Single-stranded DNA binding properties of the uvsy recombination protein of bacteriophage T41

J. Mol. Biol. (1997) 266, 927±938

Single-stranded DNA Binding Properties of the uvsY Recombination Protein of Bacteriophage T4 Mark A. Sweezy and Scott W. Morrical* Department of Biochemistry, Department of Microbiology and Molecular Genetics, and Vermont Cancer Center University of Vermont College of Medicine, Burlington, VT 05405, USA

The uvsY protein is an essential component of the bacteriophage T4 general recombination machinery. The properties of this 16 kDa protein include selective binding to ssDNA, as well as speci®c protein-protein interactions with other T4 recombination proteins including uvsX (general recombinase) and gp32 (ssDNA-binding protein). uvsY promotes the assembly of uvsX-ssDNA ®laments, the active species in uvsX-catalyzed DNA rearrangements, apparently by helping uvsX displace gp32 from the ssDNA. To better understand the role of uvsY in the T4 recombination system, here we characterize the thermodynamic and molecular properties of the interaction of the uvsY protein with a model singlestranded polynucleotide, eDNA, which is a ¯uorescent, etheno-modi®ed form of random-sequence ssDNA. We have found that the binding of uvsY protein enhances the ¯uorescence of the eDNA lattice and that the maximal amount of ¯uorescence enhancement observed is dependent on salt concentration. In addition, we have used the eDNA ¯uorescence enhancement assay to establish thermodynamic parameters of binding and to de®ne some of the molecular details of uvsY-eDNA interactions. We show that uvsY binds to eDNA in a non-cooperative manner, with a binding site size of four nucleotide residues per monomer of uvsY, and that this binding is salt-sensitive and involves the displacement of anions from the uvsY protein. We further show that uvsY protein binds preferentially to eDNA over unmodi®ed ssDNA. The signi®cance of these results is discussed in light of current models of uvsY action in the T4 recombination system. # 1997 Academic Press Limited

*Corresponding author

Keywords: bacteriophage T4; uvsY; recombination; ssDNA; af®nity

Introduction The uvsY protein is an essential component of the general recombination machinery of bacteriophage T4. Both genetic and biochemical aspects of uvsY recombination functions have been investigated. Although no enzymatic activity has yet been ascribed to the uvsY protein, it has been shown to be a stimulatory factor for virtually all reactions catalyzed by uvsX protein, the T4 recombinase (Yonesaki & Minagawa, 1989; Kodadek et al., 1989; Harris & Grif®th, 1989; Morrical & Alberts, 1990). The 16 kDa uvsY protein has been shown to reduce the concentration of uvsX protein required to catalyze in vitro DNA strand exchange and recombination-dependent DNA synthesis reactions, to stimulate the single-stranded DNA Abbreviations used: ssDNA, single-stranded DNA; dsDNA, double-stranded DNA; eDNA, etheno-modi®ed ssDNA derived from M13mp19 ssDNA circles. 0022±2836/97/100927±12 $25.00/0/mb960829

(ssDNA)-dependent ATPase activity of uvsX, and to overcome the inhibition of uvsX-catalyzed reactions caused by high salt and/or by high concentrations of gp32, the T4-encoded ssDNA1-binding protein (Formosa & Alberts, 1986; Kodadek et al., 1989; Yonesaki & Minagawa, 1989; Harris & Grif®th, 1989; Morrical & Alberts, 1990; Morrical et al., 1991). These stimulatory activities appear to be essential in vivo, since T4 uvsYÿ mutants display recombination-de®ciency phenotypes that are essentially identical with those of uvsXÿ mutants (Cunningham & Berger, 1977; Melamede & Wallace, 1977, 1978, 1980). In addition to reduced recombination frequencies, these phenotypes include DNA arrest and UV-sensitivity, corresponding to the roles of T4 recombination machinery in initiating phage DNA replication and in recombinational DNA repair (Melamede & Wallace, 1977, 1980). Evidence gathered to date suggests that uvsY affects uvsX recombination functions by acting as an # 1997 Academic Press Limited

928 assembly and stabilizing factor for uvsX ®lament formation on ssDNA (Kodadek et al., 1989; Yonesaki & Minagawa, 1989; Morrical & Alberts, 1990). The mechanism by which uvsY performs these functions is poorly understood. However, it has been demonstrated that the uvsY protein interacts with both gp32 (Hurley et al., 1993; Jiang et al., 1993) and uvsX (Formosa & Alberts, 1984). uvsY also binds to both ssDNA and dsDNA, with greater af®nity to the former (Yonesaki & Minagawa, 1989). The nature and possible functions of these interactions remain to be elucidated. However, indirect evidence suggests that uvsX and uvsY are present in approximately 1:1 stoichiometry in the mature T4 presynaptic ®lament (Kodadek et al., 1989). The interaction of uvsY with gp32 may function to allow the binding of uvsY to gp32-coated ssDNA and thus allow the loading of uvsX (Jiang et al., 1993). At this time there is no evidence that rigorously establishes the order of assembly or precise nature of the mature presynaptic ®lament. The interactions of uvsY with ssDNA have been largely overlooked in the study of uvsY recombination functions, and no detailed study of the thermodynamic or kinetic parameters of these interactions has been reported. Recent characterization of an amino-terminal fragment of the uvsY protein demonstrates that this fragment retains ssDNA-binding activity but does not interact with gp32 or uvsX (Yassa et al., 1997). This fragment retains some stimulatory activity towards the uvsXcatalyzed ssDNA-dependent ATPase and DNA strand-exchange reactions (Yassa et al., 1997), indicating that uvsY-ssDNA interactions play an important role in the recombination functions of this protein. A closer examination of the interactions of the uvsY protein with ssDNA is therefore strongly warranted. The experiments described here represent the ®rst detailed characterization of the equilibrium binding properties of uvsY protein towards a singlestranded polynucleotide. We have employed a ¯uorescent, etheno-modi®ed form of M13mp19 ssDNA (eDNA) as a model lattice for these binding studies. We have found that the binding of uvsY protein enhances the ¯uorescence of the eDNA lattice and that the maximal amount of ¯uorescence enhancement observed is dependent on salt concentration. In addition, we have used this eDNA ¯uorescence enhancement assay to establish thermodynamic parameters of binding and to de®ne some of the molecular details of uvsY-eDNA interactions. We show that uvsY binds to eDNA in a non-cooperative manner, with a binding site size of four nucleotide residues per monomer of uvsY, and that this binding is salt-sensitive and involves the displacement of anions from the uvsY protein. We further show that uvsY protein binds preferentially to eDNA over unmodi®ed ssDNA.

