Structural Features of phi 29 Single-stranded DNA-binding Protein
II. GLOBAL CONFORMATION OF phi 29 SINGLE-STRANDED DNA-BINDING PROTEIN AND THE EFFECTS OF COMPLEX FORMATION ON THE PROTEIN AND THE SINGLE-STRANDED DNA*

(Received for publication, April 2, 1996, and in revised form, October 2, 1996)

María S. Soengas Dagger §, C. Reyes Mateo , Germán Rivas par , Margarita Salas Dagger **, A. Ulises Acuña and Crisanto Gutiérrez Dagger

From the Dagger  Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM), Cantoblanco, 28049 Madrid, Spain,  Instituto de Química Física Rocasolano (CSIC), Serrano 119, 28006, Madrid, Spain, and par  Centro de Investigaciones Biológicas (CSIC), Velázquez 144, 28006 Madrid, Spain

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

The strand-displacement mechanism of Bacillus subtilis phage phi 29 DNA replication occurs through replicative intermediates with high amounts of single-stranded DNA (ssDNA). These ssDNA must be covered by the viral ssDNA-binding protein, phi 29 SSB, to be replicated in vivo. To understand the characteristics of phi 29 SSB-ssDNA complex that could explain the requirement of phi 29 SSB, we have (i) determined the hydrodynamic behavior of phi 29 SSB in solution and (ii) monitored the effect of complex formation on phi 29 SSB and ssDNA secondary structure. Based on its translational frictional coefficient (3.5 ± 0.1) × 108 gs-1, and its rotational correlation time, 7.0 ± 0.5 ns, phi 29 SSB was modeled as a nearly spherical ellipsoid of revolution. The axial ratio (p = a/b) could range from 0.8 to 1.0 (oblate model, a < b) or 1.0 to 3.2 (prolate model, a > b). Far-UV CD spectra, indicated that phi 29 SSB is highly organized within a wide range of temperatures (15 to 50 °C), being mainly constituted by beta -sheet elements (~50%, at pH 7). Complex formation with ssDNA, although inducing minimal changes on the global conformation of phi 29 SSB, had a clear stabilizing effect against pH and temperature increase of the solution samples. On the other hand, phi 29 SSB binding leads to non-conservative changes of the near-UV CD spectra of ssDNA, which are consistent with different nearest-neighbor interactions of the nucleotide bases upon complex formation. The above results will be compared to those reported for other SSBs and discussed in terms of the functional roles of phi 29 SSB.


INTRODUCTION

In all organisms studied so far, key cellular processes related to DNA metabolism, such as DNA replication, DNA repair, DNA recombination, or DNA transcription, occur via transient ssDNA1 intermediates whose structure must be properly organized. This organization is achieved and maintained by highly efficient, localized (sequence-specific), or generalized (sequence-independent) protein contacts. Noncatalytic proteins that bind ssDNA with a relatively high affinity in a sequence independent manner have been referred to as single-stranded DNA-binding proteins, SSBs, (1, 2, 3, 4). One of the most striking features of the SSBs is the lack of any clear structural common characteristics among them. Thus, the oligomeric state of these proteins in solution is highly variable. They have being found as monomers, e.g. N4 SSB (5); phi 29 SSB (6); dimers, e.g. SSBs from filamentous Fd phages (3); T7 gene 2.5 (7); tetramers, e.g. Escherichia coli SSB (8); multioligomers, T4 gp32 (1); or hetero-oligomers, e.g. human SSB (9) and Drosophila SSB (10). Moreover, although several attempts to find structural motifs in SSBs have been made (11, 12), only among SSBs of very related filamentous phages a limited sequence homology has been found within the so-called DNA binding wing (see Ref. 3, for review). NMR and crystallographic studies showed a similar three-dimensional structure of the SSBs of E. coli and Pseudomonas aeruginosa filamentous phages Ff and Pf3 (13, 14, 15), rather different from that of T4 gp32, the other SSB2 whose structure (x-ray difraction) has been reported (18). In addition, detailed analysis of a few SSB-ssDNA complexes indicate that each protein interacts with ssDNA using a particular set of residues (3, 15, 19, 20, 21) and induces different conformational changes in the DNA structure (see Refs. 2, 4, and 8, for reviews). The functional characteristics of the SSBs complicate even more this variability. Most of the SSBs activate DNA replication, e.g. E. coli SSB, T4 gp32, T7 gene 2.5 protein, phi 29 SSB, and human SSB (reviewed in Ref. 4). However, other SSBs, as those of the filamentous phages, block DNA replication (22). As a consequence of this diversity, the molecular nature of the SSB-ssDNA interactions is not yet well understood.

The requirement for a SSB is particularly important during strand-displacement DNA replication, where great amounts of ssDNA are produced. This ssDNA corresponds to the strand that is not used as template and is displaced as the DNA polymerase progresses. One of the best characterized strand-displacement DNA replication systems is that of Bacillus subtilis phage phi 29 (see Refs. 23, 24, 25, for reviews). In vivo, phi 29 DNA replication cannot procced in the absence of the viral product of gene 5, the viral SSB (12, 26). phi 29 SSB is an extremely abundant protein in infected cells (~2.5 × 106 copies/cell), representing ~3% of the total protein content (26). In vitro, phi 29 SSB stimulates DNA replication, at least at two levels: (i) by increasing the amount of dNTP incorporated (12, 26) and (ii) by increasing the DNA elongation velocity of phi 29 DNA polymerase under conditions in which the DNA opening is impaired (27). This activity has been proposed to be related to the helix-destabilizing properties of phi 29 SSB (27). phi 29 SSB becomes essential for in vitro viral DNA replication when the amount of DNA is reduced (28), the closest condition to the initial steps of the viral infection.

phi 29 SSB binds to ssDNA in a cooperative way (Keff = 105 M-1, omega  = 50-70) covering 3-4 nucleotides per monomer, likely as a single array of protein units along the ssDNA (6). When visualized by electron microscopy, the phi 29 SSB-DNA complex shows a continuous and homogeneous structure, and nucleosome-like structures were not apparent (6, 12). Protein binding induces a 50% reduction in ssDNA length (29) and a 95% reduction of the intrinsic tyrosine fluorescence emission of the protein (6). This observation has been the starting point for the analysis of phi 29 SSB ssDNA binding domain as described in the accompanying paper (30).

The functional requirement of phi 29 SSB during the DNA replication stage of phi 29 infective cycle might be related to the structural properties of phi 29 SSB that would lead to a particular organization of complex with the ssDNA of the replicative intermediates. In this paper, we have analyzed the global conformation of phi 29 SSB in solution by analytical ultracentrifugation, steady-state and time-resolved fluorescence anisotropy measurements. Conformational changes in the protein and ssDNA structure induced upon complex formation were studied by means of circular dichroism techniques. The results of these studies indicate that phi 29 SSB differs markedly from other well known SSBs.


MATERIALS AND METHODS

Buffers, Reagents, and Proteins

All the solutions were made with distilled, deionized (MilliQ) and sterilized water, and finally filtered through 0.45-µm filters using a Millex-HA Filter Unit (Millipore). The buffer used in analytical ultracentrifugation, time-resolved fluorescence, and circular dichroism (CD) analysis was 5 mM Tris-HCl, pH 7.5 (20 °C) (buffer A), while the buffer used in steady state fluorescence studies was 50 mM Tris-HCl, pH 7.5 (20 °C), 4% glycerol (buffer B). M13mp18 ssDNA was from Boehringer Mannheim and oligonucleotides dT20, dA16, and polynucleotides poly(dT300) and poly(dA300) from Pharmacia Biotech Inc. Nucleotide concentrations were quantified as indicated by the supplier. phi 29 SSB was purified by differential ammonium sulfate fractionation of phi 29-infected cellular extracts and further phosphocelulose and DEAE-cellulose chromatography, essentially as described (27). Protein concentration was estimated by the Lowry method (31), using bovine serum albumin as a standard.

