Vol. 263, No. Issue 5, pp. 4629-4640, 1988 JOURNAL THE OF ... · Q 1988 by The American Society for...

THE JOURNAL OF BIOLOGICAL CHEMISTRK Vol. 263, No. 10, Issue of April 5, pp. 4629-4640, 1988 Q 1988 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U. S. A.

Binding Mode Transitions of Escherichia coli Single Strand Binding Protein-Single-stranded DNA Complexes CATION, ANION, pH, AND BINDING DENSITY EFFECTS*

(Received for publication, August 28, 1987)

Wlodzimierz BujalowskiSQ, Leslie B. Overman$, and Timothy M. LohmanSTII From th.e Departments of $Biochemistry and Biophysics and of Whemistry, Texas A&M University, College Station, Texas 77843

We have extended our investigations of the multiple binding modes that form between the Escherichia coli single strand binding (SSB) protein and single- stranded DNA (Lohman, T. M. & Overman, L. B. (1985) J. Biol. Chem. 260, 3594-3603; Bujalowski, W. & Lohman, T. M. (1986) Biochemistry 25, 7799- 7802) by examining the effects of anions, pH, BaCl2, and protein binding density on the transitions among these binding modes. “Reverse” titrations that monitor the quenching of the intrinsic tryptophan fluorescence of the SSB protein upon addition of poly(dT) have been used to measure the apparent site size of the complex at 25 “C in pH 8.1 and 6.9 as a function of NaF, NaCl, NaBr, and MgClz concentrations. Under all conditions in which “reverse” titrations were performed, we observe three distinct binding modes with site sizes of 36 f 2, 56 f 3, and 66 f 3 nucleotides/SSB tetramer; however, the transitions among the three binding modes are strongly dependent upon both the cation and anion valence, type, and concentration as well as the pH. A net uptake of both cations and anions accompanies the transition from the (SSB),, to the (SSB),a binding mode at pH 6.9, whereas at pH 8.1 this transition is anion-independent, and only a net uptake of cations occurs. The transition from the (SSB)s6 to the (SSB),, binding mode is dependent upon both cations and anions at both pH 6.9 and 8.1 (25 “C), and a net uptake of both cations and anions accompanies this transition. We have also examined the transitions by monitoring the change in the sedimentation coefficient of the SSB protein-poly(dT) complex as a function of MgCl, concentration (20 O C , pH 8.1) and observe an increase in S Z O , ~ , which coincides with the increase in apparent site size of the complex, as measured by fluorescence titrations. The frictional coefficient of the complex decreases by a factor of two in progressing from the (SSB),, to the (SSB)sa binding mode, indicat-

* This work was supported in part by National Institutes of Health Grant GM-30498 and Robert A. Welch Foundation Grant A-898 (to T. M. L.) and National Institutes of Health Biomedical Research Support Instrumentation Grant SO1 RR01712 and Department of Defense Instrumentation Grant P-20862-LS-RI. Support from the Texas Agricultural Experiment Station is also acknowledged. A pre- liminary account of this work was presented at the 1987 Biophysical Society Meeting, New Orleans, LA (Bujalowski and Lohman, 1987~). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked ‘‘advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

5 On leave from the Institute of Biulogy, Department of Biopoly- mer Biochemistry, Poznan University, 61-701, Poznan, Poland.

(1 Recipient of American Cancer Society Faculty Research Award FRA-303. To whom reprint requests should be addressed at the Department of Biochemistry and Biophysics.

ing a progressive compaction of the complex throughout the transition. The transition between the (SSB),, and the (SSB)66 complex is dependent on the protein binding density, with the lower site size (SSB)SB complex favored at higher binding density. These results indicate that the transitions among the various SSB protein-single-stranded DNA binding modes are complex processes that depend on a number of solution variables that are thermodynamically linked. Thus, caution must be exercised when comparing data col- lected under different sets of solution conditions in any experiments involving the E. coli SSB protein in uitro.

Helix-destabilizing proteins are required for DNA replication, recombination, and a variety of repair processes (Chase and Williams, 1986) in many, if not all, organisms. These proteins seem to function by binding selectively and in some cases cooperatively to single-stranded (ss)’ nucleic acids, although there is evidence that they may also interact with other proteins involved in nucleic acid metabolism. In the case of the Escherichia coli SSB protein, several of these interactions have been identified in vitro (Sigal et al., 1972; Molineux et al., 1974; Molineux and Gefter, 1975; Low et al., 1982), and others have been inferred from genetic studies (Tessman and Peterson, 1982). The E. coli SSB protein also stimulates the DNA strand exchange activity of the recA protein under some conditions (Cox and Lehman, 1981,1982; Griffth et al., 1984).

It has recently been shown that the E. coli SSB protein forms multiple distinct complexes with ssDNA and RNA (Lohman and Overman, 1985; Griffith et al., 1984; Lohman et al., 1986a; Bujalowski and Lohman, 1986 Overman et al., 1988). Each binding mode has a distinct site size ( i e . number of nucleotides occluded by the bound SSB protein), each of which seems to involve a different degree of compaction of the ssDNA (Lohman and Overman, 1985; Griffith et al., 1984; Lohman et al., 1986a; Bujalowski and Lohman, 1986). At least one mode involves the interaction of ssDNA with all four subunits of the tetramer (Krauss et al., 1981; Lohman and Overman, 1985) and subsequent interaction of nucleic acid- bound SSB protein tetramers to form octamers (Chrysogelos and Griffith, 1982; Bujalowski and Lohman, 1987b). In studies with poly(dT) at 25 “C (pH 8.1), three binding modes possessing site sizes of 35 f 2, 56 3, and 65 f 3 nucleotides/ tetramer have been identified, whereas at 37 “C an additional binding mode is apparent covering 40 f 2 nucleotides/tetramer (Bujalowski and Lohman, 1986). The transitions among

’ The abbreviations used are: ss, single-stranded; SSB, single strand binding.

4629

4630 E. coli SSB Protein-ssDNA Binding Mode Transitions

the different binding modes are dependent upon the concentration and type of low molecular weight ions in solution (e.g. NaCl and MgC12) (Lohman and Overman, 1985; Bujalowski and Lohman, 1986) as well as the protein binding density (Griffith et al., 1984). Furthermore, the apparent cooperativity of SSB protein binding is also dependent upon ionic conditions, such that at equilibrium, a low degree of cooperativity is observed at all conditions, whereas a metastable high degree of cooperativity can form transiently under conditions of low monovalent ion concentration (Lohman et al., 1986a). Al- though the (SSB)66 binding mode is clearly a low cooperativity binding mode (Lohman et al., 1986a; Bujalowski and Lohman, 198%; Overman et d., 1988), it is not clear which mode or possible mixture of modes may be responsible for the metastable high cooperativity (Lohman et al., 1986a; Sigal et al., 1972; Ruyechan and Wetmur, 1975). The stimulatory effect of SSB protein on the DNA strand exchange activity of the recA protein seems to be correlated with structures that appear "beaded" in the electron microscope (Griffith et al., 1984). These structures are likely to be the (SSB),, or (SSB),, binding modes.

The equilibrium binding parameters of the SSB protein in its (SSB), binding mode ( i e . the intrinsic binding constant and cooperativity parameter) have been quantitatively examined as a function of a variety of monovalent salts (Lohman et al., 1986a; Bujalowski and Lohman, 1987a, 1987b; Overman et d . , 1988). One interesting feature is that the (SSB),, binding mode possesses only moderate positive cooperativity (Lohman et al., 1986a), and it is of a type that seems to limit the formation of protein clusters to two tetramers (octamers) (Chrysogelos and Griffith, 1982; Bujalowski and Lohman, 1987b), that is the (SSB),, binding mode does not form long clusters of bound protein as is the case for the bacteriophage T4 gene 32 protein (Kowalczykowski et al., 1981). The intrinsic equilibrium constant for SSB protein binding to ss nucleic acids decreases dramatically with increasing salt concentration, indicating a significant contribution of electrostatics and ion release to the free energy of binding, although the cooperativity parameter is fairly insensitive to salt concentration and type. The equilibrium constant is quite sensitive to both the cation and anion concentration, charge and type, indicating that a net release of both cations and anions occurs upon formation of the (SSB),, complex from the free protein (Over- man et al., 1988).

The transitions among the various SSB protein-ss nucleic acid binding modes are salt-dependent due to the net uptake of ions that accompanies the transitions from the lower to the higher site size binding modes (Lohman and Overman, 1985; Bujalowski and Lohman, 1986). In our previous experiments, NaCl and MgClz were used to induce the transitions and the results indicated that the effect of salt was due, at least in part, to the direct binding of cations to the SSB protein-DNA complexes. We have now extended the characterization of the SSB protein-poly(dT) binding mode transitions to include the effects of anions, pH, BaC12, and protein binding density (at 25 "C). Changes in all of these variables as well as cations and temperature (Lohman and Overman, 1985; Bujalowski and Lohman, 1986) cause dramatic effects on the binding mode transitions. We also report a hydrodynamic characterization of the MgCla-induced transitions using sedimentation velocity techniques. Our studies indicate that the effects of cations, anions, and pH (and most likely temperature) are all thermodynamically linked, so that an understanding of the effect of one variable requires detailed information about the other variables, and this will be necessary in order to understand the numerous multiple binding

equilibria that govern the interactions of the E. coli SSB protein with ss nucleic acids.