Quantitative Analysis of uvsY-ssDNA Interactions

Results Determination of the uvsY binding site size Determination of equilibrium binding parameters for non-sequence-speci®c protein-ssDNA interactions requires an accurate knowledge of the binding site size parameter (n). Previous estimates of binding site size for uvsY-ssDNA interactions have varied widely: from n ˆ 3 (Kodadek et al., 1989) to n ˆ 15 (Yonesaki & Minagawa, 1989) nucleotides bound per uvsY monomer. These estimates were derived from ®lter-binding studies, a method that introduces several potential sources of error into the determination of n for non-sequence-speci®c protein-DNA interactions (Kowalczykowski, 1990). In this study, we have employed an eDNA ¯uorescence enhancement assay, which offers numerous advantages over ®lter binding, both for the determination of binding site size and for quanti®cation of other binding parameters. The eDNA ¯uorescence assay allows protein-lattice interactions to be monitored continuously in solution, thereby avoiding ®lterinduced destabilization and/or disequilibrium problems. In addition, our assay employs an essentially in®nite lattice (eDNA derived from 7250 base M13mp19 ssDNA circles), thereby minimizing site exclusion and end effects on binding, which can skew the apparent endpoint of a titration. A further advantage of eDNA is that the etheno modi®cation disrupts base-paired secondary structures within ssDNA, allowing a more accurate determination of binding site size in cases where protein binding is excluded by secondary structure. eDNA ¯uorescence enhancement and related assays have been used successfully to determine n-values and other binding parameters for several non-sequence-speci®c ssDNA-binding proteins, including the T4 gp32 and gp59 proteins, the Escherichia coli recA protein, and the yeast RP-A protein (Kowalczykowski et al., 1981; Silver & Fersht, 1982; Menetski & Kowalczykowski, 1985; Alani et al., 1992; unpublished results). As shown in Figure 1, forward titrations of eDNA with uvsY protein result in an increase in eDNA ¯uorescence that is saturable with respect to uvsY. For each titration, the apparent endpoint was determined as the intersection of lines extrapolated through the initial points of the titration and the plateau at supersaturating concentrations of uvsY. These data demonstrate that over a fourfold concentration range of eDNA, the binding site size (n) of uvsY remains constant at 4.0(0.2) nucleotides per uvsY monomer (Figure 1). For accurate determinations of binding site size, it is desirable that titrations be performed under stoichiometric (tight) binding conditions. The titrations illustrated by Figure 1 were conducted at low salt (50 mM NaCl in chloride buffer), conditions that generally allow stoichiometric protein-ssDNA interactions. This is con®rmed by the ``salt-back titrations'' shown in Figure 2 (discussed in greater detail below) and by

Quantitative Analysis of uvsY-ssDNA Interactions

Figure 1. uvsY protein binds stoichiometrically to eDNA at low [NaCl]. Forward titrations were carried out at 25 C in 2.0 ml (starting volume) of a buffer containing 20 mM Tris-HCl (pH 7.4), 1 mM MgCl2, and 50 mM NaCl, plus either 1.88 mM (*), 3.75 mM (*) or 7.5 mM (&) eDNA. uvsY protein was added in small increments, and uvsY-eDNA binding was monitored by the enhancement of eDNA ¯uorescence as described in Materials and Methods. All data were corrected for intrinsic protein ¯uorescence, inner ®lter effects, and dilution. Titration endpoints were determined by the intersections of lines drawn through the ascending portions of the curves at low uvsY-eDNA binding density, and the plateau portions at supersaturating quantities of uvsY protein. Note the approximately sixfold enhancement of eDNA ¯uorescence at saturation under these conditions. We routeinely observed three- to sixfold enhancement effects at low salt during several repeats of these experiments involving several different batches of eDNA.

forward titrations performed at different salt concentrations (see Figure 3 and the related text, below), both of which demonstrate that stoichiometric uvsY-DNA binding occurs at salt concentrations of 200 mM NaCl or 300 mM potassium acetate (KOAc). A binding site size of about four nucleotides was con®rmed for all titrations performed under salt conditions that allow stoichiometric binding (see Figure 3 and the related text, below). Further con®rmation of the binding site size was made by performing reverse titrations (the titration of uvsY protein with eDNA lattice) under stoichiometric binding conditions (data not shown); these titrations yielded a binding site size of four as well. Therefore, n ˆ 4 was used in the evaluation of other thermodynamic parameters. The titrations illustrated by Figure 1 exhibit two other interesting features. First is the lack of a sharp break-point upon saturation of the DNA with the uvsY protein, which may be indicative of binding site exclusion (Kowalczykowski et al., 1986; Alani et al., 1992). This phenomenon is most apparent when analyzing proteins that have a large binding site size. The reason for the apparent site exclusion in this case is not known, although

929

Figure 2. NaCl and KOAc salt-back titrations of preformed uvsY-eDNA complexes. Complexes were disrupted through the step-wise addition of NaCl or KOAc and the resulting decrease in ¯ourescence was monitored. The ¯uorescence contribution of uvsY protein was subtracted from each datum point. Starting concentrations of eDNA and uvsY protein were 7.5 and 2.25 mM, respectively, representing a 1.2-fold molar excess of uvsY with respect to binding sites assuming a binding site size of n ˆ 4 nucleotides. Open circles (*) represent a NaCl titration with a starting buffer containing 20 mM Tris-HCl (pH 7.4), and 1 mM MgCl2. Filled circles (*) represent a KOAc titration with a starting buffer containing 20 mM Tris-acetate (pH 7.4), and 1 mM magnesium acetate. All other conditions were as described in Materials and Methods.