Analytical Ultracentrifugation

Analytical ultracentrifugation experiments were carried out in an Optima XL-A analytical ultracentrifuge (Beckman Instruments Inc.) equipped with a UV-VIS optical scanner, using a Ti60 rotor and double sector 12-mm centerpieces of epon charcoal. In sedimentation equilibrium measurements, samples of phi 29 SSB ranging from 10 to 30 µM were centrifuged in buffer A at 20 °C and at two speeds, 25,000 and 30,000 rpm. When sedimentation equilibrium was reached, absorbance scans (at 230 nm) were taken as a function of radial distance. The base-line offsets were determined by scanning the samples at a higher speed (42,000 rpm). To obtain the relative molecular weight (Mr) of phi 29 SSB, the experimental data were fitted to the equation which describes the radial distribution of concentration of an ideal solute at sedimentation equilibrium (see, for example, Ref. 32) using the programs XLAEQ and EQASSOC (supplied by Beckman; see Ref. 33). The value of the partial specific volume of phi 29 SSB was estimated from its amino acid composition (34) as 0.739 ml g-1. In velocity sedimentation experiments, phi 29 SSB (ranging in concentration from 10 to 30 µM) was centrifuged at 60,000 rpm and 20 °C and radial scans at 230 nm were taken at 10-min intervals during 3 h. The analysis of the experimental data was performed in two ways: (i) by taking the derivative of the absorbance data to calculate the peak of the moving boundary (XLAVEL program, supplied by Beckman), and (ii) by using the second moment analysis of Goldberg (35) to locate the boundary position (VELGAMMA program, Beckman). The rate of the motion of the boundary was used for the estimation of the sedimentation coefficient. This value was corrected to standard conditions (water, 20 °C; 33) to obtain the s20,w values.

The translational frictional coefficient, f, of phi 29 SSB was obtained by the classical Svedberg equation (see Ref. 36 for review),
f=<FR><NU>M<SUB><UP>r</UP></SUB>(1−<OVL>v</OVL>&rgr;)</NU><DE>N<SUB>A</SUB>s</DE></FR> (Eq. 1)
where Mr stands for the molecular weight of the protein obtained for equilibrium ultracentrifugation, rho  is the solvent density, and NA is the Avogadro's constant.

Hydrodynamic Analysis of the Size and Shape of phi 29 SSB

The global conformation of phi 29 SSB in solution was modeled with rigid ellipsoids of revolution (the axis being a, b = c). Two models were considered, the prolate ellipsoid, in which a b, and the oblate ellipsoid, characterized by a < b. In both cases, the rotational parameters of the protein are related to the hydrated protein volume V and asymmetry (given by the axial ratio p = a/b) in the following way,
V=<FR><NU>KT&phgr;<SUB><UP>global</UP></SUB></NU><DE>6&eegr;</DE></FR><FENCE><FR><NU>4</NU><DE>g<SUB><UP>perp</UP></SUB></DE></FR>+<FR><NU>2</NU><DE>g<SUB><UP>par</UP></SUB></DE></FR></FENCE> (Eq. 2)
where phi global stands for the rotational correlation time of the decay of the anisotropy assigned to the global motion of the entire protein (37, 38, 39), eta  is the solution viscosity, and gpar and gperp are rotational Perrin factors depending only on p (40, 41). The gpar and gperp values were calculated as,
g<SUB><UP>par</UP></SUB>=2(p<SUP>2</SUP>−1)/3p(p−s) (Eq. 3)
g<SUB><UP>perp</UP></SUB>=2(p<SUP>4</SUP>−1)/3p[(2p<SUP>2</SUP>−1)s−p] (Eq. 4)
where,
s=(p<SUP>2</SUP>−1)<SUP>−1/2</SUP><UP>ln</UP>[p+(p<SUP>2</SUP>−1)<SUP>1/2</SUP>] (Eq. 5)
for the prolate ellipsoid model, and,
s=(q<SUP>2</SUP>−1)<SUP><UP>−1/2</UP></SUP><UP>ATN</UP>(q<SUP>2</SUP>−1)<SUP>1/2</SUP> (<UP>where </UP>q=1/p) (Eq. 6)
for the oblate ellipsoid model.

The experimentally determined value of phi global (7.0 ± 0.5 ns; see Ref. 30) was introduced for axial ratio ranging from 0.1 to 1 (oblate model) and 1 to 10 (prolate model), in Equation 2 to obtain the V values of the set of ellipsoids compatible with the experimental rotational parameters. The theoretical translational frictional coefficient, ftheo corresponding to each of these ellipsoids was obtained as 6pi eta RsphF. Where Rsph stands for the radius of a sphere with the same volume V as the ellipsoid, and F is the translational Perrin factor whose value also depend on p (41). The F values for the prolate and oblate ellipsoids, Fobl and Fpro, respectively, where calculated as,
F<SUB><UP>obl</UP></SUB>=<FR><NU>(1−q<SUP>2</SUP>)<SUP>1/2</SUP></NU><DE>q<SUP>2/3</SUP><UP>ln</UP>{[1+(1−q<SUP>2</SUP>)<SUP>1/2</SUP>]/q}</DE></FR> (Eq. 7)
F<SUB><UP>pro</UP></SUB>=<FR><NU>(q<SUP>2</SUP>−1)<SUP>1/2</SUP></NU><DE>q<SUP>2/3</SUP><UP>ATN</UP>[(1−q<SUP>2</SUP>)<SUP>1/2</SUP>]</DE></FR> (Eq. 8)
where q = 1/p.

The comparison of the experimentally determined fobs and the estimated ftheo provides the range of ellipsoids compatible with the translational and rotational hydrodynamic behavior of phi 29 SSB. The hydration extent of the protein, delta  (gram of H2O/g of protein) was estimated as delta  = <OVL>&ngr;</OVL>(V - Vanh)/Vanh, where Vanh (estimated from Ranh = 0.723 × 10-8 Mr(null)/1;3; see Ref. 36 for further details) corresponds to the anhydrous volume of a sphere whose Mr is that of phi 29 SSB.