MATERIALS AND METHODS

Reagents and Buffers-All chemicals were reagent grade; all solutions were made with distilled and deionized (Milli-Q) water. The buffers used were buffer T (pH 8.1), which is 10 mM Tris and 0.1 mM Na3EDTA titrated to pH 8.1 with HCl at the temperature used in the experiments (25 or 20 "C); buffer C (pH 6.9), 3 mM sodium cacodylate, was adjusted to pH 6.9 with HCl at 25 "C. The experiments in NaF at pH 8.1 were performed using buffer T that was adjusted to pH 8.1 with concentrated HF. MgCl, stocks were prepared as described (Bujalowski and Lohman, 1986).

E. coli SSB Protein and Nucleic Acid.-The SSB protein was prepared as previously described from a strain of E. coli K12 containing the plasmid pTL119A-5 which is temperature-inducible for SSB protein overproduction (Lohman et al., 198613). The concentration of SSB protein was determined spectrophotometrically using the extinction coefficient = 1.5 ml mg" cm" (1.13 X lo6 M" (tetramer) cm") in buffer T + 0.20 M NaCl (Lohman and Overman, 1985). The poly(dT) ( ~ 2 0 , ~ = 8.4 S; -950 nucleotides (Inners and Felsenfeld, 1970)) was purchased from Pharmacia LKB Biotechnology Inc. and was extensively dialyzed before use. The concentration of poly(dT) was determined spectrophotometrically, using an extinction coefficient at 260 nm of 8100 M" (nucleotide) cm" (Kowalczykowski et ai., 1981).

Fluorescence Measurements and Site Size Determinations-"Re- verse" titrations (addition of poly(dT) to SSB protein) were performed with an SLM 8000 spectrofluorometer as described previously (Bujalowski and Lohman, 1986). An excitation wavelength of 296 nm, excitation bandpass = 1 nm (0.5-mm slit width), was used while the emission at 347 nm was monitored; emission bandpass = 4 nm (2- mm slit width). Poly(dT) was used in these experiments since the binding of E. coli SSB protein to poly(dT) is stoichiometric a t low SSB protein concentrations, even in 5 M NaCl (25 "C). The sample temperature was maintained at either 20.0 +. 0.1 or 25.0 + 0.1 "C, as indicated, with a Lauda RM-6S refrigerated circulating water bath. When necessary, inner filter corrections were applied by using the expression F,,, = Foba antilog (A,./2) (Lakowicz, 1983). The SSB protein concentration in most experiments was 5.16 X lo-* M (tetramer) (3.89 pg/ml) or as indicated in the text, although the site size determinations were checked at several concentrations to ensure that binding was indeed stoichiometric. The SSB protein concentrations used in all experiments were well below the concentrations at which precipitation of the protein may occur.

"Normal" titrations (addition of SSB protein to poly(dT)) were performed by titrating poly(dT) with a stock of SSB protein and comparing this directly with a titration of the sample buffer with the same stock of SSB protein as shown in Fig. 4. This direct comparison is necessary to correctly define the linear portion at the end of the titration of poly(dT) with SSB protein, so that a correct end point can be determined. The fluorescence of free SSB protein is independent of the NaCl concentration in buffer T, pH 8.1, 25 "C. All lines describing the site size data as a function of salt concentration reflect our representation of the data and have no theoretical basis.

Sedimentation Velocity Analysis of SSB Protein-Poly(dT) Corn- pleres-Sedimentation coefficients of SSB protein-poly(dT) complexes were determined by sedimentation velocity at 20 "C in a Beckman model E analytical ultracentrifuge equipped with a multi- plexer and photoelectric scanner. The complexes were prepared by mixing a polydisperse sample of poly(dT), ( ( s ~ ~ , ~ ) = 8.4 S), with SSB protein in buffer T (pH 8.1) + 1 M NaCl. After allowing this complex to equilibrate for approximately 1 h at 25 "C, it was extensively dialyzed versus 4 X 500-ml changes of buffer T (pH 8.1) + 0.3 mM NaCl at 4 'C (for 12 h each). The concentration of poly(dT) in the complex, after dialysis, was approximately 3.5 X M (nucleotide). This procedure was used to ensure that the complex was at equilibrium, since we have evidence that nonequilibrium complexes can form when SSB protein is directly mixed with polynucleotides at low NaCl concentrations (Lohman et al., 1986a). Complexes were formed using ratios of 101 and 87 nucleotides/SSB tetramer. Measurements of the sedimentation coefficients, as a function of MgCl, concentration, were performed with a single stock of SSB protein-poly(dT) complex by adding the required volume of a concentrated stock of MgCl, (in buffer T, pH 8.1) to a 0.5-ml aliquot of the complex in order to obtain a final MgCl, concentration in the range of 10-6-0.4 M. All sedimentation experiments were performed at 20.0 "C, and

E. coli SSB Protein-ssDNA Binding Mode Transitions 4631

sedimentation coefficients were corrected for the effects of MgCL concentration on the solution viscosity and density and are reported as SZO.~ .

Analysis of the Salt Concentration Dependences of the site Size Transitions in Terms of Preferential Zon Binding-A comparison of the site size transitions with the changes in s20,~ of the SSB protein- poly(dT) complex as a function of MgC1, concentration suggests that the site size transition curves are at or close to equilibrium. Lohman and Overman (1985) have also shown that these transitions are reversible. In order to quantitatively analyze the salt concentration dependence of the apparent site size transitions, we have used a two- state approximation, as previously described (Lohman and Overman, 1985). We can define an equilibrium constant, KAD, characterizing the transition between two SSB binding modes possessing site sizes, nA and nB, as in Equation 1.

KA/B = [(SSB)B]/[(SSB)A] (1)

where KAD is defined as

K m '(naPp - nA)/(nB - nauu) (2)

and napu is the apparent value of the site size (nA 5 napp 5 nB). If a ligand, L, preferentially interacts with either complex, (SSBh

or (SSB)A, then the dependence of KAD on the concentration of L is given by

(alOg KAp/alOg L) = (rB - FA) = ArL (3)

where FA and rB are the average degrees of binding of ligand L to the complexes with site sizes, nA and nB, respectively. If Ar > 0, then Ar mol of L are taken up in the transition, whereas if Ar < 0, then a release of Ar mol of L accompanies the transition. The magnitude and sign of Ar are obtained from the slope of a plot log KAD uersuS log L. In principle, both rA and r B depend upon ligand concentration; however, in the case of a cooperative transition, the change in ligand concentration in the transition region is generally small, and the value of Ar remains constant in this range to a good approximation. It should be noted that Equation 3 is independent of any molecular model for the binding of L to the complexes.

In our investigations, we have monitored the effects of a neutral salt on the site size transition; hence we must consider the possibility that both the cation ( M ) and anion (X) can preferentially interact with the SSB protein-poly(dT) complexes. In the case of a 1:l neutral salt, MX, using Wyman's linked function analysis (Wyman, 1964; Record et al., 1978), one obtains Equation 4.

@log KA,&log[MX]) = ArM + Arx (4)

In the case in which a 2:l salt, MX, is used to induce the transition, one obtains a similar expression for the salt concentration dependence of KAD

@log K~/~/alOg[kfxz]) = ArM + Arx (5)

RESULTS

Cation-specific Effects on the SSB Protein-Poly(dT) Binding Mode Transitions-In previous experiments with poly(dT), we have shown that the transitions among the various E. coli SSB protein-ssDNA binding modes can be induced at much lower concentrations of MgCl, than NaCl (Bujalowski and Lohman, 1986). These salt-induced effects are not the result of simple ionic strength (coulombic screening) effects; rather the transitions are facilitated by the direct binding of ions to the SSB protein-ssDNA complexes, resulting in an uptake of ions during any of the transitions from a lower to a higher site size binding mode (Lohman and Overman, 1985; Buja- lowski and Lohman, 1986).

We have extended these studies to test for cation-specific charge-independent effects on these transitions by comparing the SSB protein-poly(dT) transitions in BaC1, versus MgC1,. These two divalent cations differ significantly in their ionic and Stokes radii as well as their AH and A S of hydration (Nancollas, 1966; Nightgale, 1959), although both divalent cations interact only electrostatically with the DNA phosphate residues and show no tendency to interact specifically with the bases (Sissoeff et al., 1976).