other observations suggest that the functional unit of uvsY may be a hexamer or larger (unpublished results). If this is the case, the site size per binding unit of uvsY would be much larger. A second interesting feature of the data is the three- to sixfold relative enhancement of ¯uorescence observed (Figure 1), which is greater than is usually reported for protein-eDNA interactions. A twofold enhancement of ¯uorescence has been ascribed to the disruption of base-stacking interactions and is what is usually observed experimentally upon saturation of eDNA with a protein ligand. Examples of proteins that display this behavior are the gp32 and gp59 proteins of T4 (Kowalczykowski et al., 1981; unpublished results), the recA protein of E. coli (Silver & Fersht, 1982; Menetski & Kowalczykowski, 1986) and the RP-A protein from Saccharomyces cerevisiae (Alani et al., 1992). One possible explanation for the hyper-enhancement of ¯uorescence invokes the displacement of water from the protein-DNA complex through hydrophobic interactions. Other more trivial explanations involving protein ¯uorescence effects and light-scattering have been effectively ruled out through control titrations (see Materials and Methods).

930

Quantitative Analysis of uvsY-ssDNA Interactions

Salt-back titrations of uvsY-ee DNA complexes The determination of af®nity (K) and cooperativity (o) parameters for protein-ssDNA interactions generally must be performed under non-stoichiometric (weak) binding conditions, which are usually attainable at higher concentrations of salt. In order to de®ne an effective range of salt concentrations in which to analyze non-stoichiometric uvsY-eDNA binding, salt-back titrations were performed. In these experiments, eDNA was incubated with a slight excess of uvsY (1.2-fold saturating, assuming n ˆ 4) in either chloride or acetate buffer. Complexes were allowed to form and then disrupted by titrating with NaCl or KOAc; data from these experiments are shown in Figure 2. The decrease in ¯uorescence as salt is added is attributed to the dissociation of uvsY-eDNA complexes and provides a measure of the range of salt concentration in which non-stoichiometric binding occurs. Figure 2 shows that uvsY-eDNA complexes dissociate with salt midpoints of approximately 440 mM NaCl and 720 mM KOAc, respectively. Complexes are completely dissociated at [NaCl] > 600 mM and [KOAc] > 1000 mM, respectively, whereas stoichiometric binding occurs at [NaCl] 4 200 mM and [KOAc] 4 300 mM, respectively. The increased stability of uvsY-eDNA in the presence of KOAc versus NaCl suggests that anion displacement plays an important role in uvsYeDNA interactions, an aspect of binding that we will explore in greater detail in a later section. From the data in Figure 2, we selected salt concentration ranges of 200 to 500 mM NaCl and 300 to 750 mM KOAc, respectively, in which to conduct forward titrations under non-stoichiometric binding conditions. Binding isotherms and determination of K The af®nity (K) and cooperativity (o) parameters for uvsY-eDNA interactions were determined by performing forward titrations of eDNA with uvsY protein under non-stoichiometric binding conditions (salt concentration ranges determined above), and ®tting data to theoretical isotherms generated by the McGhee-von Hippel equation (McGhee & von Hippel, 1974; Kowalczykowski et al., 1986). Data from forward titration experiments are shown in Figure 3. Initial analyses of these data revealed three features: (1) a lack of sigmoidicity in the data, indicative of non-cooperative binding; (2) a salt-dependent decrease in the maximal ¯uorescence signal at saturation; and (3) stoichiometric binding conditions up to 200 mM NaCl and 300 mM KOAc, respectively, consistent with results of salt-back titrations (Figure 2). It is evident from Figure 3 that at relatively low concentrations of salt (50 to 200 mM NaCl and 100 to 300 mM KOAc) there is a signi®cant decrease in the plateau or maximal ¯uorescence signal (Fmax) obtained with increasing salt, while the positions of the titration endpoints do not appear to change

Figure 3. Forward titrations of the uvsY protein onto eDNA were performed at a variety of salt concentraitons, as described in Materials and Methods. a, Titrations in chloride buffer containing the following NaCl concentrations: 50 mM (*), 150 mM (*), 250 mM (&), 300 mM ( & ), 350 mM (~), 400 mM (~), 450 mM (^), 500 mM (^). b, Titrations in acetate buffer containing the following KOAc concentrations: 100 mM (*), 300 mM (*), 400 mM (&), 500 mM ( & ), 600 mM (~), 700 mM (~), 750 mM (^). Corrections for dilution, inner ®lter effects, and protein ¯uorescence were as described for Figure 1 and in Materials and Methods.

appreciably with increasing salt. Thus, the observed changes in Fmax appear to re¯ect a salt-dependent decrease in the degree of eDNA ¯uorescence enhancement induced by uvsY, rather than a change in uvsY site size or binding density on eDNA. This interpretation agrees with the determinations of stoichiometric binding conditions obtained from the salt-back titrations in Figure 2, which indicate that the uvsY-eDNA complexes are