Circular Dichroism

CD spectra were recorded on a Jasco Model J-700, at 0.2-nm intervals and, unless otherwise stated, at 25 °C and at a scan speed of 20 nm/min. The absorbance of the samples was kept below 0.1 over the measuring range. phi 29 SSB (6.5 µM) CD spectra were obtained with 0.1-cm cells in the absence and presence of an excess of ssDNA (350 µM nucleotide). DNA binding was tested by parallel measurements of the quenching of the intrinsic protein fluorescence upon DNA addition as described previously (6). When required, small amounts of 2.5 M NaOH from a concentrated stock were added to the indicated amount of free or bound phi 29 SSB (the volume change at the end of the titration was <1%), the pH being simultaneously measured. The effect of temperature on the protein conformational stability was analyzed by recording the spectra after maintaining the protein or protein-ssDNA solution for 5 min at the different temperatures. Controls were made to ensure that the variations in the pH due to temperature change did not affect the far-UV CD spectra of the protein. Plotted CD data, as mean residue ellipticity, corresponded to an average of three scans. Corrections for the contribution of the buffer and DNA to the optical activity were made when necessary. The relative amount of alpha -helix, beta -sheet, and beta -turns of phi 29 SSB where estimated with the CONTIN (version 2) program (42) by fitting the phi 29 SSB spectra, between 190 and 240 nm, to those of 16 proteins of known secondary structure (43) as outlined by Provencher and Glöckner (44). Alternatively, convex constraint analysis, as described by the Fasman group (45, 46), were also made, but the best fits were not so accurate. ssDNA spectra (dT20, dA16, poly(dT), or poly(dA)) were recorded in buffer A, with 1-cm path length cells. Five scans were averaged and the data were plotted as epsilon L - epsilon R (Delta epsilon ) per mol of nucleotide. Stacking interactions between neighboring nucleotides were analyzed by increasing the temperature of the solution. Spectra were taken after keeping ssDNA samples 5 min at the desired temperature. To analyze the effect of phi 29 SSB binding on the ssDNA structure, spectra were recorded after adding small aliquots of a concentrated protein stock (~7 mg/ml) to a fixed amount of the indicated ssDNA (~110 µM nucleotide). Buffer and protein optical activity were substracted to obtain the corrected ssDNA spectra. Saturation was considered to be reached when further protein addition did not induce any change in CD spectra. The nucleotide site size, that is, the number of nucleotides covered by a protein monomer, was calculated from the ratio DNA/SSB that induced the maximal change at wavelengths corresponding to the maximal activity of free ssDNA.

Fluorescence Spectroscopy

Steady-state measurements of the intrinsic fluorescence of phi 29 SSB were performed at 25 °C in a SLM-8000D photon counting L-format fluorimeter fitted with Glan-Thompson polarizers at lambda exc = 276 nm and lambda em = 308 nm (slits set to 2 and 4 nm, respectively; Ref. 47). The anisotropy, <r>, of phi 29 SSB fluoresence emission was obtained as described in the accompanying paper (30). Complex formation with ssDNA was monitored by measuring the increase in steady-state anisotropy together with the quenching of the fluorescence emission at magic angle (I54) and during reverse titrations of a fixed amount of phi 29 SSB (10 µM) with increasing quantities of ssDNA. Corrections for dilution and inner filter effect due to ssDNA absorption were made when necessary as described previously (6).


RESULTS

Structural Features of Free and Bound phi 29 SSB

The Hydrodynamic Parameters of phi 29 SSB Are Consistent with a Monomeric Globular Conformation

The solution conformation of phi 29 SSB was approached by analyzing hydrodynamic parameters directly dependent on the protein aggregation state, size, and shape. The initial step in the modeling process was the estimation, by analytical ultracentrifugation techniques (sedimentation equilibrium and sedimentation velocity), of the molecular weight and the sedimentation and frictional coefficients of phi 29 SSB. Table I summarizes the results obtained.

Table I.

Hydrodynamic parameters of phi 29 SSB


Mr
  Sequence 13,343
  Sedim. equilibrium 13,600  ± 300
Ranha 16 Å 
Vanh 17 × 103 Å3
 <OVL><IT>v</IT></OVL>b 0.739 ml g-1
D20,w (1.18 ± 0.07) × 10-6 cm2 s-1
s20,w  (1.7 ± 0.1) × 10-13 s
fobs (3.50 ± 0.10) × 10-8 g s-1
 phi globalc        (7.0 ± 0.5) × 10-9 s
Oblate model (a < b)d
  p 0.9  ± 0.1
  V  (25 ± 3) × 103 Å3
  a' 17 (13)e ± 1 Å 
  b' 19 (16)e  ± 2 Å 
  delta 0.33  ± 0.01 g H2O/g protein
  ftheo (3.41 ± 0.10) × 10-8 g s-1
Prolate model (a > b)d
  p 2  ± 1
  V (23 ± 1) × 103 Å3
  a' 28 (25)e  ± 1 Å 
  b' 14 (13)e  ± 2 Å 
  delta 0.25 ± 0.01 g H2O/g protein
  ftheo (3.36 ± 0.10) × 10-8 g s-1

a  Radius of a sphere with the same Mr as phi 29 SSB estimated from (6.723 × 10-8). Mr1/3 (34).
b  Partial specific volume of the protein estimated from phi 29 SSB aminoacid composition according to Laue et al. (31).
c  Rotational correlation of the time-resolved fluorescence anisotropy of phi 29 SSB, corresponding to the global motion of the entire protein (see Soengas et al. (30)).
d  Average values of the axial ratio (p), volume (V), semiaxis (a', b'), hydration factor (delta ), and theoretical translational frictional coefficient (ftheo) of the revolution ellipsoids compatible with the hydrodynamic behavior of phi 29 SSB.
e  Values of the semiaxis that could correspond to the anhydrated phi 29 SSB.

The experimental data corresponding to the radial distribution of phi 29 SSB concentration after sedimentation equilibrium were best fitted to equations described for a monomeric behavior of a protein. An example for 12 µM SSB centrifuged at 30,000 rpm is depicted in Fig. 1. The fitting procedures (chi 2 = 6.4 × 10-3) gave an average of 13,600 ± 400, for the Mr of phi 29 SSB, very close to the calculated from its amino acid composition (13,343; see Ref. 26). This confirms the results previously obtained with indirect techniques (gel filtration chromatography; Ref. 6), and therefore, phi 29 SSB can be confidently considered as a monomer in solution, at least in the concentration range used for the analysis of its functional activities and ssDNA binding properties.


Fig. 1. Analytical ultracentrifugation analysis of the oligomeric state of phi 29 SSB in solution. A solution containing 12 µM phi 29 SSB was centrifuged at 30,000 rpm in buffer A until sedimentation equilibrium was reached. Data from absorbance scans at 230 nm were plotted (open circles) as a function of the radial distance (A). The line corresponds to the best fit to equations describing the ideal distribution of ideal monomeric solutes whose residuals are shown in B. The Mr calculated in this case was 13,600.
[View Larger Version of this Image (29K GIF file)]


Sedimentation velocity experiments were carried out to determine the sedimentation and diffusion coefficients, s20,w and D20,w, respectively. The sedimentation process of a 60,000 rpm centrifugation of phi 29 SSB was analyzed by following the movement of the boundary as described elsewhere (see Ref. 48, for a review). For the above indicated protein range, the estimated s20,w and D20,w (procedures not shown) were 1.7 ± 0.1 S and (1.18 ± 0.07) × 10-6 cm2 s-1, respectively. By including the Mr and s20,w values in the Svedberg equation (see "Materials and Methods," Equation 1) the translational frictional coefficient, fobs, was computed as (3.5 ± 0.1) × 108 gs-1 (Table I).

It is well known that the value of the frictional coefficient does not allow, by itself, the distinction between a symmetric but rigid structure from a highly asymmetric but flexible conformations (see for examples, Refs. 36, 48, and 49). Therefore, another hydrodynamic parameter was considered to restrict the size and shape of phi 29 SSB, the rotational depolarization of its intrinsic fluorescence anisotropy. As described in the accompanying paper (30), the decay of phi 29 SSB anisotropy is well described by two widely separated rotation correlational times, phi 1 = 0.14 ± 0.06 ns (beta 1 = 0.24 ± 0.03) and phi 2 = 7.0 ± 0.5 ns (beta 2 = 0.76 ± 0.03). Since phi 1 <<  phi 2, the second one can be considered as corresponding to the global motion (phi 2 triple-bond  phi global) of the entire protein.