A comparison of the site size transitions induced by BaCL and MgCl,, in Fig. 1, indicates that cation-specific charge- independent effects also contribute to the effects of salt on the site size transitions. With respect to the transitions in MgCl,, the (SSB)s5 to (SSB),, transition in BaC1, is shifted to a lower salt concentration, whereas the (SSB),, to (SSB),, transition is shifted to a higher salt concentration. The midpoints of the two transitions in BaClz occur at approximately 0.2 mM and 0.3 M, as compared to 0.6 mM and 66 mM in MgCI,. Consequently, in the presence of BaCl,, the two transitions are better separated than in MgC1, (pH 8.1, 25 "C); the (SSB),, binding mode is stable over a wider salt concentration range in BaCl,, from 1 to 100 mM. This result clearly demonstrates that the two site size transitions ((SSBs5 to (SSB)56 and (SSB)56 to (SSB)65) do not reflect the same molecular process. Rather, each transition must be viewed as a separate process, and the interactions of the cations with the SSB protein-poly(dT) complex that induce the two transitions must be different (e.g. different cation binding sites with different affinities). Furthermore, although the cation charge is clearly important in determining the effectiveness of the salt to induce these transitions, there are also specific chemical effects which contribute to the stability of each binding mode.

Anion and p H Effects on the SSB Protein-Poly(dT) Binding Mode Transitions-The equilibrium binding of E. coli SSB protein to form the (SSB)65 binding mode is dependent upon both cation and anion type and concentration, as well as pH (Overman et al., 1988 and unpublished experiments). There- fore, it was of interest to examine the effects of anion type and concentration as well as pH on the SSB protein-poly(dT) binding mode transitions using a number of 1:1 sodium salts which differ in the monovalent anion. These experiments were performed at pH 8.1 and 6.9 at 25 "C. The dependence of the SSB protein-poly(dT) site size on NaBr, NaC1, and NaF concentration at pH 8.1 is shown in Fig. 2 A . Within experimental error, the (SSB),, to (SSB),, transition is independent of the anion type at pH 8.1, with a midpoint of 17 mM Na' as previously reported (Bujalowski and Lohman, 1986). However, there is a dramatic effect of anion type on the (SSB),, to (SSB),, transition; the midpoint of the transition is shifted to higher salt concentration following the order Br- < c1- < F-. The midpoints of the (SSB),6 to (SSB),, transition occur at approximately 80, 160, and 600 mM for

7 0

30 I I I . I I I . . , , , , , I , , , , , I , , , , ,d , , L , , , , , , , , , , . , . 4

10" 10" ('0-1 ('0" (0" 1

CMCIJ (M)

FIG. 1. Effects of BaClz concentration on the apparent site size of the SSB protein-poly(dT) complex. The apparent site size (nucleotides/SSB tetramer) of SSB protein-poly(dT) complexes, determined from a series of reverse titrations, monitoring the quenching of the SSB protein tryptophan fluorescence is plotted as a function of BaC1, concentration ( M ) (logarithmic scale) in buffer T, pH 8.1, 25.0 "C. The dashed line represents data of Bujalowski and Lohman (1986), showing the dependence of the site size on the MgC1, ( M ) , under the identical conditions.

4632 E. coli S S B Protein-ssDNA Binding Mode Transitions

-b

io-. 10.3 10” 10-1 10

CNaXl (M)

FIG. 2. Anion and pH effects on the SSB protein-poly(dT) binding mode (apparent site size) transitions. A, pH 8.1. Appar- ent site sizes (nucleotides/SSB tetramer), determined from reverse titrations performed in buffer T, pH 8.1, 25.0 “C, are plotted as a function of NaF (O), NaCl (O), and NaBr (A) concentrations ( M ) (logarithmic scale). The Tris buffer used in the NaF experiments was titrated to pH 8.1 with HF. B, pH 6.9. Apparent site sizes (nucleotides/SSB tetramer), determined from reverse titrations performed in 3 mM sodium cacodylate/HCl, pH 6.9, 25.0 ‘C, are plotted as a function of NaF (O), NaCl (O), and NaBr (A) concentrations ( M ) (logarithmic scale). All experiments were performed with an SSB protein concentration of 5.16 X lo-’ M (tetramer), except for the experiment in 0.6 M NaBr at pH 6.9, which used an SSB protein concentration of 1.79 X M (tetramer), since binding at this NaBr concentration was not stoichiometric at the lower SSB concentration.

NaBr, NaCl, and NaF, respectively. Moreover, the steepness of the transition (cooperativity) is also a function of the anion type, decreasing in the order Br- > C1- > F-. These data provide further evidence that the two salt-induced SSB protein binding mode transitions reflect different processes and that the effects of ions on the two site size transitions have different molecular origins. Clearly these salt effects do not result from simple ionic strength effects but rather must reflect the direct preferential interaction of anions with the different SSB protein-poly(dT) complexes. The anion-dependent shifts in the (SSB),, to (SSB),, transition presumably reflect different affinities of these anions for binding sites on the two complexes. As discussed below, these data indicate that a net uptake of anions accompanies the (SSB),, to (SSB),, transition.

The effects of anions on the binding mode transitions are quite different at pH 6.9 (25.0 “C) as shown in Fig. 2B. In this case, both the (SSB),, to (SSB)56 transition as well as the (SSB)6B to (SSB),, transition are affected by the anion type. The (SSB),, to (SSB),, transition in NaBr is essentially unchanged at pH 6.9; however, the transition is shifted to a higher salt concentration in NaCl (midpoint - 25 mM) and still higher in NaF (midpoint - 70 mM). In addition, the steepness (cooperativity) of the (SSB),, to (SSB),, transition at pH 6.9 is greatest in the presence of Br- and decreases

substantially in the order Br- > C1- > F- (see Table I). The (SSB),, to (SSB)65 transition is also affected by anions at pH 6.9 in a manner similar to that observed for pH 8.1. The midpoints are 0.14 M in NaBr and 0.5 M in NaCl; however, the cooperativity of the transitions decreases only slightly upon substituting chloride for bromide. We were not able to follow the (SSB),, to (SSB),, transition in NaF at pH 6.9, since the solubility limit of NaF (0.9 M) was reached before attaining the second transition. A comparison with pH 8.1 (Fig. 2 . 4 ) indicates that the transitions in all of the salts are shifted to higher concentrations at pH 6.9, with the exception of the (SSB),, to (SSB)56 transition in NaBr, which remains unchanged. Upon lowering the pH from 8.1 to 6.9, the (SSB),, to (SSB)56 transition becomes sensitive to anions, so that three separate transition curves are observed for NaF, NaC1, and NaBr at pH 6.9. The (SSB),, to (SSB),, transition is sensitive to anions at both pH 8.1 and 6.9; however, at the lower pH, the transition midpoint for a given salt occurs at a higher salt concentration (see Table 11). Clearly, the effects of pH and anions on these binding mode transitions are thermodynamically linked. The effect of cations may also be coupled to the pH, although we have no direct evidence for this.

We have also examined the effects of MgC1, on the binding mode transitions at pH 8.1 and 6.9 (25 “C), and these data are shown in Fig. 3, along with the experiments in NaCl, described above. For each salt, the (SSB),, to (SSB)BB transition is shifted slightly to higher salt concentration at pH 6.9, as compared to pH 8.1. On the other hand, the (SSB),, to (%%),e transition is dramatically shifted to higher salt concentrations for both MgClz and NaCl at pH 6.9. Similar direct comparisons of data in NaF and NaBr at pH 8.1 and 6.9 show the same qualitative behavior as for NaCl; however, the effect of a change in pH is most dramatic for the NaF experiments (see Fig. 2). These direct comparisons at pH 6.9 and 8.1 also indicate that there is an increase in site size with increasing pH at some salt concentrations, i.e. (dn.,,/dpH) > 0, which indicates that the transitions to the higher site sizes are accompanied by a release of protons into solution. However, the data are insufficient to quantitatively evaluate this proton release.

The Transition from the (SSB),, to the (SSB),, Binding Mode Is Dependent on the SSB Binding Density-The SSB protein site sizes that we have previously reported (Lohman and Overman, 1985; Bujalowski and Lohman, 1986) as well as those discussed above were obtained from “reverse” titrations, that is solutions of SSB protein were titrated with aliquots of DNA. In the initial stages of such “reverse” titrations, the SSB protein is in large excess over the nucleic acid; hence the site sizes are determined in the limit of high protein binding density in these experiments. Since E. coli SSB protein binds to ss nucleic acids in a number of binding modes that differ dramatically in site size, it was of interest to investigate the binding density dependence of the binding mode transitions.

