating these experiments in the presence of Eco SSB shows that (SSB)caused only 20% of the polymerase molecule to dissociate from the template, suggesting that this binding mode is increasing the affinity of the polymerase for the template under these high salt conditions. Interestingly, at high polymerase to template ratios, Eco SSB had no effect on the polymerase transfer in either of the binding modes. Under all conditions, approximately 15-19% of the polymerase molecules were free to transfer. These results are in good agreement with prior studies, which showed that Eco SSB increased the affinity of the T7 DNA polymerase for the template only when the polymerase was limiting (Myers and Romano, 1988).


Figure 7: Effect of Eco SSB binding mode on transfer of T7 DNA polymerase to single-stranded competitor DNA. Stage I, reaction mixtures (43 µl) contained the indicated concentration of KGB, either 0 µg (% free) or 0.15 µg of pentadecamer-primed M13mp9, either 0 µg (-SSB) or 3.19 µg (+SSB) of Eco SSB, and either 0.083 units (2pol:1 mp9) or 0.0028 units (1pol:15 mp9) of T7 DNA polymerase. Incubation was at 4 °C for 3 min, allowing a polymerase-template complex to form. Stage II, 2 µl containing dATP, dCTP, dGTP, and dTTP were added to each reaction, yielding a concentration of dNTPs of 250 µ M each. Incubation was at 25 °C for 1 min, to initiate synthesis. Stage III, 5-µl mixtures containing 0.019 µg (1pol:15 mp9), 0.076 µg (2pol:1 mp9 +SSB), or 1.15 µg (2pol:1 mp9-SSB) of EcoRI-cut P-labeled pentadecamer-primed M13mp7L1 DNA, either 0 µg (-SSB), 0.44 µg (1pol:15 mp9+SSB), or 0.87 µg (2pol:1 mp9+SSB) Eco SSB, and dATP, dCTP, dGTP, and dTTP were added to each reaction. Final concentrations of dNTPs were 500 µ M each. Incubation was at 25 °C. 10-µl aliquots were removed over a range of time points. Reactions were stopped by addition of 2.5 µl of 0.5 M EDTA (pH 8). A, autoradiogram of the DNA synthesis products after they were digested with proteinase K, denatured, and resolved on a 15% denaturing polyacrylamide gel. The control reactions (% free) were without pentadecamer-primed M13mp9 DNA, using the indicated percentage of total T7 DNA polymerase added to each reaction in presence of primed M13mp9 DNA. The 42-mer bands present at each time point were quantitated using Molecular Dynamics PhosphorImager SF, as described under ``Experimental Procedures.'' For each percentage transfer reaction, synthesis was plotted versus time. An average of plateau synthesis values was then used to generate a standard curve for synthesis in each reaction condition, and this standard curve used to determine the percentage of free polymerase in the presence of M13mp9 template and M13mp7L1 trap. The percentage of free polymerase represents the average of at least three experiments.



Effect of Eco SSB Binding Mode on T7 DNA Polymerase Synthesis Rate

Finally, we determined if the Eco SSB binding modes have a differential effect on the rate of synthesis by the T7 DNA polymerase. To measure this property, T7 DNA polymerase was preincubated with a primed ssM13 template to allow a DNA-protein complex to form under conditions of limiting polymerase. A level of approximately 1 polymerase molecule/15 template molecules was chosen. Although some strand displacement synthesis does occur in 0.8 KGB at this polymerase per template level, it comprises only a small percent of total synthesis (Fig. 5). dNTPs were added to initiate DNA synthesis simultaneously with excess oligo(dT)-primed poly(dA) challenger DNA. Polymerase dissociating from the template after partially or completely copying the M13 DNA is trapped by the challenger DNA and does not reinitiate synthesis on a second M13 template. This is indicated by a plateau of synthesis reached in presence of trap. In the absence of trap synthesis continues to increase over the time course of the experiment. When Eco SSB is not present (Fig. 8, -SSB) a plateau synthesis level is reached within 4 min at both ionic strengths (Fig. 8). This is additional evidence that polymerase sensitivity to changes in ionic strength necessary to form the different Eco SSB binding modes is not playing a significant role in the effects that are measured. The elongation rate was calculated assuming that the full 7200 nucleotides of M13mp9 circular DNA was replicated. Even in the absence of Eco SSB the majority of synthesis products on M13mp9, which lacks strong secondary structure, are full-sized (data not shown). In the presence of Eco SSB (Fig. 8, +SSB) a plateau is reached within 1 min in the presence of both (SSB)and (SSB)indicating that the Eco SSB binding mode does not have a significant effect on the ability of Eco SSB to stimulate the rate of DNA synthesis.