Quantitative Analysis of uvsY-ssDNA Interactions

stable in 50 to 200 mM NaCl and 100 to 300 mM KOAc. However, the salt-back titrations show constant ¯uorescence in these salt ranges, in contrast to the salt-dependency of Fmax observed in the forward titrations of Figure 3. We have obtained similar results through several repetitions of both salt-back and forward titrations, and with several different preparations of uvsY protein and eDNA. One possibility to consider is that the decrease in maximal ¯uorescence observed in the forward titrations is due to a salt-dependent change in the conformation of the uvsY-eDNA complex, and that complexes formed in low salt exist in a metastable state that is not easily overcome through the subsequent addition of salt. We discuss this possibility in light of information on the aggregation state of uvsY protein in a later section (see Discussion). Since the values of K and o are calculated from the point at which the DNA lattice is half saturated (Kowalczykowski et al., 1986), a reasonable estimate of maximal ¯uorescence at saturation (Fmax) is needed to determine the fractional saturation of the DNA with protein. For some of the titrations at higher concentrations of salt, it was not possible to measure Fmax directly, since the binding curves did not reach a clear plateau, indicating that saturation of the eDNA with uvsY was not achieved. We therefore took advantage of the essentially hyperbolic nature of the titration curves, a consequence of the apparent non-cooperativity of binding, to perform extrapolations to Fmax via double reciprocal plots of the data near the endpoints of each curve (Figure 4){. The estimated Fmax values were then replotted in terms of fold-enhancement of eDNA ¯uorescence at saturation (Fmax/F0) as a function of salt concentration (Figure 5). The data in Figure 5 indicate that the majority of the salt effect on eDNA ¯uorescence enhancement occurs in the lower salt concentration range where uvsYeDNA binding is stoichiometric. The signi®cance of this effect is not yet understood; however, it is interesting to note that the salt concentration (200 mM NaCl) at which ¯uorescence enhancement approaches the expected twofold is equivalent to the salt concentration at which larger complexes of uvsY are dissociated into hexamers in solution (unpublished results). While there is no direct evidence for a correlation between these phenomena, the possibility of two different conformations of the uvsY-ssDNA complex related to different aggregation states of uvsY is intriguing. Once the maximal ¯uorescence signal for each salt concentration was estimated, the forward titrations were plotted in terms of fractional saturation (y) of eDNA as a function of uvsY concentration (Figure 6). The lack of sigmoidicity in these curves { The extrapolated Fmax values were used as initial estimates only, since the binding curves may not be truly hyperbolic. Some Fmax values were adjusted in order to optimize ®ts between experimental data and theoretical binding isotherms. See the text.

931

Figure 4. Double reciprocal plots of the ®nal eight datum points of the curves in Figure 3 were constructed for the experiments conducted at salt concentrations in which eDNA saturation was not achieved. Extrapolation of these plots to the vertical axis yields the reciprocal of the estimated maximum ¯uorescence at that salt concentration. a, Double-reciprocal plots derived from titrations performed in Figure 3a, representing NaCl concentrations of 300 mM (*), 350 mM (*), 400 mM (&), 450 mM ( & ), and 500 mM (~) respectively. b, Doublereciprocal plots derived from titrations performed in Figure 3b, representing KOAc concentrations of 400 mM, 500 mM (*), 600 mM (&), 700 mM ( & ), and 750 mM (~). See the text for details.

indicates that binding is essentially non-cooperative, thus o can be set equal to 1. With the values for n and o established, equation (4) of Kowalczykowski et al. (1986) allows the calculation of K. The calculated values of K at different salt concen-

932

Quantitative Analysis of uvsY-ssDNA Interactions

Figure 5. The effect of salt concentration on maximal ¯uorescence (Fmax) of the uvsY-eDNA complex. Experimental and extrapolated Fmax values are shown here replotted in terms of fold enhancement of ¯uorescence (Fmax/F0) as a function of salt concentration in both chloride (*), and acetate (*) buffer/salt systems. The Fmax values at lower salt concentrations (from the curves in Figure 3 that reached saturation) were taken from experimentally measured values. The Fmax values from the titrations in Figure 3 that did not reach saturation were derived from extrapolation of the double reciprocal plots shown in Figure 4.

trations are presented in Table 1. These K values were inserted into the McGhee-von Hippel equation for non-cooperative binding systems (equation (1) of Kowalczykowski et al., 1986) and used to generate theoretical binding isotherms. These theoretical isotherms are shown in comparison with experimental data as continuous lines in Figure 6. Reasonable ®ts of experimental data to the theoretical isotherms were obtained in all but two cases; only the 500 mM NaCl and 600 mM KOAc data sets displayed signi®cant deviations from the theoretical values. It was assumed that these deviations were due to errors in estimating the Fmax values for these data sets, leading to subsequent errors in the determination of K. For each of these two data sets, the value of K was adjusted until a reasonable theoretical ®t to the data was obtained, then Fmax was recalculated from the new K value via equation (1) of Kowalczykowski et al. (1986). The 500 mM NaCl and 600 mM KOAc data in Figure 6 were adjusted according to the new Fmax values{. The values of K presented here (Table 1) include both observed and Mg2‡-corrected values. The latter were calculated as de{ It should be noted that as a result of these manipulations, there is a slight variation in these two data sets as presented in Figure 6 from what would be obtained from replotting the data from Figure 3 in terms of y versus [uvsY] using the Fmax values obtained from Figures 4 and 5.

Figure 6. The data presented in Figure 3 are replotted here in terms of fractional saturation of the lattice (y), using the relative enhancement values presented in Figure 5 as representing 100% saturation. a, Titrations in the chloride buffer system with NaCl concentration equal to 300 mM (*), 350 mM (*), 400 mM (&), 450 mM ( & ), and 500 mM (~) respectively. b, Titrations in the acetate buffer system with KOAc concentration equal to 400 mM (*), 500 mM (*), 600 mM (&), 700 mM ( & ), and 750 mM (~), respectively. Continuous lines represent theoretical isotherms generated from the McGhee-von Hipple equation for non-cooperative binding systems, using the observed K values (not Mg2‡corrected) shown in Table 1. The values of the n and o parameters were set at 4 and 1, respectively.

scribed by Menetski & Kowalczykowski (1985); however, the corrections are minor due to the low Mg2‡ concentration (1 mM) and relatively high M‡ concentrations employed in our experiments. The af®nity data (Table 1) indicate a high degree of dependency on salt concentration for the ssDNAbinding activity of the uvsY protein, as a 200 mM

933

Quantitative Analysis of uvsY-ssDNA Interactions Table 1. The values of the af®nity constant, K, for uvsY-eDNA interactions at different salt concentrations, as calculated from the binding data shown in Figure 6 and from equations derived for the non-sequence-speci®c binding of a protein to an in®nite polynucleotide lattice (Kowalczykowski et al., 1986) [salt] (mM) 300 350 400 450 500 600 700 750