In order to probe this question, we have measured the apparent site size for the SSB protein-poly(dT) interaction by titrating poly(dT) with SSB protein. We refer to this as a “normal” titration, since it involves the addition of protein (ligand) to the nucleic acid lattice (macromolecule). In this type of experiment, the DNA is in excess over the protein in the initial stages of the titration; hence the measurements reflect the site size that is favored at low binding densities. Clearly, if there is no effect of binding density on the site size transitions, the experiments should yield identical results regardless of the method used. Fig. 4 shows an example of two


TABLE I Preferential ion binding parameters for the (SSB)s, to (SSB),, binding mode transition with poly(dT)

Site sizes were monitored by "reverse" titrations. NaBr NaCl NaF MgCL BaCll

pH 8.1, 25 "C ArM + Ar= 2.8 f 0.5 2.8 f 0.5 2.8 f 0.5 1.6 * 0.3 1.6 & 0.3 Transition midpoint 17 mM 17 mM 7 mM 0.6 mM 0.2 mM

pH 8.1, 20 "C + Arx 0.95 k 0.1

Transition midpoint 0.8 mM pH 6.9, 25 'C

ArM + Ar= 2.1 f 0.5 1.6 & 0.3 1.0 k 0.3 1.1 & 0.2 Transition midpoint 16 mM 25 mM 70 mM 0.45 mM

TABLE I1 Preferential ion binding parameters for the (SSB),, to (SSB), binding mode transition with poly(dT)

Site sizes were monitored by "reverse" titrations. NaBr NaCl NaF MgCL BaC1,

pH 8.1, 25 "C ATM + Ar,

pH 8.1, 37 "C Transition midpoint

ArM + Ar, Transition midpoint

pH 8.1, 20 "C ArM + Ar,

pH 6.9, 25 "C Transition midpoint

ArM Ar, Transition midpoint

6.5 f 1.5 5.5 f 1.5 3.1 f 1 4.8 f 1 2.0 f 0.5 0.08 M 0.16 M 0.6 M 0.064 M 0.35 M

6.4 & 1 0.174 M

2.6 f 1 0.046 M

5.3 & 2.3 0.05 M

5.2 -C 1.2 4.5 f 1.2 0.14 M

4.1 f 1 0.50 M 0.10 M

7 0 1 ' ' " " " I ' ' " " " 1 ' ' " ' " ' 1 ' ' " ' ' ' 7 ' ' " " T ' ' " " " ' ' ' " "

30 I * , , , , , , , I , , , , , , / , , , , , , , , , , I , , , , , , , , 1 , , , , , , , ( I , , , , , , ,

10- 10-5 io-4 10" 1'0-2 io-? 1 I

FIG. 3. A direct comparison of the effects of pH on the SSB protein-poly(dT) binding mode transitions induced by NaCl and MgC1, concentrations. Apparent site sizes (nucleotides/SSB tetramer), measured by reverse titrations at 25 "C, are plotted as a function of salt concentration (M) (logarithmic scale). 0, MgCl,, 3 mM sodium cacodylate, pH 6.9; 0, NaC1, 3 mM sodium cacodylate, pH 6.9. The dashed line (- - -) and the dotted line (. . . .) describe the site size data as a function of MgCl, and NaC1, respectively, in buffer T, pH 8.1 (25 "C) (data from Bujalowski and Lohman, 1986). All experiments were performed with an SSB protein concentration of 5.16 X lo-' M (tetramer), except for the experiment in 0.4 M MgCL at pH 6.9, which used an SSB protein concentration of 1.79 X 10" M (tetramer), since binding at this MgC1, concentration was not stoichiometric at the lower SSB concentration.

normal titrations (addition of protein to poly(dT)) performed at NaCl concentrations of 1 mM and 1 M in buffer T (pH 8.1), 25 "C. Under these conditions, the SSB protein-poly(dT) affinity is extremely high so that there is no detectable free SSB protein in the initial stages of the titration. In each case, when poly(dT) is present, the change in the fluorescence upon addition of SSB protein is lower in the initial stages of the titration, since the tryptophan fluorescence is quenched. After saturation of the poly(dT), a much larger increase in fluorescence is observed upon further addition of the SSB protein, since this additional protein remains free, and, therefore, no further quenching occurs. For each experiment, the slope of

the titration (dF,./d[SSB]) at high SSB concentrations is parallel to the slope for the blank titration of buffer T with SSB protein in the absence of poly(dT). The intersection of the initial linear portion of the titration with the final linear portion of the titration yields the SSB concentration at which saturation of the poly(dT) occurs. The molar ratio of the total nucleotide concentration to the total SSB tetramer concentration at that point is defined as the apparent site size. The two normal titrations shown in Fig. 4 yield site sizes of 52 2 3 and 70 +- 3 nucleotides/SSB tetramer in 1 mM and 1 M NaC1, respectively (25 "C, pH 8.1).

The results of a series of normal titrations to determine the


0 I I I 0 0.4 0.8 1.2 1.6 2.0

[:SSBlxlO‘(M(tetramer)) FIG. 4. Normal titrations of poly(dT) with SSB protein to

determine the apparent site size in the limit of low SSB protein binding density. Aliquots of a stock of SSB protein were added to solutions containing poly(dT) (7.415 X M (nucleotide)) in buffer T, pH 8.1,25.0 “C containing (0) 1 mM NaCl, (m) 1 M NaCl. The straight lines drawn through the’data a t high SSB concentrations are parallel to the straight line describing a blank titration, with SSB protein, of buffer T, pH 8.1,l mM NaCl, containing no poly(dT) (0).

30

1 o - ~ 10-2 IO“ 1

CNaCll 0” FIG. 5. Only two discrete binding modes, with site sizes of

52 f 3 and 70 f 3 nucleotides/SSB tetramer, are observed as a function of NaCl concentration when the site sizes are measured in the limit of low SSB protein binding density. Apparent site sizes (nucleotides/SSB tetramer), determined from a series of normal titrations (0) (addition of SSB protein to poly(dT)), in buffer T, pH 8.1, 25.0 ‘C, are plotted as a function of NaCl concentration ( M ) (logarithmic scale). For comparison we also show apparent site sizes, determined from reverse titrations (0) (addition of poly(dT) to SSB protein), as a function of NaCl concentration ( M ) (Bujalowski and Lohman, 1986).

apparent site size of the SSB protein-poly(dT) complex as a function of NaCl concentration are shown in Fig. 5. For comparison, we have also included our previous results of site size determinations using reverse titrations (Bujalowski and Lohman, 1986). The data from the normal titrations indicate the presence of only a single transition between two binding modes with apparent site sizes of 52 f 3 and 70 f 3 nucleotides/SSB tetramer over the salt concentration range from 1 mM to 1 M NaC1. We do not observe the presence of the (SSB),, binding mode at low NaCl concentration in a normal

titration. Within experimental error, the site sizes in the two plateau regions observed in the normal titrations (52 f 3 and 70 f 3) are equivalent to the two higher site size binding modes observed using the reverse titrations (56 +- 3 and 65 f 3), and it is most likely that they correspond to the (SSB)56 and (SSB)65 binding modes, as we have previously designated them. However, there is one important difference between the two complexes having napp = 56 f 3 and 52 f 3. The extent of quenching of the SSB protein tryptophan fluorescence is lower for the complex with an apparent site size of 52 f 3, as determined by the normal titration (69 f 2% quenching), than for the (SSB)56 binding mode, as determined by a reverse titration (89 f 2% quenching). For the binding modes with high site sizes of 65 f 3 and 70 +- 3, we measure 89 f 2% quenching independent of the method of titration. The dependence of the extent of SSB protein fluorescence quenching on the method of titration for the intermediate site size mode, nepp = 52-56, indicates that these complexes are slightly different. Alternatively, the “mode” with nap, = 52 may actually be a mixture of the (SSB),, and (SSB)56 modes, which possess -55 f 2% and 89 f 2% fluorescence quenching, respectively, thereby explaining the intermediate quenching of 69%. The fact that the (SSB), mode is induced by salt, whereas the (SSB),, mode is not, also suggests a basic difference between the two modes.

We suggest that the absence of the site size plateau at 35 nucleotides/SSB tetramer in the normal titrations indicates that the transition between the (SSB)s5 and the (SSB)56 binding modes is dependent upon the SSB protein binding density. That is, the (SSB)s5 binding mode is favored at high binding density; hence it is observed in a reverse titration at low [NaCl], whereas the (SSB)56 binding mode is favored at low binding density, even at low [NaCl]; hence the (SSB),, binding mode is not observed in a normal titration. The electron microscope experiments of Griffith et al. (1984) have previously demonstrated that an increase in the SSB protein to ss M13 DNA ratio induces a transition between complexes with a “beaded” morphology and those with a smoother morphology, having approximately twice the contour length of the beaded complexes. Hence our observations and those of Griffith et al. (1984) seem consistent, since it is likely that the “smooth” morphology structures correspond to the (SSB),, mode, whereas the beaded structures correspond to the (SSB)65 and possibly the (SSB), binding modes, as previously suggested (Lohman and Overman, 1985; Bujalowski and Lohman, 1986).