The values of phi global, within the experimental uncertainties, were used to simulate (using Equations 2, 3, 4, 5, 6, 7, 8 under "Materials and Methods") a set of rotationally compatible ellipsoids of revolution with axial ratios p (=a/b) ranging from 0.1 to 1 (oblate mode) or 1 to 10 (prolate mode). The theoretical f, ftheo, of each of these ellipsoids are represented in Fig. 2, A and C. Those values of p giving ftheo in the range of fobs could account for the shape of phi 29 SSB. Thus, a nearly spheric oblate ellipsoid, p = 0.8 to 1 (Fig. 2A), and a set of prolate ellipsoids up to p ~ 4.5 (Fig. 2C) would satisfy simultaneously the experimental phi global and fobs. Thus, both, a disc-shape and a fibrillar shape can be excluded for phi 29 SSB solution conformation. Since the dry volume of the protein can be easily estimated from the Mr (Table I), it is possible to compute the corresponding hydration factors delta  (see "Materials and Methods") from the hydrodynamic volume of each of the ellipsoids. All of the compatible oblate ellipsoids (Fig. 2B) and the prolate ellipsoids with p < ~3.2 (Fig. 2D) would have a reasonable hydration (delta  values below 0.15 g of water/g of protein were considered as higly unlikely). The best approximation to the dimensions (a × b) of phi 29 SSB, corresponding to the mean values of the compatible p, would be a = 34 ± 2 Å, b = c = 38 ± 4 Å (V = 25 × 103 Å3) for the oblate ellipsoid (p = 0.9 ± 0.1), and a = 56 ± 16 Å, b = c = 28 ± 8 Å (V = 23 × 103 Å3) for the prolate ellipsoid (p = 2 ± 1).


Fig. 2. Determination of the set of revolution ellipsoids compatible with the translational and rotational parameters of phi 29 SSB. Two models, oblate (A and B) and prolate ellipsoids (C and D), were considered. The solid lines in A and C correspond to the set of ellipsoids whose rotational correlation time would be equal to phi global of phi 29 SSB, measured as 7.0 ± 0.5 ns form the decay of its intrinsic fluorescence (30). The theoretical frictional coefficient, f, of these ellipsoids is indicated as a function of their axial ratio, p. Those values of p compatible with the experimental fobs (3.5 ± 0.1) × 10-8 gs-1 are indicated by an open rectangle on the p axis. B and D, further restriction of the plausible p values based on the hydration factors of the ellipsoids in A and C. Values below 0.15 g of H2O/g of protein were considered as unlikely (see text). The filled rectangles on the p axis correspond to the translationally and rotationally compatible values of p, the averaged values being 0.9 ± 0.1 (oblate model) and 2 ± 1 (prolate model).
[View Larger Version of this Image (23K GIF file)]


phi 29 SSB Is a Highly beta -Structured Protein

Circular dichroism (CD) spectroscopy is a powerful tool for the analysis of the global secondary structure of proteins in solution due to its dependence on the type and amount of alpha -helical, beta -sheets, and unordered conformations (50, 51). Moreover, aromatic amino acids have also a characteristic CD activity toward the near UV region of the spectra, that is more evident when these residues are not free to rotate (52, 53).

Fig. 3A shows the far UV CD spectrum at neutral pH and 25 °C of free and bound phi 29 SSB. A maximum at 196 nm and two minima at 210 and 223 nm can be clearly identified in both cases. These spectra were analyzed by the comparative method of Provencher (42, 44) using as reference proteins whose structure is known (43). The goodness of the fitting procedure is shown in Fig. 3, B and C, as the random distribution of the weighted residuals. Both forms of phi 29 SSB were found to be highly structured. The free protein contains a relatively high amount of beta -structures (47% beta -sheet and 23% beta -turn) and a low amount of alpha -helix (10%).


Fig. 3. Secondary structure analysis of free and bound phi 29 SSB. A, far UV spectra of 6 µM free (thick line) and bound phi 29 SSB (thin line) in buffer A and 25 °C. In each case, the plotted mean residue ellipticity (theta ) corresponds to the average of three scans corrected for background emission. The relative amount of the structural secondary elements alpha -helix (alpha -H), beta -sheet (beta -S), beta -turn (beta -T), and the remainder (REM) obtained by the Provencher (1984) CONTIN (version 2) program (43) are indicated as percentages of the total protein contain. B and C, distribution of the weighted residuals for free and bound phi 29 SSB, respectively.
[View Larger Version of this Image (37K GIF file)]


Complex formation with ssDNA had minimal effects on the CD spectra of phi 29 SSB: the difference on the content of the secondary structure elements of phi 29 SSB between the free and poly(dT)-bound forms of the protein being within the experimental error (Fig. 3A). As expected, since phi 29 SSB has no sequence specificity (6), similar results were observed with ssDNA of heterogeneous sequence, M13 ssDNA (not shown). Nonconformational changes upon complex formation have been also reported for SSBs of filamentous phages fd (54), Ike (55), or Pf3 (56). The ellipticity in the near UV region of the spectrum (wavelengths above 250 nm) which has been described (52, 53) to correspond to the emission of the aromatic residues of the protein (in the case of phi 29 SSB, 3 Tyr and 7 Phe), was found almost negligible for both, free and complexed phi 29 SSB (not shown). Therefore, and in agreement with the results obtained by analyzing the intrinsic features of phi 29 SSB fluorescence (30), phi 29 SSB tyrosines could not be strongly restricted by interactions with surrounding residues.

ssDNA Binding Stabilizes phi 29 SSB Secondary Structure

Although ssDNA binding did not apparently change the global conformation of phi 29 SSB, it had, however, a clear effect on its stability. Changes in the far UV spectra at different pH monitored for free and bound phi 29 SSB in buffer Tris-HCl are shown in Fig. 4. It should be mentioned that reliable spectra could not be recorded at pH < 6.0 because phi 29 SSB became insoluble. A pH increase up to 7.0 had no effect on the protein structure, as indicated by the invariability of the CD spectra. In the absence of ssDNA (Fig. 4A), from pH 7.0 to 9.3 the same molar ellipticity (theta ) was observed for wavelengths ranging from 190 to 210 nm, although great changes were observed in the region spanning between 210 and 240 nm. Thus a 20% reduction respect to the values at pH 7.5 was recorded for pH 8.0, while a 30% increase was recorded for pH 9.3. These oscillations likely correspond to less stable conformations of phi 29 SSB. Higher pH values greatly affected the protein structure. The apparition of a minimum around 200 nm and the progressive anulation of theta from this wavelength are consistent with a mostly random coiled organization, which reaches the maximum value at pH 11.5. Similar results were obtained with sodium cacodylate-HCl or glycine-NaOH buffers (not shown). The increase in the salt concentration (due to the NaOH added to modify de pH) was discarded as responsible of those structural changes, since additions of even a 3-fold excess of NaCl did not induce any effect on the phi 29 SSB CD spectra at neutral pH (results not shown).