Kobserveda (Mÿ1) NaCl KOAc 3.00  106 5.83  105 2.59  105 1.35  105 9.50  104 ND ND ND

S ND 9.30  106 ND 1.35  106 5.64  105 2.94  105 1.70  105

KMg-correctedb (Mÿ1) NaCl KOAc 3.22  106 6.16  105 2.71  105 1.40  105 9.78  104 ND ND ND

S ND 9.72  106 ND 1.39  106 5.76  105 2.99  105 1.73  105

A binding site size parameter of n ˆ 4 was used based on results obtained in Figure 1. Since the curves in Figure 6 show no sigmoidicity, the binding is assumed to be non-cooperative (o ˆ 1). Under these conditions, K ˆ (1 ‡ 1/n)n ÿ 1/n L0.5, where L0.5 is the molar concentration of unbound uvsY at half-saturation (y ˆ 0.5; Figure 6). S, stoichiometric binding; ND, not determined. a Observed af®nity constant at the given salt concentration, before correction for Mg2‡ effects. b Af®nity constants corrected for Mg2‡ effects as described in the text. These values are plotted in Figure 7.

increase in NaCl or KOAc concentration leads to a greater than tenfold reduction in K. This dependency of binding af®nity on salt concentration is indicative of a high degree of electrostatic stabilization of uvsY-ssDNA complexes. Quantification of salt effects on K The number of ionic interactions involved in uvsYeDNA binding can be estimated from a log-log plot of K versus [salt] (Record et al., 1976, 1977; deHaseth et al., 1977; Menetski & Kowalczykowski, 1985), which has a slope described by the equation:

lattice, eDNA. The etheno-modi®cation of a polynucleotide frequently alters the af®nity of a protein for that polynucleotide, as has been established for the T4 gp32 and E. coli recA proteins, which bind with higher af®nity to the etheno form (Newport et al., 1981; Kowalczykowski et al., 1981; Silver & Fersht, 1982; Menetski & Kowalczykowski, 1985), and for the S. cerevisiae RP-A and T4 gp59 proteins, which bind with higher af®nity to the unmodi®ed form (Alani et al., 1992; unpublished results). In order to determine the relative af®nity of uvsY for unmodi®ed ssDNA with respect to eDNA, compe-

d log K=d log‰saltŠ ˆ ÿm0 c ÿ a where m0 is the number of ionic contacts formed between the bound protein and the DNA phosphate groups, c is the fraction of ions thermodynamically bound to DNA (equal to 0.71 for ssDNA: Record et al., 1976; Menetski & Kowalczykowski, 1985), and a is the number of anions displaced through binding. The slopes of the log-log plots of the data obtained from the NaCl and KOAc experiments are ÿ6.80 and ÿ6.10, respectively (Figure 7). If we assume that the maximum possible value for m0 is 4 (since n ˆ 4) and that no anions are displaced (e.g. a ˆ 0), then theoretically the greatest magnitude that d log K/ d log[salt] can have is ÿ2.84. This dictates that the minimum value for a is 3, and suggests that the release of at least three anions accompanies uvsYeDNA binding. The notion that anion release plays a role in the uvsY-eDNA interaction is consistent with the greater stability of uvsY-eDNA in the presence of acetate versus chloride, as observed in Figure 2 (von Hipple & Schleich, 1969). Binding of uvsY to unmodified M13mp19 ssDNA The results presented thus far deal with the interaction of uvsY with an etheno-modi®ed ssDNA

Figure 7. A log-log plot of K (Mg2‡-corrected, from Table 1) versus salt concentration allows quanti®cation of electrostatic effects in the interaction of uvsY with eDNA. The slopes of these curves for data obtained in the chloride/NaCl (*) and acetate/KOAc ( & ) buffer/ salt systems are evaluated in terms of electrostatic parameters m0 and a as described in the text. The best-®t (least-squares) lines to these data give slopes of d logK/ d log[NaCl] ˆ ÿ6.8 for the NaCl data and d logK/ d log[KOAc] ˆ ÿ6.1 for the KOAc data. See the text for details.

934 tition titrations were performed. In these experiments, forward titrations were carried out under the stoichiometric binding conditions described for Figure 1, and binding to eDNA with or without equimolar amounts of native DNA present in the reaction was monitored. Analysis of these data (Figure 8a) suggests that since there is little differ-

Quantitative Analysis of uvsY-ssDNA Interactions

ence in the lower portions of these curves (e.g. below 50% saturation of eDNA with uvsY protein), the af®nity of uvsY is greater for eDNA than for unmodi®ed M13mp19 ssDNA. In an effort to produce quantitative data for the difference in af®nities of uvsY for the two lattices, the data were treated in the following manner. It was assumed that under these conditions binding is stoichiometric to both lattices; thus, prior to saturation the concentration of free uvsY is negligible. Construction of fractional saturation (y) curves for each substrate is possible by taking the difference in the amount of uvsY required to reach a given value of y in each of the curves. This difference is then used to calculate the degree of saturation of unmodi®ed ssDNA (Figure 8b), assuming random, noncooperative binding and equal binding site size on both lattices. Approximately twofold more uvsY protein is required to reach the half-saturation point with unmodi®ed ssDNA than with eDNA (Figure 8b); this translates into an approximately sixfold higher af®nity for the etheno-modi®ed lattice under these conditions, based on apparent K values calculated from equation (4) of Kowalczykowski et al. (1986).

Discussion

Figure 8. Competition titration to estimate the relative af®nity of uvsY for unmodi®ed ssDNA versus eDNA. a, Forward titration of uvsY into a solution containing either 7.5 mM eDNA alone (*) or 7.5 mM eDNA plus 7.5 mM unmodi®ed M13mp19 ssDNA (*), both in a buffer containing 20 mM Tris-HCl (pH 7.4), 1 mM MgCl2, and 50 mM NaCl. The data were corrected for dilution, protein ¯uorescence, and inner ®lter effects as described in Materials and Methods. b, The data from the mixed lattice (competition) experiment in a was replotted in terms of fractional saturation (y) of eDNA (*) and unmodi®ed ssDNA (*) as described in the text.