The midpoint of the (SSB)52 to (SSB), binding mode transition occurs at -70 mM NaCl in the low binding density limit (normal titrations), which is approximately a factor of 2 lower than the midpoint for the (SSB)s6 to (SSB)65 transition in the high binding density limit. This difference may reflect a slight binding density dependence for this transition since a binding density dependence should exist for any binding mode transition in which the binding modes differ in site size (Schwarz and Stankowski, 1979). Fig. 5 also indicates that this transition is significantly broader when it is determined using normal titrations.

Hydrodynamic Characterization of the SSB Protein- Poly(dT) Binding Mode Transitions-Previously, the change in site size of the SSB protein-ss nucleic acid complex has been used to monitor the SSB protein binding mode transitions as a function of salt concentration (Lohman and Over- man, 1985; Bujalowski and Lohman, 1986). The transitions have also been observed using the change in the contour length and morphology of SSB protein-ss M13 DNA complexes as a function of the SSB protein to DNA ratio using


electron microscopy (Griffith et al., 1984). In order to further characterize the salt-induced binding mode transitions by an independent technique, we have monitored changes in the sedimentation coefficient of the complex to determine if the frictional coefficient of the complex changes as a function of the binding mode. We have used MgC12 to induce the transitions in a preformed SSB protein-poly(dT) complex, since the site size plateaus, representing the (SSB)BB, (SSB)5B, and (SSB)6s binding modes, are better separated as a function of [MgC12] than [NaCl] (Bujalowski and Lohman, 1986). The SSB protein-poly(dT) complexes used in the sedimentation velocity experiments were initially formed in 1 M NaCl and subsequently dialyzed to a low salt buffer (10 mM Tris, pH 8.1, 0.3 mM NaCl; see “Materials and Methods”). This procedure was used to ensure that the complex was in its equilibrium form in this low salt buffer, since we have previously noted nonequilibrium behavior when SSB protein is directly mixed with ss M13 DNA at low salt (Lohman et al., 1986a). We used an excess of poly(dT) to form the complex (101 nucleotides/SSB tetramer) in order to be certain that dissociation did not occur upon inducing the increase in SSB site size with MgC12. If there is insufficient free DNA available, dissociation will occur during the transition to the higher site size binding modes (Lohman and Overman, 1985).

A plot of szo,w as a function of the logarithm of the MgC12 concentration is shown in Fig. 6A, where it is directly compared with measurements of the apparent site size of the SSB protein-poly(dT) complex, also determined at 20 “C (pH 8.1). The comparison was made by normalizing the two sets of data, such that the limiting values of each quantity at low and high MgClz concentrations were constrained to be coincident. Fig. 6A indicates that s20,w increases dramatically as a function of MgClz concentration from 12.5 S in the absence of MgClz (0.3 mM NaCl) to 23.7 s in 240 mM MgC12 (0.3 mM NaC1). Since dissociation does not occur under these conditions, the increase in s20,w must reflect a decrease in the frictional coefficient of the SSB protein-poly(dT) complex, indicating a significant compaction of the complex as it pro- gresses from the (SSB)35 to the (SSB),, binding mode. This direct evidence for compaction of the complex in solution is consistent with the proposal that the increase in SSB protein site size reflects additional wrapping of DNA around the SSB tetramer so that all four SSB subunits are contacted by ss DNA, rather than just two (Lohman and Overman, 1985; Lohman et al., 1986a). The formation of SSB octamers on the DNA may also contribute to the compaction (Chrysogelos and Griffith, 1982; Griffith et al., 1984; Bujalowski and Loh- man, 1987b).

The comparison of the site size and sedimentation measurements also indicates that the two transitions are super- imposable within experimental error. The slight difference in the first transition, as measured by the two techniques, is within experimental error, since if the s20,w data are renor- malized to a high site size limit of 67 rather than 65, the first transitions are coincident for both techniques. The major difference is the absence of a defined plateau in the szo,w transition corresponding to the site size plateau at 56 nucleotides/SSB tetramer, although there are clearly plateau regions corresponding to the (SSB)35 and (SSB), binding modes. It may be that the larger relative error in the sedimentation experiments as well as the narrower range of MgClz concentrations over which the (SSB)56 plateau occurs at 20 “C (see Fig. 1 and Bujalowski and Lohman, 1986 for the data at 25 and 37 “C) makes this intermediate plateau more difficult to detect by monitoring changes in sz0,,,.

We also performed a second set of sedimentation velocity

-10

- 5 - 4 -3 -2 - 1 0

log [MgCI,]

30t k 1 - k 4 -4 -6 -; I

0

log CMgCI, 1 FIG. 6. An increase in the sedimentation coefficient is coin-

cident with the MgCla-induced increase in the apparent site size of the SSB protein-poly(dT) complex. Sedimentation coefficients (Svedberg units) of SSB protein-poly(dT) complexes (A), prepared as described under “Materials and Methods,” were measured at 20.0 ‘C in buffer T, pH 8.1, and corrected to are plotted as a function of MgCl, concentration (M) (logarithmic scale). Apparent site sizes of SSB protein-poly(dT) complexes (0) were determined as a function of MgCl, concentration (M) using reverse titrations at 20.0 ‘C in buffer T, pH 8.1. A, the ratio of poly(dT) to SSB protein was 101 nucleotides/tetramer; B, the ratio of poly(dT) to SSB protein was 87 nucleotides/tetramer.

experiments on an SSB protein-poly(dT) complex at a lower ratio of 87 nucleotides/SSB tetramer in order to examine the effects on szO.,,, of a slight increase in the protein binding density. These results are shown in Fig. 6B. At this higher binding density, a plateau in szo,w is apparent in the same range of MgClz concentrations in which the (SSB), site size plateau is observed (10-30 mM). However, in these experiments we did not observe a strict plateau for ~ ~ 0 , ~ above 0.1 M MgClz, as was observed for the experiments performed with 101 nucleotides/tetramer (Fig. 6A). The szo,u, reaches an initial plateau of 26.5 S at 10 mM MgCl,, corresponding to the site size plateau of 56 nucleotides/tetramer. After this plateau, a further increase to 30 S is observed at -80 mM MgCl,; however, this is followed by a slight decrease in s20,w, leveling at -29 S. This slight drop in s20,w, above 0.1 M MgC12, may reflect some dissociation of the complex due to the increase in site size from 56 to 65 nucleotides, since the nucleotide to SSB tetramer ratio is lower in this set of experiments. As a result, the plot in Fig. 6B was made by normalizing the szo,w data to the intermediate plateau corresponding to the (SSB), binding mode. We conclude from these two sets of sedimentation velocity experiments (Fig. 6, A and B ) that the three


plateau regions observed for correlate well with the three site size plateaus at 35, 56, and 65 nucleotides/SSB tetramer. Therefore, the transitions from the (SSB),, to the (SSB),, and finally to the (SSB),, binding modes are each accompanied by a compaction of the complex, with the major compaction occurring for the (SSB),, to (SSB),G transition. The sedimentation coefficient, s , ~ . ~ , for poly(dT) alone increases only slightly from 7.5 to 8.7 S over the range from 0 to 51.6 mM M&1, in buffer T + 0.3 mM NaC1.

Analysis of the Extent of Ion Uptake Accompanying the (SSB),, to (SSB),, Binding Mode Transition for the SSB Protein-Poly(dT) Complex-The effects of salt concentration on the transitions among the different SSB protein-ss nucleic acid binding modes described in this paper and previously (Lohman and Overman, 1985; Bujalowski and Lohman, 1986) indicate that a net uptake of ions accompanies each transition to a higher site size binding mode. One can analyze the salt dependence of the transition equilibrium constants to obtain information about the preferential interaction of cations and anions with each of the SSB protein-poly(dT) binding modes, using a standard two-state analysis outlined previously (Loh- man and Overman, 1985) and summarized above. The equilibrium between the (SSB),, and the binding modes can be described by the following equilibrium transition constant,

(SSB),s +-, (SSB)sS ( 6 4

K 3 5 / 5 e = (napp - 35)/(56 - n w p ) (6b)

where naPp is the apparent site size measured for the SSB protein-poly(dT) complex at a given salt concentration. Using Equations 4, 5, and 6b, we have analyzed the effects of salt concentration on the (SSB)35 to (Sf%),, binding mode transition in NaBr, NaCl, NaF, MgCl,, and BaCl, at pH 8.1 and 6.9, and these are summarized in Table I. At pH 8.1, the data in NaBr, NaCl, and NaF (Fig. 2 A ) indicate that Ar = 2.8 f 0.5 ions, i.e. a net uptake of at least 3 ions/SSB tetramer accompanies the transition from the (SSB),, to the (SSB),B binding mode. Since at pH 8.1, the (SSB),, to (SSB),, transition is independent of the anion for the SSB protein- poly(dT) complex (see Fig. 2), we conclude that a net binding of at least 3 sodium ions/SSB tetramer accompanies the transition from the (SSB),, to the (SSB)56 binding mode at this pH.