Fig. 4. Effect of the pH on phi 29 SSB CD. Stabilizing effect of poly(dT). Far UV spectra of 6 µM phi 29 SSB in buffer A at 25 °C and the indicated pH, in the absence (A) or presence of saturating amounts (350 µM) of poly(dT) (B). Plotted data, as mean residue ellipticity (theta ), correspond to the average of three scans and were corrected for background emission.
[View Larger Version of this Image (32K GIF file)]


CD spectra of the bound phi 29 SSB (at a binding density of ~1) was, on the contrary, relatively insensitive to the solution pH (Fig. 4B). The same maxima and the same theta  values were recorded between pH 6.0 and pH 10.5. A further increase in the solution pH started to induce changes in the global protein structure, as indicated by the reduction of the optical activity values around 196 nm, which, nevertheless, are still less affected respect to those in the absence of ssDNA. pH 11.5 also denatured the protein structure.

Temperature had a less dramatic effect on phi 29 SSB structure. The far UV spectra of phi 29 SSB was almost constant from 15 °C up to 55 °C (Fig. 5A). Even at temperatures as high as 70 °C the protein retained a large fraction of its tertiary structure. Again, ssDNA had a stabilizing effect, alleviating the effects of temperature increase (Fig. 5B).


Fig. 5. Temperature dependence of phi 29 SSB CD spectra. Stabilizing effect of poly(dT). Far UV spectra of 6 µM phi 29 SSB free (A) or complexed with poly(dT) (B) in buffer A, at the indicated temperatures. Plotted data, as mean residue ellipticity (theta ), correspond to the average of three scans and were corrected for background emission.
[View Larger Version of this Image (22K GIF file)]


The Steady-state Intrinsic Anisotropy of phi 29 SSB Increases Upon Complex Formation

Although ssDNA binding did not significantly change the global phi 29 SSB structure, it could restrict the rotational properties of the protein. To test this possibility, binding was followed by monitoring the differential changes in the vertical and horizontal components of the protein emission during reverse titrations of a constant amount of protein with poly(dT). Reduction of fluorescence intensity at magic angle orientation of the polarizers, I54 was recorded in parallel to follow ssDNA binding. An example (20 µM phi 29 SSB) is shown in Fig. 6. The anisotropy, increased from 0.174 to 0.214, a value very close to the limiting anisotropy of phi 29 SSB, that is, the remaining anisotropy that would be recorded by time-resolved techniques if the protein was immobile (rinfinity = 0.210; 30). In addition, this maximal value is still far from the expected value for immobilized tyrosines (ro = 0.32; 57-58). Changes in the anisotropy of a single molecule of phi 29 SSB could not be analyzed due to the special binding features of this protein: it cannot bind to short oligonucleotides (see above and Ref. 6).


Fig. 6. Effect of complex formation on the steady-state fluorescence emission features of phi 29 SSB. Binding isotherms (25 °C) obtained by reverse titrations of a solution of 6 µM phi 29 SSB in buffer B with increasing amounts of poly(dT). Samples were excited with a vertically polarized light (lambda exc = 276 ± 4 nm) and the fluorescence was recorded at 308 ± 2 nm for emission polarizer orientations of 54 ° (I54, open circles), 0 ° (Ipar ), and 90 ° (Iperp ), and corrected for dilution and inner filter effects (see "Materials and Methods"). The anisotropy values (closed circles) were obtained as the ratio (Ipar  - GIperp )/(Ipar  + 2GIperp ), were G stands for the factor of the photodetection set-up accounting for the differential polarization sensitivity. The maximal change in the anisotropy was achieved at 100 µM poly(dT). At this concentration the quenching of the I54 emission was ~74%.
[View Larger Version of this Image (15K GIF file)]


Effects of Complex Formation on ssDNA Structure

Complex formation between phi 29 SSB and ssDNA was also studied by analyzing the changes in the CD spectra of DNA induced by protein binding and comparing them with those induced by temperature increase. The spectra were recorded above 250 nm, where, as already mentioned, phi 29 SSB has a negligible optical activity. As it can be deduced form Fig. 7A, phi 29 SSB induced a great alteration of the M13 ssDNA secondary structure. Both a progressive red shift of the spectra and a reduction of the circular dichroism (Delta epsilon ) values took place when increasing amounts of protein were added. At the saturation point, the two exciton maxima moved from 249.0 to 251.0 nm and 276.0 to 281.0 nm, the activities being reduced by ~2.2-fold (negative band) and by ~0.1-fold, 10% (positive band). The crossover moved from 261.5 to 266.5 nm. Those effects could be reflecting reorganizations of the contiguous nucleotides. To distinguish unstacking from other orientation effects, spectra of free M13 ssDNA were recorded at higher temperatures, since, as described for different ssDNAs, temperature decreases the stacking degree of neighbor nucleotides (59, 60, 61, 62). The conservative changes (no displacement of the maxima and the crossover) induced by temperature increase (Fig. 7A, inset) were clearly different from the effects of phi 29 SSB. In addition, binding was tested on poly(dT) and poly(dA), ssDNAs described as having low and high, respectively, degree of stacking (63, 64). In the case of poly(dT), again, a red shift and a reduction (~35% in this case) of the Delta epsilon intensities were detected upon complex formation (Fig. 7B). Therefore, it can be concluded that the ssDNA in the complex with phi 29 SSB still remains organized, although the nearest-neighbor interactions differ from the ones established in the free DNA, likely reflecting, apart from a different degree of stacking, a different geometry of the contiguous bases. These changes should be corresponding to the factors leading to a global 50% reduction of the ssDNA length measured by electron microscopy techniques (12). phi 29 SSB did not induce any change in the poly(dA) spectra (not shown), suggesting an impairment of intercalating interactions between the aromatic ring of phi 29 SSB tyrosines and the bases of the ssDNA, reinforcing the hypothesis presented in the accompanying paper (30) of the involvement of these residues in complex formation.


Fig. 7. Secondary structure of free and bound ssDNA. A, near UV spectra of M13mp18 DNA, 112 µM (thick line), at 25 °C in the presence of increasing amounts of phi 29 SSB: 6, 16, 29, 46, and 60 µM (thin lines from upper to bottom curve, respectively). Inset, near UV spectra of free M13 mp18 (112 µM) at 25 °C (thick line) and at 60 °C (thin line). The exciton bands and the crossover remained in both cases, at the same position, 249.0, 276.0, and 261.5 nm, respectively. B, near UV spectra of poly(dT), 110 µM, at 25 °C (thick line), in the presence of the following amounts of phi 29 SSB (thin lines from upper to bottom curve: 12, 20, 27, 33, 39, and 47 µM). Inset, near UV spectra of 110 µM poly(dT) at 25 °C (thick line) and at 60 °C (thin line). The exciton bands and the crossover were located in both cases at 253.0, 276.0, and 264.0 nm, respectively.
[View Larger Version of this Image (19K GIF file)]


We also analyzed whether the changes in the CD intensity (Delta epsilon ) were compatible with the results obtained from the analysis of the changes in the intrinsic phi 29 SSB fluorescent emission upon complex formation. Thus, the value of the nucleotide site size, n, the number of nucleotides covered by a protein monomer, was computed from the relationship between the changes in Delta epsilon at the wavelengths corresponding to the exciton bands of the free DNA and the phi 29 SSB/poly(dT) ratio (Fig. 8). Saturation, given by a maximum change of Delta epsilon , started at a phi 29 SSB/poly(dT) ratio of 0.28-0.29 which gave a value of n of about 3.4-3.6, in agreement with the results from fluorescent techniques (6). No effects on the conformation of short oligonucleotides dT20, dA16 were detected even at very high doses of phi 29 SSB (not shown), confirming the impairment of phi 29 SSB to bind these DNAs, that was also observed by fluorescent techniques (6).