Our major conclusions are the following. (1) The uvsY protein binds to an ``in®nite'' eDNA lattice with a binding site size of four nucleotides per monomer of uvsY. (2) The degree of enhancement of eDNA ¯uorescence upon uvsY binding is dependent on salt concentration, with an unusually large enhancement observed at lower salt concentrations. (3) uvsY binds to eDNA in a non-cooperative manner (o ˆ 1). (4) uvsY-eDNA interactions are highly electrostatic in nature, with a strong salt effect on the K parameter observed, and with a minimum of three anions displaced per binding event. (5) uvsY binds with higher af®nity to an eDNA lattice than to an equivalent unmodi®ed ssDNA lattice. These ®ndings constitute the ®rst detailed biochemical characterization of uvsY interactions with a single-stranded polynucleotide. Many questions remain about the mechanisms utilized by bacteriophage T4 in the replication and recombination of its DNA. Clearly, protein complexes involved in DNA metabolism must compete effectively with E. coli proteins for the DNA substrates. One means used by the T4 phage to accomplish this is through the high af®nity and cooperativity of interactions between gp32, the T4encoded ssDNA-binding protein, and ssDNA. These binding properties allow gp32 to compete effectively with the abundant E. coli SSB protein for ssDNA-containing intermediates of phage DNA metabolism (Kodadek, 1990). The high af®nity and cooperativity of gp32 creates an additional problem for the phage, however, in that the enzymes essential for metabolic activity can be excluded from their DNA binding sites in the presence of sa-

Quantitative Analysis of uvsY-ssDNA Interactions

turating amounts of gp32. In at least two different instances, T4 overcomes this problem through the use of accessory factors. The gp59 protein acts as a loading factor for gp41, the T4 replicative DNA helicase, onto gp32-ssDNA complexes (Morrical et al., 1994, 1996; Barry & Alberts, 1994). Likewise, the uvsY protein appears to speci®cally load the uvsX recombinase onto gp32-covered ssDNA (Kodadek et al., 1989; Yonesaki & Minagawa, 1989; Harris & Grif®th, 1989; Morrical & Alberts, 1990; Jiang et al., 1993). In an effort to better understand how this loading process occurs, we have characterized the binding properties of uvsY towards ssDNA. It should be mentioned that the use of assembly factors for the loading of recombinases onto proteinssDNA matrices is not unique to the T4 phage. Recently, studies characterizing the recO/R proteins of E. coli have shown that this complex can overcome the inhibition of recA-ssDNA ®lament formation by SSB (Umezu et al., 1993). Further studies have shown that recO/R accomplishes this through interactions with SSB and ssDNA (Umezu & Kolodner, 1994). Similarly, the RAD52 protein of S. cerevisiae has been shown to interact with the RAD51 recombinase and RP-A, the yeast SSB, and these interactions have been proposed to play a similar role in that system (Firmenich et al., 1995). The results of our uvsY-eDNA binding studies raise several points of interest. First, the observation that the site size of uvsY binding is four nucleotides per protein monomer is identical with the value proposed for uvsX protein (Grif®th & Formosa, 1985). This provides further evidence to support the notion of a one-to-one binding stoichiometry between uvsY and uvsX in the mature presynaptic ®lament (Kodadek et al., 1989). The site size determined in our experiments differs slightly from the value of n ˆ 3 determined by Kodadek et al. (1989), and greatly from the value of n ˆ 15 determined by Yonesaki & Minagawa (1989). These differences are probably due to the different assay methods employed (see below). Second, the lack of cooperativity in the uvsYeDNA interaction suggests either that uvsY does not require large contiguous ®laments for its function, or that heterologous protein-protein interactions with gp32 and/or uvsX provide the required ®lament-forming properties. The observed lack of cooperativity in uvsY-eDNA binding is contrary to previously published results (Kodadek et al., 1989; Yonesaki & Minagawa, 1989). The differences could be due to variations in salt, buffer, temperature, and/or ssDNA species used in the three studies; however, it appears more likely { With the nitrocellulose ®lter-binding method, it is inherently dif®cult to accurately measure protein binding density on a long lattice where many nonsequence-speci®c interactions occur. This dif®culty may account for the anomolously large binding site size of n ˆ 15 nucleotides obtained by Yonesaki & Minagawa (1989) in ®lter-binding studies of uvsY interactions with large phage ssDNA molecules.

935 that the differences are due to the assay methods employed. The two earlier studies employed ®lterbinding assays to monitor the binding of uvsY to either oligonucleotide (Kodadek et al., 1989) or phage (Yonesaki & Minagawa, 1989) ssDNA molecules. The apparent cooperativity seen in these assays is limited to slight sigmoidicity through one or two experimental points early in the titrations. This effect could be explained by site exclusion and/or end effects in the experiments with oligonucleotides (see Kowalczykowski et al., 1986), or by the inability of the ®lter-binding method to accurately quantify the degree of saturation of the ssDNA molecules being retained on the ®lter (Kowalczykowski, 1990){. It is highly probable that the eDNA ¯uorescence enhancement assay we have employed gives a truer picture of the equilibrium binding properties of uvsY protein, since it allows direct measurements of fractional saturation of the lattice in solution. Third, the K parameter of uvsY for unmodi®ed ssDNA appears to be comparable with that of gp32, and perhaps even greater than that of gp32 at physiological ionic strengths. This conclusion is based on an extrapolation of the logK versus log[NaCl] plot shown in Figure 7 to 200 mM NaCl, and on the assumption that the af®nity of uvsY for unmodi®ed ssDNA is 6-fold greater than that for eDNA as estimated in Figure 8b. These manipulations result in an estimated K-value (Mg2‡-corrected) of 1  107 Mÿ1 for uvsY in 200 mM NaCl. In comparison, Ko for gp32-ssDNA interactions is reported to be 1  109 Mÿ1 at 200 mM NaCl (Newport et al., 1981), with o  1000 and therefore K  1  106 Mÿ1. This admittedly crude comparison nevertheless suggests that the uvsY protein may not be an intrinsically weaker ssDNAbinder than gp32, but that the greater binding energy of gp32 may result solely from its high cooperativity, which uvsY lacks. If true, this conclusion could have important implications for models of presynaptic ®lament formation in the T4 system. If the role of uvsY protein is to help uvsX protein displace or partially displace gp32 from ssDNA (Yonesaki & Minagawa, 1989; Morrical & Alberts, 1990; Hashimoto & Yonesaki, 1991; Jiang et al., 1993), then perhaps a disruption of gp32's cooperativity is all that is required for displacement, since uvsY has suf®cient intrinsic af®nity to compete with gp32 for binding sites. However, it appears likely that uvsY and gp32 bind to different surfaces of the ssDNA and do not compete directly for binding sites. Results of hydroxyl radical ®ngerprinting, DNase l protection, co-precipitation, and protein cross-linking experiments all support this notion (Jiang et al., 1993; Yonesaki & Minagawa, 1989; Hashimoto & Yonesaki, 1991; unpublished results), and a discrete uvsY-gp32ssDNA complex containing stoichiometric amounts of both proteins has been proposed as an early intermediate in presynaptic ®lament formation (Jiang et al., 1993; unpublished results). Presumably both