Further support for this conclusion comes from the analysis of the (SSB),, to (SSB),, transition in the presence of the divalent cations Mg2' and Ba2+. Analysis of the data in Fig. 1 (see Table I) reveals that Ar = 1.6 f 0.3/SSB tetramer for both BaC1, and MgCl,, indicating a net uptake of only 1.5-2 divalent cations. Within experimental error, this value is one- half the value found for Na+ uptake. This suggests that the uptake of a single M P or Ba2+ ion/SSB tetramer can substi- tute for the uptake of 2 Na' ions in the (SSB),, to (SSB),, transition, further suggesting that cation uptake during this transition is required for charge neutralization. The different midpoints for Ba" and Mg2' presumably reflect differences in the binding affinities of these cations for binding sites on the SSB protein-poly(dT) complex.

At pH 6.9, the salt dependence of the (SSB),, to (SSB), binding mode transition has contributions from both anions and cations as can be seen in Fig. 2B. The data in NaBr, NaCl, and NaF indicate that Ar = 2.1 +- 0.5, 1.6 f 0.3, and 1.0 f 0.3, respectively (see Table I). Clearly, the net ion uptake is anion-dependent and decreases in the order Br- > C1- > F; however, we cannot determine from this data whether the extent of cation uptake differs from that observed at pH 8.1.

Analysis of the Extent of Ion Uptake Accompanying the (SSB),, to (ssB)~, Binding Mode Transition for the SSB Protein-poly(dT) Complex-The salt dependence of the (SSB),, to (SSB),, binding mode transition can be analyzed as above with the following definition of the transition equilibrium constant.

(SSB)m * (SSB)6s (6c)

K M , ~ = (napp - 56)/(65 - napp) (6d)

AS shown in Fig. 2, both the midpoint as well as the steepness of this transition are functions of the anion type at both pH 8.1 and 6.9. Analysis of the transitions, according to Equations 4,5, and 6d, yields the following parameters for net ion uptake at pH 8.1, 25 "C (see Table 11): Ar = 6.5 2 1.5 in NaBr, Ar = 5.6 f 1.5 in NaCl, and Ar = 3.1 f 1 for NaF. Therefore, it is apparent that in addition to a net uptake of cations, there is also a net uptake of anions accompanying the transition from the (SSB),, to the (SSB),, binding mode at pH 8.1. The values of Ar also indicate that more bromide ions are taken up than chloride ions and the uptake of fluoride ions is smaller still. The ordering of these three anions, Br- > C1- > F-, follows that found by von Hippel and Schleich (1969) in their studies of anion binding to models of the peptide bond in proteins. We have additional evidence from equilibrium binding studies of the (SSB)B6 complex with poly(U) that F- binds only weakly if at all to the E. coli SSB protein (Overman et al., 1988).' Therefore, as a first approximation, we interpret the value of Ar = 3.1 for the (Sf%),, to (SSB), transition induced by NaF as having contributions only from Na' uptake (i.e. ArF = 0). One can further estimate the anion contribution to ion uptake, in the case of Br- and C1-, from the difference in the values of Ar determined for the sodium salt of that anion and Ar for

ArN,cl = 5.5 2 1.5, we estimate ArBr = 3.4 f 1.5 and Arcl = 2.4 f 1.5. Thus, the (SSB),, to (SSB), binding mode transition is accompanied by the net uptake of approximately 3-4 cations and 2-3 anions.

An estimation of Ar for the (SSB),, to (St%),, transition in the presence of BaClz (pH 8.1, 25 "C) has been made assuming that the plateau at high BaC1, concentration is identical to that determined in MgCl, (see Fig. 1). Besides the shift of the transition to higher salt concentration, there is also an apparent decrease in the cooperativity in the presence of BaC1, as compared to MgC12. We calculate values of Ar = 4.8 f 1 and 2.0 f 0.5 for MgCl, and BaC12, respectively. It is interesting to compare these values with the results obtained for the sodium salts. The uptake of sodium ions in the (SSB)BB to (SSB)6s transition is 3.1 at pH 8.1. If charge compensation is responsible for this cation uptake, as it seems to be for the (SSB),, to (SSB),, transition, then the uptake of divalent cations would be predicted to be approximately 1.6. Subtract- ing this number from the Ar obtained for MgC12 yields a value of 3.2. Therefore, the contribution of chloride, Arcl, to the (SSB),, to (SSB),, transition induced by MgCl, is 3.2 (Equa- tion 5). This value is slightly larger than the value of Arc1 = 2, obtained from the data in NaCl (see Table 11), although it is probably the same within experimental error. The data for BaC1, could not be analyzed quantitatively; however, the shift in the transition to higher salt concentrations and the lower slope suggest that Ba2+ ions bind with lower affinity than M g + to the cation binding site(s) on the (SSB),, complex.

Analysis of the (SSB),, to (SSB)65 transition in Fig. 2B at pH 6.9 also indicates a net ion uptake for both NaBr and NaCl, with Ar = 5.2 f 1.2 and 4.5 k 1.2, respectively. Since

NaF, e.g. Arcl = (ArNacl - ArN,F). Since ArNaBr = 6.5 f 1.5 and

* L. B. Overman, unpublished data.

E, coli SSB Protein-ssDNA Binding Mode Transitions 4637

the error limits on these values overlap those obtained at pH Low Binding Density at Low NaCl Concentrations-For any 8.1, it seems that the net ion uptake is essentially independent ligand (protein) that is capable of binding to a linear lattice of pH for the (SSB),, to (SSB),, transition, although the salt (DNA) in two or more binding modes that differ in site size, concentration midpoints of this transition are clearly depend- the higher site size mode will be favored at lower binding ent on pH (see Fig. 2 and Table 11). density and the lower site size mode will be favored at higher

binding density, if the binding constants for the two modes DISCUSSION are comparable (Schwarz and Stankowski, 1979). This is the

The SSB Protein-ssDNA Complex Undergoes Successive Compactions in Progressing from the (SSB),, through (SSB)II to the (SSB),, Binding Modes-We have shown that a substantial increase in s , ~ , ~ , reflecting a decrease in the frictional coefficient of the SSB protein-poly(dT) complex of approximately a factor of 2, occurs upon increasing the MgC1, concentration over the range from 10 pM to 0.1 M. This increase in szo,w exactly coincides with the MgC1,-induced increase in binding site size from the (SSB),, complex through the (SSB),, to the (SSB), complex. A compaction of SSB protein- ss M13 DNA complexes was first suggested by electron microscopic studies (Griffith et al., 1984) and presumably reflects the additional wrapping of the ssDNA around the SSB tetramer as well as the aggregation of the DNA-bound tetramers to form octamers as discussed below (Chrysogelos and Grif- fith, 1982; Lohman and Overman, 1985; Lohman et al., 1986a; Bujalowski and Lohman, 1987b). The fact that the MgC1,- induced transition of s , ~ , ~ exactly coincides with the site size transition of the SSB protein-poly(dT) complex also suggests that these transitions are at or close to equilibrium. This result, as well as the fact that these transitions are reversible (Lohman and Overman, 1985), suggests that we can treat them as equilibrium transitions.

We have previously proposed that the (SSB),, binding mode represents a complex in which ssDNA binds to and wraps around only two subunits of the SSB tetramer, whereas the (SSB),, and (SSB), modes involve the interaction of ssDNA with all four subunits of the SSB tetramer (Lohman and Overman, 1985; Lohman et al., 1986a; Bujalowski and Loh- man, 1986). A transition between complexes such as these would lead to a substantial compaction of the ssDNA. An additional compaction could also result from the fact that the SSB protein can form octamers (dimers of tetramers) when bound to ssDNA under some conditions (Chrysogelos and Griffith, 1982). From an examination of the extent of tryptophan fluorescence quenching of the different SSB protein binding modes (Lohman and Overman, 1985); it is likely that the ssDNA interacts with all four subunits in both the (SSB),, and (SSB),, binding modes. The (SSB),, mode seems to involve an equilibrium mixture of nucleic acid-bound tetramers and octamers (Bujalowski and Lohman, 1987b); however, the quaternary structure of the (SSB),, mode is not known. The (SSB),, binding mode that has been observed at 37 "C (Bujalowski and Lohman, 1986) may represent a form with an intermediate extent of wrapping between that proposed for the (SSB),, and (SSB),, modes.

The salt-induced compactions of the SSB protein-ssDNA complexes, which accompany the SSB binding mode transitions, seem to be qualitatively similar to the salt-induced compaction of chromatin from the 10-nm filament to the 30- nm higher order structure (Butler, 1983; Widom, 1986; Greu- lich et al., 1987). However, one major difference is that no anion effects have been reported for the chromatin compaction, whereas we see substantial anion effects on the SSB protein-ss nucleic acid transitions.