Fig. 8. Quantification of the changes in poly(dT) structure upon binding. Estimation of the nucleotide site size (n) of phi 29 SSB-ssDNA complex. Changes in the Delta epsilon of poly(dT) upon phi 29 SSB addition (see B) were measured at the wavelengths corresponding to the location of the exciton bands of the free poly(dT), 253 nm (A) and 276 nm (B), respectively. Data were plotted against the molar ratio, R, of phi 29 SSB and poly(dT) at each point of the titration. The observed plateau indicate saturation of all of the possible binding sites in the ssDNA. The number of nucleotides covered by a monomer of phi 29 SSB was calculated from the inverse of R at the beginning of the saturation as 3.6 (A) and 3.4 (B).
[View Larger Version of this Image (12K GIF file)]



DISCUSSION

SSBs are critical for the proper metabolism of ssDNA. Although they have been found in all the organisms studied so far (4), and different efforts have been made to understand their mechanism(s) of action, only very few3 of them have been well characterized in terms of the molecular interactions that contribute to complex formation with ssDNA: filamentous phages Ff gene V proteins (3, 13, 15, 19), E. coli SSB (see Ref. 21, for review), and T4 gp32 (18, 19, 20). A certain conservation in the global folding of the ssDNA binding domain (beta -sheet strands connected by loops) has been proposed (16, 17). However, only among the SSBs of very related organisms (E. coli Ff phages and P. aeruginosa phage Pf3) is this conservation clear (3, 15). Therefore, the particular residues involved in SSB-ssDNA contacts can be considered as being rather specific, likely as a consequence of their differences in the global structure of the SSBs and in their DNA binding parameters.

B. subtilis phage phi 29 DNA replication occurs via a strand displacement mechanism which takes place with the transient formation of high amounts of ssDNA (see Refs. 23 and 24, for reviews). The elongation step of phi 29 DNA replication requires a viral-encoded SSB (phi 29 SSB), very abundant in infected cells (4% of the total protein content; 26). The participation of phi 29 SSB in viral DNA replication seems to be rather specific in vivo, since it cannot be substituted by the host SSB. The thermodynamic parameters defining complex formation between phi 29 SSB and ssDNA, i.e. maximal quenching, nucleotide site size, affinity, cooperativity, have been described in a wide variety of experimental conditions (6, 30). However, very little information was available with respect to the structural features of the free and bound phi 29 SSB and to the effects of binding on the ssDNA structure.

In this paper, we propose that the hydrodynamic behavior of phi 29 SSB in solution (see Table I), analyzed by translational (sedimentation equilibrium and sedimentation velocity centrifugations) and rotational measurements (time-decay of its intrinsic fluorescence anisotropy), is compatible with a monomeric protein of ~13,600 kDa that could be modeled as a nearly spherical ellipsoid of revolution. The axial ratio p (=a/b) of phi 29 SSB could range from 0.8 to 1.0 (oblate model) or 1.0 to 3.2 (prolate model). CD analysis of phi 29 SSB spectra between 190 and 240 nm allowed us to estimate that phi 29 SSB is highly structured (47% beta -sheets, 10% alpha -helices, 23% beta -turns, at neutral pH). This high amount of beta -structures is in the order of that derived from the CD of T4 gp32 (18) or Ff SSBs (see Ref. 3, for review) and much higher than the ~20% reported for E. coli SSB (67). In the case of M13 and Pf3 SSB or T4 gp32, the high amount of beta -elements has been confirmed by three-dimensional analysis (13, 14, 15, 18). No positive band was detected at 230 nm, a difference with the filamentous phages fd and Ike SSBs, which was proposed to derive from interactions between the phenolic group of certain tyrosines and local amides (55, 56, 57).

Complex formation induces minimal changes in phi 29 SSB spectra, the global conformation of the bound protein remaining essentially unchanged. However, ssDNA had a significant stabilizing effect against denaturation induced by pH and temperature increase. The major structural effects induced upon complex formation seem to occur on the ssDNA structure. Thus, great non-conservative changes (59, 61), defined by a red shift and a reduction of the Delta epsilon values, occur in the CD spectra of the ssDNA. These changes likely correspond, besides a decrease of the stacking, to a different spatial geometry of the neighbor nucleotides in the bound ssDNA. This different geometry might be related to the 50% reduction of the ssDNA length determined by electron microscopic visualization of the phi 29 SSB-ssDNA complexes (12).

In the absence of three-dimensional data, the results presented here constitute the first approximation to the global conformation of phi 29 SSB and its complex with ssDNA. A hypothetical model to illustrate the ratio between the size of the protein and the ssDNA in the complex is presented in Fig. 9. The protein is depicted as a revolution ellipsoid assuming an axial ratio p = 2. Taking a standard nucleotide interspace of 4 Å, and the calculated value for the nucleotide site size (n) of phi 29 SSB, 3-4 nt (6), it is possible to discard phi 29 SSB-ssDNA interactions as ssDNA wrapping around the protein core upon complex formation, as it has been proposed for E. coli SSB (8). Rather, it is likely that, in agreement to the relatively low ssDNA binding constant (Keff = 1-3 × 105 M-1), ssDNA contacts with a limited region of phi 29 SSB. The location of this DNA binding domain is not presently known. However, consistent with the steady-state and time-decay fluorescence analysis presented in the accompanying paper (30), it could contain the three tyrosines.


Fig. 9. Model for phi 29 SSB in solution to show the relationship between the protein and DNA size. phi 29 SSB is depicted as a prolate ellipsoid using the average values for p = 2 ± 0.2 (see text and also Fig. 3). The values of the semiaxis a', b'(=c'), were 14 and 28 Å, respectively, for the hydrated molecule (delta  = 0.25 g of H20/g of protein), and 12 and 25 Å, respectively, for the unhydrated molecule. The estimated error in the dimensions is ~20%. ssDNA is drawn at the same scale as phi 29 SSB, assuming an internucleotide distance of 4 Å. Considering a reduction of 50% in the ssDNA length upon complex formation (12), the 3-4 nucleotides covered by protein monomer (6) would correspond to 8 Å. The localization of the ssDNA binding domain in the tertiary structure of the protein is still unknown, and therefore, the bound DNA is arbitrarily placed onto phi 29 SSB only to show the relative size of both molecules and therefore to discard protein-DNA interactions, i.e. ssDNA wrapping around the phi 29 SSB core.
[View Larger Version of this Image (23K GIF file)]


Certain features of the SSBs from the above indicated filamentous phage SSBs, their small size (~9.7 kDa), small n (4 nt/monomer), a not so high Keff (105-107 M-1), and a high contain of beta -structure elements (3) that remain essentially unchanged upon ssDNA binding (54, 55, 56), are close to those of phi 29 SSB. However, a number of significant differences can be found that strongly suggest that phi 29 SSB may have a particular structure and a particular DNA binding domain. First, those SSBs form dimers in solution and the ssDNA binding domain is shared by both monomers (13, 15, 19) and therefore each dimer can contact two strands of ssDNA. Second, although all contain high amounts of beta -elements, the CD spectra of those SSBs is quite different from that of SSB phi 29 SSB. Third, ssDNA binding of these SSBs is moderately salt-dependent (68), indicating that ionic interactions are mediating complex formation. An opposite situation was found for phi 29 SSB-ssDNA complex, where the ionic composition of the reaction buffers had small effects on its Keff (6). Fourth, significant functional differences exist, since binding of Ff SSB to ssDNA leads to an inhibition of the phage DNA replication process (22) while complex formation between the phi 29 SSB and the displaced ssDNA has an stimulatory effect on phi 29 DNA replication (23, 24).