936 uvsY-ssDNA and uvsY-gp32 interactions play important roles in the formation of this complex. Other aspects of the data presented here are not readily interpreted. The signi®cance of the extensive enhancement of eDNA ¯urorescence observed with saturating amounts of uvsY at low salt concentrations is unclear. Also unexplained is the apparent ``metastability'' of the high-Fmax uvsY-eDNA complexes observed during salt-back titrations (see Figures 2 and 3, and the related text). There are, however, two indirect lines of evidence that suggest that these effects may be related to the polymeric state of uvsY upon binding. As mentioned previously, evidence exists that the uvsY protein exists as a hexamer at NaCl concentrations greater than 200 mM and as larger complexes at lower concentrations of salt (unpublished results). Also, our observation of apparent binding site exclusion under stoichiometric uvsY-eDNA binding conditions (Figure 1) is suggestive of a protein that possesses a large site size, indicating that uvsY does not bind as a monomer. The fact that the degree of enhancement of eDNA ¯uorescence is saltdependent and changes most dramatically over a salt concentration range in which uvsY hexamers appear to assemble into larger complexes suggests that the degree of eDNA ¯uorescence enhancement could be affected by the size of the uvsY binding unit. Efforts are underway to characterize the oligomeric structure of the uvsY ssDNA-binding unit.

Materials and Methods Reagents and buffers All chemicals used were reagent grade and aqueous solutions were made with deionized, glass-distilled water. All buffers used in ¯uorescence experiments were sterile®ltered through a 0.45 mm membrane. Chromatography buffers were as follows: for phosphocellulose chromatography, PC-100 and PC-1000 buffers contained 20 mM TrisHCl (pH 7.4), 1 mM EDTA, 5 mM b-mercaptoethanol, and 10% (w/v) glycerol plus either 100 or 1000 mM NaCl, respectively. For ssDNA-cellulose chromatography, DC100, DC-200, DC-600 and DC-2000 contained 20 mM TrisHCl (pH 8.1), 5 mM EDTA, 5 mM b-mercaptoethanol, and 10% (w/v) glycerol, plus either 100, 200, 600, or 2000 mM NaCl, respectively. For hydroxyapatite chromatography, HAP-100 buffer contained 100 mM potassium phosphate (pH 6.8), 5 mM b-mercaptoethanol, and 10% (w/v) glycerol, while HAP-900 contained 900 mM potassium phosphate (pH 6.8), 5 mM b-mercaptoethanol, and 10% (w/v) glycerol. uvsY storage buffer contains 20 mM Tris-HCl (pH 7.4), 0.2 mM EDTA, 1 mM b-mercaptoethanol, 100 mM NaCl, and 60% (w/v) glycerol. Buffers used in ¯uorescence experiments included a chloride buffer system (20 mM Tris-HCl (pH 7.4), 1 mM MgCl2, plus variable NaCl) and an acetate buffer system (20 mM Tris-acetate (pH 7.4), 1 mM MgOAc2, plus variable KOAc). Proteins and nucleic acids uvsY protein was overexpressed by temperature induction in an E. coli strain harboring plasmid pTL251W,