Figure 8: Effect of Eco SSB binding mode on rate of DNA synthesis by T7 DNA polymerase. T7 DNA polymerase was preincubated at 25 °C for 1 min with primed M13mp9 at the indicated concentration of KGB in the presence or absence of Eco SSB to form a polymerase-template complex. DNA synthesis reactions at 25 °C were initiated by simultaneous addition of dATP, dCTP, dGTP, and dTTP. Aliquots were removed at the indicate time points and synthesis stopped with excess EDTA. A 10-fold excess of oligo(dT)-primed poly(dA) challenger DNA molecules was either present or absent during the DNA synthesis reactions. Polymerase-template association reactions contained either 0.0078 units or T7 DNA polymerase, 0.72 µg of primed M13mp9 DNA, and 19.0 µg of Eco SSB (+SSB) or 0.023 units of T7 DNA polymerase, and 2.16 µg of primed M13mp9 DNA (-SSB). Challenge reactions contained 0 µg of poly(dA) (-trap), 1.4 µg of poly(dA) and 13.2 µg Eco SSB (+trap, +SSB), or 4.19 µg of poly(dA) (+trap, -SSB). Synthesis was labeled with [-P]dATP, and measured on DE81 filter discs as described under ``Experimental Procedures.''




DISCUSSION

Eco SSB Binding Site Sizes

The distinct binding modes for the interaction of Eco SSB and single-stranded DNA are the first example of multiple types of interactions for a single protein binding to single-stranded DNA (Bujalowski and Lohman, 1986). Although the salt concentration range in which these binding modes were observed encompasses that routinely used for in vitro studies of DNA replication, recombination, and repair (Bujalowski and Lohman, 1986; Morrical and Cox, 1990; Greip and McHenry, 1989; Richey et al., 1987; Leirmo et al., 1987) as well as the ranges estimated for E. coli in vivo (Bujalowski and Lohman, 1986; Leirmo et al., 1987; Kuhn and Kellenberger, 1985), there are no reports of differential effects of Eco SSB binding modes on DNA replication.

Using native ssM13 DNA and a potassium glutamate buffer system, we find two distinct Eco SSB binding modes with sizes of 72 and 91 nucleotides. It is likely that these binding modes correspond to the two largest Eco SSB binding modes determined for Eco SSB-poly(dT) complexes (Bujalowski and Lohman, 1986) of n = 56 and 65 nucleotides/ Eco SSB tetramer. Several pieces of evidence support this conclusion. First, these are the two highest binding site sizes we have observed; we saw none higher even at salt concentrations as high as 2.5 KGB. Second, the quenching of intrinsic tryptophan fluorescence measured for the two highest binding Eco SSB-poly(dT) complexes was 89 ± 2% (Bujalowski and Lohman, 1986, 1988; Lohman and Overman, 1985), in agreement with the values we determined for (SSB)and (SSB). Third, others have seen higher values for the binding site sizes using native (non-homopolymer) DNA (Lohman and Overman, 1985; Morrical and Cox, 1990) and suggested that these differences are due to the fact that native DNA has duplex regions that do not bind Eco SSB resulting in a smaller pool of bound nucleotides and therefore a larger average calculated binding site size. Fourth, others have reported that there are ion-specific effects on the transition between Eco SSB binding modes, and that the transitions depend on the identity and charge of cations and anions, as well as electrostatic interactions (Bujalowski and Lohman, 1986; Lohman and Overman, 1985; Overman et al., 1988). Finally, we have also verified that Eco SSB-poly(dT) complexes undergo a transition in binding site size over the same range of KGB concentrations as Eco SSB-M13mp9 (Table II) and exhibit larger binding site sizes, but approximately the same percentage of fluorescence quenching as observed for Eco SSB-M13mp9 complexes at these concentrations of KGB. In addition, our determinations of binding sites sizes with both poly(dT) and single-stranded M13mp9 in NaCl were in agreement with previous reports (). Taken together, these results suggest that the larger binding site sizes we observe for Eco SSB-M13mp9 complexes in KGB are due to two effects: the use of native DNA rather than homopolymers, and ion-specific effects because of our having used a potassium glutamate buffer system.