The (SSB),, Binding Mode Is Favored at High Binding Density, Whereas the Higher Site Size Modes Are Favored at

W. Bujalowski, unpublished experiments.

situation that seems to exist for the SSB protein at low salt concentrations. As can be seen in Fig. 5, the apparent site size of an SSB protein-poly(dT) complex is dependent upon the binding density at low NaCl concentration (CO.2 M). The (SSB),, binding mode is favored at high binding density, as seen in the titrations of protein with DNA (reverse titrations), whereas a complex with napp = 52 nucleotides/tetramer is favored at low binding density, as seen in the titrations of DNA with protein (normal titrations). As a result, only the (SSB),, to (SSB),, binding mode transition is observed when a series of normal titrations is performed as a function of NaCl concentration, whereas the (SSB),, to (SSB)B6 transition is also observed when reverse titrations are performed. We stress that the (SSB),, mode (normal titration) has different fluorescence characteristics than the (SSB),, mode ( eg . 69% versus 89% fluorescence quenching); hence the (SSB),, mode may actually represent a mixture of the (SSB),, and (SSB),, modes.

We have not directly measured the transition between the (SSB),, and the (SSB),, binding modes as induced by a decrease in SSB binding density, and hence we do not know the extent to which this transition, as induced by binding density changes at low salt, is at equilibrium or whether kinetic limitations dominate the transition. The previous results of Lohman and Overman (1985) suggest that the (SSB),, mode can be formed from the (SSB),, mode by the addition of excess DNA. The observed fluorescence quenching of an SSB-poly(dT) complex, formed under low salt conditions that favor the (SSB),, binding mode, will increase slowly upon addition of excess poly(dT), suggesting the slow conver- sion of some (SSB),, complexes into (SSB),, complexes. Nevertheless, these data still indicate that the (SSB),, binding mode is favored at high binding densities.

This binding density effect was first observed by Griffith et al. (1984) in their electron microscopic studies of SSB protein- ss M13 DNA complexes at low salt concentration (10 mM Tris, pH 7.5, 37 "C). At low SSB/DNA ratios, a beaded morphology was observed; however, upon increasing the SSB/ DNA ratio, a sharp transition to a "smooth" contoured structure possessing twice the contour length of the beaded structures was observed. The contour lengths of both of these complexes were shorter than naked ssDNA. It is possible that the data in Fig. 5 reflect the same binding density-dependent transition between the (SSB),, and (SSB),, binding as was observed by Griffith et al. (1984). As mentioned above, it is also possible that the binding mode transitions that are induced by salt may be different in detail than the transitions that are induced by protein-binding density.

Cation, Anion, and pH Effects on the SSB Protein-Poly(dT) Binding Mode Transitions Are Thermodynamically Linked- The two major binding mode transitions that we observe at 25 "c, (SSB),, to (SSB),, and (SSB),, to (SSB),, are each affected in a qualitatively different manner by changes in the cation or anion of the salt used to induce the transitions. For example, at pH 8.1, the (SSB),, to (SSB),, transition is unaffected by changes in the anion, whereas the (SSB),, to (SSB),, transition is quite sensitive to the anion concentration. This clearly indicates that these transitions reflect different molecular processes and cannot be considered as a


single process. Analysis of previous experiments (Bujalowski and Lohman, 1986) and those reported here indicates that the transitions from the lower to the higher site size binding modes are accompanied by the following: 1) net cation uptake which is dependent upon cation valence, concentration, and type; 2) net anion uptake which is dependent upon anion type, concentration, and pH (and presumably valence, although this has not been examined). At pH 8.1 (25 "C), only the 56 to 65 transition is anion-dependent, whereas at pH 6.9, both the 56 to 65 as well as the 35 to 56 transitions are anion- dependent. In general, the salt concentrations that are required to induce the transitions are higher for NaF than for NaCl than for NaBr. Anion uptake is greater for Br- than for C1- than for F-. 3) Net proton release (deprotonation). Al- though we have only investigated the transitions at pH 8.1 and 6.9, it is clear that the effects of pH are linked to the anion effects. The transitions become more sensitive to anion effects upon lowering the pH. This suggests that protonation of groups on the SSB protein enhances the preferential interactions of anions with the protein in the different binding modes. With the exception of the NaBr data, fewer ions are taken up in the 35 to 56 transition as the extent of protonation increases at lower pH; however, at this time, we cannot determine whether this reflects a reduction in cation or anion uptake, or both.

We do not yet have sufficient information to allow a defin- itive dissection of the salt dependences at the two pH values in terms of the separate effects of cations and anions on the binding mode transitions. However, we can dissect the salt dependences further if we make one of the following simpli- fying assumptions, which are mutually exclusive. 1) Cation uptake is pH-independent, and hence this term is assumed to be constant for the data at pH 6.9 and pH 8.1. Therefore, the reduction in the value of Ar, obtained in NaF upon lowering the pH from 8.1 to 6.9, represents a reduction in the net anion uptake at pH 6.9. 2) Fluoride does not preferentially interact with the SSB protein-poly(dT) complexes at either pH. Therefore, the reduction in the value of Ar, obtained in NaF upon lowering the pH from 8.1 to 6.9, represents a reduction in the net cation uptake.

Based on the first assumption that cation uptake is independent of pH, it follows that the reduction in Ar at pH 6.9 reflects a reduction in the net uptake of anions. This could result from either an increase in the extent of anion release or a decrease in the extent of anion uptake for the (SSB)35 to (SSB),, transition. Our measurements of the equilibrium binding constant for formation of the (SSB)65 complex from free SSB and poly(U) indicate that the net anion release increases upon lowering the pH.4 If we view the (SSB)35 to (SSB),, transition as an intermediate step in the formation of the (SSB)65 complex from free SSB protein, then these observations seem qualitatively consistent. The additional observation that a net release of protons accompanies the transitions from the lower to the higher site size complexes may relate to why the anion effects are thermodynamically linked to pH effects. If preferential protonation of free SSB protein and the (SSB)35 complex provides additional anion binding sites, then the release of these protons upon forming the higher site size complexes would also result in the release of anions. However, this assumption leads to the conclusion that the net uptake of fluoride ion increases by -2 ions upon increasing the pH from 6.9 to 8.1, whereas the equilibrium binding data for (SSB)65 complex formation suggest that there is very little preferential interaction of fluoride (Overman et al., 1988).4

' L. B. Overman, unpublished experiments.

If we make the second assumption that there is no preferential interaction of fluoride ions throughout these transitions, then the data indicate that fewer Na+ ions are taken up at pH 6.9 than at pH 8.1. It then follows that either more anions are taken up or fewer anions are released in the (SSB)35 to (SSB)s6 transition at pH 6.9. This conclusion is difficult to understand if the residues that become protonated at pH 6.9 also become sites for anion binding. Further experiments will be necessary to differentiate among these and other possibilities.

Relationship of the Binding Mode Transitions to the Equi- librium Binding of SSB Protein to ss Nucleic Acids in the fSsBj6, Binding Mode-The salt dependence of the equilibrium binding constant, Kg5,, for E. coli SSB protein binding to ss homopolynucleotides to form the binding mode has been measured as a function of a number of monovalent salts differing in the anion (pH 8.1, 25 "C) (Overman and Lohman, 1986; Lohman et al., 1986a; Overman et al., 1988). Forpoly(U), (dlogK,%/~3log[NaCl]) = -7.4 f 0.5, for poly(dT), (dog Ktz,/dlog[NaBr]) = -5.7 f 0.7, whereas for poly(A) and poly(dA), (dog K%,./dlog[NaCI]) = -6.1 f 0.6. This indicates that a net release of ions occurs upon formation of the (SSB),, binding mode from the free protein. Furthermore, the effects of different anions indicates a net release of both cations and anions. Approximately four Na' and three C1- are released over the range from 0.2 to 0.4 M NaCl at 25 "C (pH 8.1); however, the extent of anion release is anion-dependent, increasing for Br- and decreasing for F-, with respect to C1- (Overman et al., 1988). On the other hand, the binding mode transitions occur with a net uptake of cations (Lohman and Overman, 1985; Bujalowski and Lohman, 1986) and anions, in progressing from the (SSB),, through the (SSB)56 to the

binding modes, as shown here. Therefore, although a net release of cations and anions occurs upon formation of the (SSB)65 binding mode from the free protein, this net release is composed of separate contributions from cation release and uptake as well as anion release and uptake.

Possible Roles of Ions in the Compaction of the SSB Protein- ss Nucleic Acid Complex-The effects of ions on protein- nucleic acid interactions can originate from a variety of sources: 1) binding of cations to the nucleic acid 2) binding of ions (cations and/or anions) to the protein or protein- nucleic acid complex; 3) coulombic screening or "ionic strength" effects as described by Debye-Huckel theory. Quan- titative theories of the effects of salt on the equilibrium binding and kinetics of protein-nucleic acid interactions have been described (Record et al., 1976,1978; Lohman et al., 1978; Lohman, 1986; Manning, 1978). However, the experiments described in this paper deal with ion effects on conformational changes in a protein-nucleic acid complex, rather than on the equilibrium binding affinity of the complex.