Quantitative Analysis of uvsY-ssDNA Interactions which was a generous gift from Dr T. C. Lin of Yale University. Cell induction, lysis, and protein puri®cation steps represent a slight modi®cation of the procedures described by Kodadek et al. (1989). Brie¯y, uvsY puri®cation from a high-speed supernatant was performed at 4 C as follows: the lysate was exhaustively dialyzed against PC-100 and loaded onto a phsophocellulose column equilibrated in the same buffer. The column was washed with PC-100 then eluted with a 15 column volume gradient from PC-100 to PC-1000 (100 to 1000 mM NaCl). The uvsY protein eluted with a peak at approximately 400 mM NaCl. Fractions were analyzed by staining of SDS/polyacrylamide gels with Coomassie brilliant blue, and uvsY-containing fractions were pooled and dialyzed into DC-100 buffer. The resulting dialysate was loaded onto a ssDNA-cellulose column (prepared as described by Alberts & Herrick, 1971) equilibrated in DC100, which was then washed with DC-100 and subsequently step-eluted with DC-200, DC-600 and DC2000. The DC-600 (600 mM NaCl) fraction contained the uvsY protein. This fraction was loaded directly onto a hydroxyapatite column equilibrated in HAP-100 buffer. After washing with HAP-100, the column was gradienteluted from HAP-100 to HAP-900 (100 to 900 mM potassium phosphate) over ten column volumes. The uvsY protein eluted with a peak at approximately 750 mM potassium phosphate. Analysis of SDS/polyacrylamide gels stained with Coomassie brilliant blue revealed a single band corresponding to the uvsY protein. Purity was estimated at >98%. The protein was dialyzed into uvsY storage buffer and stored at ÿ20 C. The puri®ed uvsY protein was tested for nuclease contamination by incubating protein samples (sub-saturating amounts with respect to DNA) with supercoiled dsDNA, with linear dsDNA, and/or with both circular and linear ssDNA species. The DNA molecules were examined for nucleolytic degradation by electrophoresis in agarose gels. All uvsY stocks used in this study were judged to be nuclease-free by these criteria. The concentrations of uvsY stocks were determined by the absorbance at 280 nm, using an extinction coef®cient at 280 nm of 0.83 mg/ml per absorbance unit for uvsY. This extinction coef®cient was calculated from the amino acid sequence of uvsY using the method of Gill & von Hippel (1989). Circular single-stranded DNA from the bacteriophage M13mp19 was isolated from puri®ed phage particles as described by Yamamoto et al. (1970) and Miller (1987). The M13mp19 ssDNA concentration, in nucleotides, was determined by phosphate analysis as described by Ames (1966). Brie¯y, a small portion of the sample was combined with a drop of magnesium nitrate solution and taken to dryness over a ¯ame. The ashed phosphates were mixed with HCl to hydrolyze any pyrophosphate formed. The solution was then mixed with an ascorbic acid solution and an absorbance at 820 nm was taken, using a conversion factor of 0.260 A820 unit ˆ 0.01 micromole of nucleotides. A portion of the M13mp19 ssDNA was used to make etheno-DNA (eDNA) by modi®cation with chloracetaldehyde (obtained from Aldrich), according to the method of Menetski & Kowalczykowski (1985). The integrity of the eDNA was veri®ed by agarose gel electrophoresis. The eDNA had a slightly decreased electrophoretic mobility compared with unmodi®ed M13mp19 ssDNA, consistent with previous reports (Menetski & Kowalczykowski, 1985), and no fragmentation of the eDNA was observed. The concentration of eDNA (in nucleotides) was determined by the Ames (1966) method as described above for the unmodi®ed ssDNA.

937

Quantitative Analysis of uvsY-ssDNA Interactions e DNA fluorescence titrations All ¯uorescence data were gathered on an SLM model 8000 spectro¯uorimeter. All titrations were performed at a constant temperature of 25 C, maintained by a circulating waterbath. All experiments had a starting volume of 2.0 ml prior to the addition of titrant. For salt-back titrations (salt added incrementally to preformed uvsYeDNA complexes; Kowalczykowski et al., 1986), titrant consisted of concentrated NaCl or KOAc. For forward titrations (uvsY added incrementally to eDNA) performed at different salt concentrations, the titrant consisted of uvsY protein in storage buffer supplemented with NaCl or KOAc such that total salt concentration did not change during the titration. For all titrations, the total volume of titrant added was between 50 and 200 ml depending on the experiment. Titrant was added in 5 to 9 ml aliquots at one minute intervals with constant stirring. The excitation and emission wavelengths for eDNA were 300 and 405 nm, respectively; bandpass was 5 nm. All datum points were obtained as an average of ®ve to ten readings of the ¯uorimeter, set on continuous mode. Corrections of ¯uorescence data were made for dilution, protein ¯uorescence, and inner ®lter effects. Corrections for protein ¯uorescence were made by titrating the uvsY protein into a mock reaction containing no DNA; ¯uorescence was monitored as described above. The resulting data were corrected for dilution then subtracted from the experimental data. Corrections for inner ®lter effects were made according to the method of Birdsall et al. (1983). No correction for photobleaching was made, since the exposure of eDNA to the excitation beam for extended periods of time resulted in negligible decreases in ¯uorescence. As a general precaution against photobleaching, samples were exposed to the beam only during data acquisition. Controls for possible salt-dependent variations in protein ¯uorescence and/or light-scattering were carried out as follows. First, forward titrations were performed as described above while exciting at 300 nm and monitoring emission at 405 nm, except that unmodi®ed M13mp19 ssDNA was used in place of eDNA as the lattice for uvsY binding. These titrations were performed at multiple salt concentrations in both the chloride and acetate buffer systems; no signi®cant difference in uvsY protein ¯uorescence was observed between the various salt conditions. These controls rule out the possibility of salt-dependent changes in protein ¯uorescence affecting ¯uorescence readings in the eDNA ¯uorescence titrations. Second, light-scattering controls were conducted by repeating the above uvsY/unmodi®ed ssDNA titrations at various salt concentrations while monitoring 90 scattering at 405 nm (excitation and emission) or at 300 nm (excitation and emission). Bandpass, photomultiplier, and other machine settings were identical with those used in uvsY-eDNA ¯uorescence titrations. In all cases, the light-scattering signals obtained were less than 10% of the protein ¯uorescence signals from identical samples, and less than 4% of the uvsY-eDNA ¯uorescence signals obtained from equivalent samples after all corrections were made. Furthermore, no signi®cant difference in scattering intensity was observed between samples at different salt concentrations. These controls demonstrate that sample light-scattering at both the emission and excitation wavelengths is negligible compared to sample ¯uorescence intensities, and rule out the possibility that salt-dependent changes in light-scattering affect ¯uorescence readings in the eDNA ¯uorescence titrations. As a general precaution against light-scattering

effects, all samples were inspected before and after titrations for visible turbidity; no visible turbidity was noted under any salt condition.

Acknowledgements We thank David S. Yassa and Yu Jie Ma for their technical assistance. We thank Dr Stephen C. Kowalczykowski for his helpful comments and advice, and for a critical reading of the manuscript prior to its submission. We thank Dr Tom R. Tritton for the use of his ¯uorimeter. This work was supported by research grant GM48847 from the National Institutes of Health. M.A.S. was supported in part by a Minority Graduate Research Fellowship from the National Science Foundation.

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Edited by P. E. Wright (Received 4 October 1996; received in revised form 22 November 1996; accepted 26 November 1996)