Effect of Binding Site Size on DNA Synthesis

In this study we have determined that the two highest Eco SSB binding modes have different effects on DNA synthesis by the T7 DNA polymerase, a polymerase that is one of the best studied replication enzymes. For these experiments we have used uniquely primed single-stranded DNA as a model for RNA-primed lagging strand synthesis in the bacteriophage T7 replication system. Addition of Eco SSB to a T7 in vitro replication system has been shown to result in a substantial stimulation of DNA synthesis by the T7 DNA polymerase (Scherzinger et al., 1977; Romano and Richardson, 1979a; Fuller and Richardson, 1985; Myers and Romano, 1988).

The two Eco SSB binding modes show substantial differences in some of the mechanisms of stimulation of T7 DNA polymerase, while some of the properties of each form are shared. For example, both (SSB)and (SSB)reduce polymerase pausing at secondary structures (Fig. 3), and both provide a 4-fold enhancement in the rate of DNA synthesis (Fig. 8). The differences include the ability of (SSB)to stimulate DNA synthesis on a poly(dA) template, which lacks the ability to form hairpin structures, while (SSB)does not stimulate on this template, and the fact that only (SSB)is able to stimulate strand displacement synthesis (Fig. 5).

The ability of Eco SSB to stimulate DNA synthesis on templates that do not form base-paired secondary structure has been shown before and cited as evidence that the mechanism for stimulation involves properties other than destabilizing secondary structure (Myers and Romano, 1988). One property was identified as an ability to increase the binding strength of the polymerase for the template (Myers and Romano, 1988). In this present study, we find that when the template is present in large molar excess, (SSB)can increase the affinity of the polymerase for the template (Fig. 7), resulting in a greater than 3-fold decrease in its dissociation from a primed ssDNA template over that observed in the absence of Eco SSB. Interestingly, it is only under these conditions of limiting polymerase that (SSB)can stimulate DNA synthesis ( cf. Figs. 1 and 2). suggesting that this increased affinity of the polymerase for the template may be responsible for the stimulation observed for (SSB).

On the other hand, (SSB)is able to stimulate T7 DNA polymerase synthesis even though we cannot show an increased affinity of the polymerase for the template when Eco SSB is present in this binding mode. The fact that (SSB)is unable to stimulate synthesis on poly(dA) is also evidence that the effect of this form of Eco SSB has a fundamentally different effect on the T7 DNA polymerase. Interestingly, (SSB)induces a 4-fold stimulation of DNA synthesis, which is independent of the polymerase concentration (Figs. 1 and 2). Prior studies have shown that the effect of Eco SSB on the polymerase template interaction is very dependent on the polymerase concentration, with no differences observed at high polymerase to template ratios. Taken together, these facts suggest that (SSB)is not altering the affinity of the polymerase for the template. Therefore, the mechanism of stimulation afforded by (SSB)most likely involves either its effect on secondary structure or on strand displacement synthesis.

Other studies have suggested differences in the biological properties of the Eco SSB binding modes. Recent in vitro studies with RecA protein have suggested that the higher Eco SSB binding modes might specifically be involved in recombination. The higher binding modes displays a limited cooperativity, which may result in the formation of clusters of dimers of Eco SSB tetramers leaving regions where other proteins may interact with the DNA (Lohman et al., 1988; Lohman and Ferrari, 1994). In addition, solution conditions that favor the formation of the higher Eco SSB binding modes have been shown to be more favorable for formation (Griffith et al., 1984) and stabilization (Morrical and Cox, 1990) of RecA protein filaments on ssDNA than solution conditions that favor the lowest Eco SSB binding mode (Griffith et al., 1984; Bujalowski and Lohman, 1986; Lohman and Bujalowski, 1990). Finally, in the lowest binding mode Eco SSB ((SSB)) has been shown to displace RecA from ssDNA (Griffith et al., 1984), presumably because the unlimited cooperativity associated with this binding mode allows it to saturate a region of single-stranded DNA, even at low protein concentrations (Lohman et al., 1988). Unfortunately the lowest binding mode of Eco SSB requires MgClconcentrations less than 1 m M (Bujalowski and Lohman, 1986), and therefore we were unable to study this binding mode because of requirement of the T7 DNA polymerase for Mgconcentrations greater than this. However, the free Mgconcentration in vivo has been estimated to be in the range of 1-4 m M (Lusk et al., 1968; Alatossava et al., 1985), while the total (bound + free) intracellular concentration of Mgin E. coli has also been shown to be higher than this (Kuhn and Kellenberger, 1985). Therefore, it is unlikely that this form plays a significant role in vivo, since even in 2 m M Mgand the absence of other ions the intermediate binding mode of Eco SSB has been shown to be induced on poly(dT) (Bujalowski and Lohman, 1986).