From the specific effects of different cations and anions on the SSB protein-poly(dT) binding mode transitions, we can rule out that the observed ion effects are solely due to ionic strength effects, but rather they must be due to the direct binding of cations and anions. As we have shown, an uptake of both anions and cations accompanies the transitions to the higher site size complexes. Lohman and Overman (1985) have proposed that the complex involves the interaction of ssDNA with only two subunits of the SSB tetramer, whereas it is likely that the (SSB)56 and (SSB)c5 complexes involve the interaction of ssDNA with all four subunits. In view of this proposal, it is possible that some of the cation uptake is required to reduce the repulsion between the ssDNA phosphate residues that occupy the first two subunits of the SSB tetramer and the phosphate residues that are brought in


close proximity upon binding to the third and fourth subunits. The fact that Mg2' is much more effective than Na' in facilitating the site size transitions (Bujalowski and Lohman, 1986) is consistent with this hypothesis since Mg' has a higher affinity for DNA than does Na' (Krakauer, 1971, 1974). In this context, we note that additional ionic interactions should be formed between the DNA and the third and fourth SSB subunits upon forming the (SSB)5s complex, and this should result in the release of Na+ from the DNA (Over- man et al., 1988; Record et al., 1976). Therefore, there is likely to be both uptake and release of Na' in this step, with a net uptake of Na+, as observed experimentally. Alternatively, the need for ion uptake may result from a conformational transition in the SSB protein upon binding of the DNA to the first two SSB subunits, and ion uptake may be required to affect another conformational transition to allow further DNA binding to the second two SSB subunits.

The observation of anion effects on these transitions clearly indicates that preferential interactions of anions with the SSB protein or the SSB protein-DNA complex must accompany the transitions; however, the molecular basis for these effects could involve any number of possibilities including aggregation of the tetramers to form octamers on the DNA in one of the transitions (Chrysogelos and Griffith, 1982; Buja- lowski and Lohman, 1987b). It is clear that these effects do not reflect simple ionic strength effects nor are they only the result of interactions of cations with the nucleic acid.

Comparisons with Site Size Measurements from Other Stud- ies-Some SSB site size measurements have been reported (Krauss et al., 1981; Greipel et al., 1987) that differ with those determined in our laboratory (Lohman and Overman, 1985; Bujalowski and Lohman, 1986; this work). We feel that most of these discrepancies, although not all, can be explained by the fact that different extinction coefficients have been used to determine the SSB protein concentration, as we have discussed (Lohman and Overman, 1985). Maass and col- leagues (Krauss et al., 1981; Greipel et al., 1987) have reported using two different extinction coefficients for SSB protein (ezm = 1.22 versus 0.98 ml mg" cm"), with resulting site sizes of 37 & 2 and 40 & 2 in 0.2 M KC1 (pH 7.4,8 "C) and 0.3 M NaCl (pH 7.4, temperature unspecified), respectively. We have used an extinction coefficient of tZrn = 1.5 ml mg" cm" (1.13 X lo5 M" (tetramer) cm-l), which agrees with four independent determinations (Ruyechan and Wetmur, 1975; Williams et al., 1983; Lohman and Overman, 1985; Shima- mot0 et al., 1987) and have found that nepp is pH-dependent at 0.2-0.3 M NaC1, 25 "C (see Fig. 2), although fairly independent of temperature.

We have made a direct measurement of the SSB protein- poly(dT) site size under the identical conditions reported by Krauss et al. (1981) (20 mM "potassium phosphate," pH 7.5, 8 "C). These are the same buffer conditions used by Greipel et ai. (1987), although they did not specify the temperature. Under these conditions, we measure nap, = 60 f 3 nucleotides/ tetramer (data not shown) using our extinction coefficient of 1.5 ml mg" cm"; hence it is within the 56-65 transition. If we recalculate the site size data of Greipel et al. (1987) and Krauss et al. (1981), using our extinction coefficient of 1.5 ml mg" cm", then we estimate napp = 49 f 3 and 56.6, respectively, rather than 40 k 2 and 37 2 2. Hence a considerable discrepancy still exists between our findings and the report by Greipel et al. (1987), even after correcting for the different extinction coefficients that have been used. However, the site size data of Krauss et al. (1981) is in closer agreement with our findings, after recalculation using our extinction coefficient.

All of the measurements discussed in the above comparisons were made with poly(dT) (Lohman and Overman, 1985; Bu- jalowski and Lohman, 1986; this work). We have also reported "apparent" site sizes using ss M13 DNA at four NaCl concentrations, 1 mM, 12 mM, 0.1 M, 0.3 M, and obtained values of 35, 44, 62, and 77 nucleotides/tetramer, respectively, at 25.0 "C, pH 8.1 (Lohman and Overman, 1985). These data allowed comparisons to be made with our poly(dT) measurements, and we stated that the ss M13 DNA values were overestimates, presumably due to the inability of E. coli SSB protein to melt stable secondary structure in the ss M13 DNA (Lohman and Overman, 1985). Greipel et al. (1987) have attempted to explain the discrepancy between our site size measurements and theirs by comparing their measurement of 40 & 2 nucleotides/tetramer, made in 0.3 M NaCl with poly(dT), to our measurement of 62 nucleotides/tetramer, made with ss M13 DNA in 0.1 M NaC1, concluding that our measurement of 62 is an overestimate because we used ss M13 DNA. This comparison is improper. The measurements of site sizes using ss M13 DNA do overestimate the site size in 0.3 M NaCl; however, with poly(dT), we measure a site size of 65 f 3 nucleotides/SSB tetramer (Lohman and Overman, 1985; Bujalowski and Lohman, 1986).

Possible Effect of the Different SSB Binding Modes on Functions Involving the SSB Protein-Most proteins that bind to linear nucleic acids form unique well defined complexes with a specific site on the nucleic acid or, if they bind nonspecifically, with a defined length of nucleic acid. Many nucleic acid binding proteins, such as repressors, function by occupying a specific site on the DNA in a unique binding mode. The occupancy of that site is controlled by the free protein concentration and the competition for nonspecific binding sites (von Hippel et al., 1974). Of course, the distribution of bound protein may be under thermodynamic (equilibrium) or kinetic control, depending on the process. On the other hand, the E. coli SSB protein can bind in multiple modes and can form higher order complexes with ss nucleic acid; hence it is not simply a matter of whether the protein is bound but in what mode it is bound. It is possible that the different modes of interaction may selectively facilitate or stimulate different processes that are required during the life cycle of the cell. In any event, there are multiple equilibria involved in the interaction of this protein with nucleic acids, since the equilibria among the different forms of bound protein must be considered in addition to the equilibria involving free protein. As a result, it is necessary to understand the effects of solution conditions on the relative distribution of the different binding modes and the interconversion among the modes.

The E. coli SSB protein is involved in DNA replication, recombination, and repair processes, all of which are being studied intensively in vitro. Many times, in such studies, the E. coli SSB protein is considered to be merely an "ingredient" in the reaction, much like Mg', ATP, or pH, with the view that the E. coli SSB protein binds to ss nucleic acids in a manner that is independent of solution conditions; however, it has now been well documented that this is not the case (Lohman and Overman, 1985; Griffith et al., 1984; Bujalowski and Lohman, 1986; this work). As a result, it is extremely likely that the activities of other proteins and enzymes that require or are facilitated by the E. coli SSB protein are dependent upon the particular binding mode that the E. coli SSB protein forms with ssDNA. In fact, Griffith et al. (1984) have demonstrated that the DNA strand exchange reaction of the recA protein is selectively stimulated by the beaded mode of the SSB protein, although it is not clear whether this


morphology describes only the (SSB)65 or also the (SSB),, binding mode (Bujalowski and Lohman, 198713). From a purely practical point of view, it is clearly important to define the variables that determine the relative stabilities of the various SSB protein binding modes on ssDNA, so that these can be correlated with the results of replication, recombination, and repair studies. As yet, no systematic attempt has been made to compare the effects of the different SSB protein binding modes on replication, recombination, and repair re- actions in vitro. Our studies indicate that the E. coli SSB protein interacts differently with ssDNA, depending on a wide variety of conditions, and the potential effects of these different binding modes must be considered in any interpretations of experiments that include the SSB protein. An additional complication that must also be considered in studies in uitro is the observation that the highly cooperative binding of the E. coli SSB protein is not an equilibrium phenomenon; rather it forms only transiently under conditions of low monovalent salt concentrations in uitro (Lohman et al., 1986a). As a result, the procedures that are used to form SSB protein-DNA complexes must also be carefully examined, since these are also likely to influence the results of an experiment.

Acknowledgments-We thank Mike Green for technical assistance and Lisa Lohman for preparing the figures.

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