The structural and functional differences discussed above strongly suggest that phi 29 SSB may have a unique organization. The absolute requirement of phi 29 SSB during in vivo viral DNA replication could be reflecting these particularities.


FOOTNOTES

*   This work has been supported in part by Grants 5RO1 GM 27242-16 from the National Institute of Health, CHRX-CT93-0248 from European Union and PB93-0176 form Dirección General de Investigación Científica y Técnica (DGICYT) (to M. S.), PB93-126 from DGICYT (to A. U. A.) and PB92-007 form DGICYT (to G. R.). An institutional grant from Fundación Ramón Areces to Centro de Biología Molecular "Severo-Ochoa" is acknowledged. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§   Recipient of a predoctoral fellowship from Comunidad Autónoma de Madrid.
**   To whom correspondence should be addressed. Tel.: 1-3978435; Fax: 1-397-4799.
1    The abbreviations used are: ssDNA, single-stranded DNA; SSB(s), single-stranded DNA-binding protein(s); Ff SSB, SSBs of Escherichia coli phages M13, fd, and pf1; T4 gp32, phage T4 SSB; T7 gp2.5, phage T7 SSB; N4 SSB, phage N4 SSB; Keff, effective ssDNA binding constant; omega , cooperativity parameter of ssDNA binding process.
2    Adenovirus DBP, whose three-dimensional structure has been recently resolved by x-ray difraction (16) is not considered as a SSB sensu stricto since is able to bind both, ssDNA and dsDNA, with high affinity (17).
3    Studies foccused on the characterization of the thermodynamic parameters that define ssDNA binding have been done for other SSBs, e.g. T7 gene 2.5 (65, 66); Drosophila SSB (10); phi 29 SSB (6); human SSB (7, 9); N4 SSB (5), however, they are still less well characterized as Ff SSBs, E. coli SSB or T4 gp32.

Acknowledgments

We are indebted to J. M. Lázaro and L. Villar (Centro de Biología Molecular "Severo-Ochoa," Spain) for the purification of phi 29 SSB and Drs. M. P. Lillo and R. Dale (Instituto de Química Física Rocasolano, Spain) for their invaluable contribution in fluorescence and analytical ultracentrifugation data analysis and interpretation. We also thank Drs. C. W. Hilbers and B. J. M. Harmsen (University of Nijmegen, The Netherlands) for their training in the circular dichroism techniques, Dr. A. J. W. G. Visser (Wageningen Agricultural University, The Netherlands) for the analysis of the secondary structure of free and bound phi 29 SSB, and Dr. J. M. Andreu and J. Evangelio for their help with the J700 CD apparatus.