At present it is not known whether these various binding modes play a specific role in DNA replication in general and T7 replication specifically. However, it is tempting to speculate how the presence of either (SSB)or (SSB)might fit in the model proposed for T7 RNA-primed DNA synthesis (Nakai and Richardson, 1986a, 1986b, 1988a). The T7 in vitro replication system requires only three proteins to catalyze the basic reactions necessary for leading and lagging strand synthesis on a duplex DNA molecule (Richardson, 1983). These proteins are the T7 gene 4 protein and a 1:1 complex of T7 gene 5 protein and E. coli thioredoxin, which function together as a high processivity complex referred to as T7 DNA polymerase (Richardson, 1983). The gene 4 protein binds to single-stranded DNA and translocates 5` to 3`, unwinding duplex DNA (Matson and Richardson, 1983) for T7 DNA polymerase to catalyze leading strand synthesis (Lechner and Richardson, 1983). Both of these reactions are highly processive (Nakai and Richardson, 1988a, 1988b). In lagging strand synthesis, the gene 4 protein primase activity catalyzes the synthesis of tetraribonucleotide primers at recognition sites on single-stranded DNA, and these primers are extended by T7 DNA polymerase (Romano and Richardson, 1979a). Lagging strand synthesis is not processive, requiring multiple association and dissociation steps by the gene 4 protein (Nakai and Richardson, 1988a, 1988b).

A proposal for recycling of DNA polymerase for lagging strand synthesis was not included in these models (Nakai and Richardson, 1988a). Myers and Romano (1988) have suggested that the ability of Eco SSB to increase binding of the T7 DNA polymerase to a single-stranded template at low polymerase concentrations may provide this mechanism. We have shown that (SSB)is able to increase polymerase-template binding, and we present evidence that (SSB)does not. Alternatively, (SSB)is specifically able to stimulate strand displacement synthesis by the T7 DNA polymerase. These differences suggest that (SSB)may be more effective at recycling the T7 DNA polymerase for lagging strand synthesis by promoting a tighter binding of the polymerase at the primer terminus. On the other hand, the presence of (SSB)at a replication fork, with its ability to promote strand-displacement DNA synthesis, could result a more efficient progression of the leading strand.

Is it possible that either or both of these Eco SSB binding modes might specifically form in vivo? The intracellular ionic environment of E. coli includes a mixture of organic and inorganic species but the primary ionic osmolytes are Kand glutamate (Richey et al., 1987; Measures, 1975). The levels of these ions in E. coli vary widely enough in response to external osmotic strength so one potassium glutamate concentration has not been assigned as physiological (Leirmo et al., 1987; Kuhn and Kellenberger, 1985). Moreover, the intracellular ionic environment of both prokaryotes and eukaryotes is known to vary in response to changes in the external environment, the rate of cell growth, or the developmental stage of the cell (Epstein and Schultz, 1965; Cameron et al., 1980; Epel, 1982; Steinhardt, 1982; Richey et al., 1987); therefore, it is possible that any of the binding modes may form and that some may be used preferentially in different DNA metabolic processes. Whether both forms of Eco SSB could form in close proximity to a replication fork is less likely, although it is possible that other factors, such as interactions with other replication proteins or the release of ions caused by the binding of these proteins (Record et al., 1985) to the DNA in close proximity to the Eco SSB-DNA complex, could play a role.

  
Table: Eco SSB binding site size and quenching determined in KGB


  
Table: This study


FOOTNOTES

*
This investigation was supported by Public Health Service Grants CA35451 and CA40605 from the NCI, National Institutes of Health, Department of Health and Human Services. 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.

§
Present address: Dept. of Chemistry, California Polytechnical State University, San Luis Obispo, CA 93407.

To whom correspondence should be addressed. Tel.: 313-577-2584; Fax: 313-577-8822; E-mail: ljr@chem.wayne.edu.

The abbreviations used are: Eco SSB, E. coli single-stranded DNA-binding protein; ssDNA, single-stranded DNA.


ACKNOWLEDGEMENTS

We thank Dr. Thomas M. Lohman for helpful discussions and Mubashir Sabir and Shuaib Malik for invaluable technical assistance.


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