REFERENCES

  1. 14, 373-444Kowalczykowski, S. C., Bear, D. G., and von Hippel, P. H. (1981) The Enzymes, 3rd Ed., 14, 373-444
  2. Chase, J. W., and Williams, K. R. (1986) Annu. Rev. Biochem. 55, 103-136 [CrossRef][Medline] [Order article via Infotrieve]
  3. Kneale, G. G. (1992) Curr. Opin. Struct. Biol. 2, 124-130
  4. Kornberg, A., and Baker, T. A. (1992) in DNA Replication (Kornberg, A., and Baker, T. A., eds), pp. 323-354, W. H. Freeman & Co., San Francisco
  5. Lindberg, G., Kowalczykowski, S. C., Rist, J. K., Sugino, A., and Rhotman-Denes, L. (1989) J. Mol. Biol. 264, 12700-12708
  6. Soengas, M. S., Esteban, J. A., Salas, M., and Gutiérrez, C. (1994) J. Mol. Biol. 239, 213-226 [CrossRef][Medline] [Order article via Infotrieve]
  7. Kim, C., Synder, R. O., and Wold, M. S. (1992) Mol. Cell. Biol. 12, 3050-3059 [Abstract]
  8. Meyer, R. R., and Laine, P. S. (1990) Microbiol. Rev. 54, 342-380
  9. Wold, M. S., and Kelly, T. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 2523-2527 [Abstract]
  10. Mitsis, P. G., Kowalczykowski, S. C., and Lehman, I. R. (1993) Biochemistry 32, 5257-5266 [Medline] [Order article via Infotrieve]
  11. Prasad, B. V. V., and Chiu, W. (1987) J. Mol. Biol. 193, 579-584 [CrossRef][Medline] [Order article via Infotrieve]
  12. Gutiérrez, C., Martín, G., Sogo, J. M., and Salas, M. (1991) J. Biol. Chem. 266, 2104-2111 [Abstract/Free Full Text]
  13. Skinner, M. M., Zhang, H., Leschnitzer, D. H., Guan, Y., Bellamy, H., Sweet, R. M., Gray, C. W., Konings, R. N. H., Wang, A. H. J., and Terwilliger, T. C. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 2071-2075 [Abstract]
  14. Folkers, P. J. M., Nilges, M., Folmer, R. H. A., Konings, R. N. H., and Hilbers, C. W. (1994) J. Mol. Biol. 236, 229-246 [CrossRef][Medline] [Order article via Infotrieve]
  15. Folmer, R. H. A., Nilges, M., Konings, R. N. H., and Hilbers, C. W. (1995) EMBO J. 14, 4132-4142 [Abstract]
  16. Stuiver, M. H., Bergsma, W. G., Arnber, A. C., van Amerongen, H., van Grondelle, R., and van der Vliet, P. C. (1992) J. Mol. Biol. 255, 999-1011
  17. Tucker, P. A., Tsernoglou, D., Tucker, A. D., Coenjaerts, F. E. J., Leenders, H., and van der Vliet, P. C. (1994) EMBO J. 13, 2994-3002 [Abstract]
  18. Shamoo, Y., Friedman, A. M., Parsons, M. R., Konigsberg, W. H., and Steitz, T. A. (1995) Nature 376, 362-366 [CrossRef][Medline] [Order article via Infotrieve]
  19. Folmer, R. H. A., Nilges, M., Folkers, P. J. M., Konings, R. N. H., and Hilbers, C. W. (1994) J. Mol. Biol. 240, 341-357 [CrossRef][Medline] [Order article via Infotrieve]
  20. Casas-Finet, J. R., and Karpel, R. L. (1993) Biochemistry 32, 9735-9744 [Medline] [Order article via Infotrieve]
  21. Lohman, T. M., and Ferrari, M. E. (1994) Annu. Rev. Biochem. 63, 527-5701 [CrossRef][Medline] [Order article via Infotrieve]
  22. Salstrom, J. S., and Pratt, D. (1971) J. Mol. Biol. 61, 489-501 [Medline] [Order article via Infotrieve]
  23. Salas, M. (1991) Annu. Rev. Biochem. 60, 39-71 [CrossRef][Medline] [Order article via Infotrieve]
  24. Salas, M., Freire, R., Soengas, M. S., Esteban, J. A., Méndez, J., Bravo, A., Serrano, M., Blasco, M. A., Blanco, L., Gutiérrez, C., and Hermoso, J. M. (1995) FEMS Microbiol. Rev. 17, 73-82 [CrossRef][Medline] [Order article via Infotrieve]
  25. Salas, M., and Rojo, F. (1993) in Bacillus subtilis and Other Gram-positive Bacteria: Biochemistry, Physiology and Molecular Genetics (Hoch, J. A., and Losick, R., eds), pp. 843-845, American Society for Microbiology, Washington, D. C.
  26. Martín, G., Lázaro, J. M., Méndez, E., and Salas, M. (1989) Nucleic Acids Res. 17, 3663-3672 [Abstract]
  27. Soengas, M. S., Gutiérrez, C., and Salas, M. (1995) J. Mol. Biol. 253, 517-529 [CrossRef][Medline] [Order article via Infotrieve]
  28. Blanco, L., Lázaro, J. M., de Vega, M., Bonnin, A., and Salas, M. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 12198-12202 [Abstract/Free Full Text]
  29. Gutiérrez, C., Sogo, J. M., and Salas, M. (1991b) J. Mol. Biol. 222, 983-994 [Medline] [Order article via Infotrieve]
  30. Soengas, M. S., Mateo, R. C., Salas, M., Acuña, A. U., and Gutiérrez, C. (1997) J. Biol. Chem. 272, 295-302 [Abstract/Free Full Text]
  31. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275 [Free Full Text]
  32. Minton, A. P. (1994) in Modern Analytical Ultracentrifugation (Schuster, T. M., and Laue, T. M., eds), pp. 81-93, Brikhäuser, Boston
  33. van Holde, K. E. (1985) Physical Biochemistry, 2nd Ed., pp. 225-291, Prentice-Hall, Inc., New York
  34. Laue, T. M., Shah, B. D., Ridgeway, T. M., and Pelletier, S. L. (1993) in Analytical Ultracentrifugation in Biochemistry and Polymer Science (Harding, S. E., Horton, H. C., and Rowe, A. J., eds), pp. 90-125, Royal Society of Chemistry, Cambridge
  35. Goldberg, R. J. (1953) J. Phys. Chem. 57, 194
  36. Waxman, E., Laws, W., Laue, T. M., Emerson, Y., and Ross, A. B. A. (1993) Biochemistry 32, 3005-3012 [Medline] [Order article via Infotrieve]
  37. Wahl, P. H. (1983) in Time-resolved Fluorescence Spectroscopy in Biochemistry and Biology (Cundall, R. B., and Dale, R. E., eds) Nato Asci Series A, Vol. 69, Plenum Press, New York
  38. Montejo, J. M., Navqi, K. R., Lillo, M. P., González, J., and Acuña, A. U. (1992) Biochemistry 31, 7580-7586 [Medline] [Order article via Infotrieve]
  39. Steel, C., and Naqvi, R. K. (1995) J. Phys. Chem. 95, 10713-10718
  40. Perrin, F. (1936) J. Phys. Radium. 7, 1-11
  41. Cantor, C. R., and Shimmel, P. R. (1980) Biophysical Chemistry, Part II, Freeman W. H. and Co., San Francisco
  42. Provencher, S. W. (1984) EMBL Technical Report DAO7
  43. Chang, T. C., Wu, C. C.-S., and Yang, J. T. (1978) Anal. Biochem. 91, 13-31 [Medline] [Order article via Infotrieve]
  44. Provencher, S. W., and Glöckner, J. (1981) Biochemistry 20, 33-37 [Medline] [Order article via Infotrieve]
  45. Perczel, A., Hollósi, M., Tusnády, G., and Fasman, G. D. (1991) Protein Eng. 4, 669-679 [Abstract]
  46. Perczel, A., Park, K., and Fasman, G. D. (1992) Proteins Struct. Funct. Genet. 13, 57-69 [Medline] [Order article via Infotrieve]
  47. Mateo, C. R., Lillo, M. P., González-Rodríguez, J., and Acuña, A. U. (1991) Eur. Biophys. J. 20, 41-52 [Medline] [Order article via Infotrieve]
  48. Hansen, J. C., Lebowitz, J., and Demeler, B. (1994) Biochemistry 33, 13155-13163 [Medline] [Order article via Infotrieve]
  49. Stafford, W. F., III, and Szent-Györgyi, A. G. (1978) Biochemistry 17, 607-614 [Medline] [Order article via Infotrieve]
  50. Hirs, C. H. W., and Timasheff, S. N. (1973) Methods Enzymol. 27, 675-735 [Medline] [Order article via Infotrieve]
  51. Yang, J. T., Wu, C-S. C., and Martínez, H. M. (1986) Methods Enzymol. 130, 208-269 [Medline] [Order article via Infotrieve]
  52. Baley, P. (1980) in An Introduction to Spectroscopy for Biochemists (Brown, S. B., ed), pp. 148-233, Academic Press, London
  53. Anderson, R. A., and Coleman, J. E. (1975) Biochemistry 14, 5485-5491 [Medline] [Order article via Infotrieve]
  54. Day, L. A. (1973) Biochemistry 12, 5329-5339 [Medline] [Order article via Infotrieve]
  55. Sang, B.-C., and Gray, M. (1989) Biochemistry 28, 9502-9507 [Medline] [Order article via Infotrieve]
  56. Powell, M. D., and Gray, D. M. (1993) Biochemistry 32, 12538-12547 [Medline] [Order article via Infotrieve]
  57. Lakowicz, J. R., Laczko, G., and Gryzynski, I. (1986) Biophys. Chem. 24, 97-100 [CrossRef][Medline] [Order article via Infotrieve]
  58. Gryzynski, I., Steiner, R. F., and Lakowicz, J. R (1991) Biophys. Chem. 39, 69-78 [CrossRef][Medline] [Order article via Infotrieve]
  59. Cantor, C. R., Warshaw, M. M., and Shappiro, H. (1970) Biopolymers 9, 1059-1077 [Medline] [Order article via Infotrieve]
  60. Catlin, J. C., and Guschlbauer, W. (1975) Biolopymers 14, 51-72
  61. Olsthoorn, C. S. M., Haasnoot, C. A. G., and Altona, C. (1980) Eur. J. Biochem. 106, 85-95 [Abstract]
  62. Riley, M., Maling, B., and Chamberlin, M. J. (1966) J. Mol. Biol. 20, 359-389 [Medline] [Order article via Infotrieve]
  63. Dewey, T. G., and Turner, D. H. (1980) Biochemistry 19, 1681-1685 [Medline] [Order article via Infotrieve]
  64. Kim, C., Paulus, B., and Wold, M. S. (1994) Biochemistry 33, 14197-14206 [Medline] [Order article via Infotrieve]
  65. Kim, Y, T., Tabor, S., Churchich, J. E., and Richardson, C. C. (1992) J. Biol. Chem. 167, 15032-15040
  66. Kim, Y. T., and Richardson, C. C. (1994) J. Biol. Chem. 269, 5270-5278 [Abstract/Free Full Text]
  67. Anderson, R. A., and Coleman, J. E. (1975) Biochemistry 14, 5485-5491 [Medline] [Order article via Infotrieve]
  68. Alma, N. C. M., Harmsen, B. J. M., de Jong, E. A. M., Ven, J., and Hilbers, C. W. (1983) J. Mol. Biol. 163, 47-62 [Medline] [Order article via Infotrieve]

©1997 by The American Society for Biochemistry and Molecular Biology, Inc.