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J. Biol. Chem., Vol. 281, Issue 36, 26308-26319, September 8, 2006
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From the Departments of Biochemistry and Microbiology and Molecular Genetics, Vermont Cancer Center, University of Vermont College of Medicine, Burlington, Vermont 05405
Received for publication, May 8, 2006 , and in revised form, July 5, 2006.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Studies of bacteriophage T4 recombination proteins UvsX (recombinase, 44 kDa), UvsY (recombination mediator protein, 16 kDa), and Gp32 (SSB, 34 kDa) have provided important insights on the biochemical mechanism of presynaptic filament assembly (6-11). UvsX, the RecA ortholog of T4 phage, exhibits both ssDNA-dependent ATPase and DNA strand exchange activities. UvsY, the prototype recombination mediator protein, stimulates enzymatic activities of UvsX by promoting assembly onto ssDNA of the latter protein. Gp32, the prototype SSB, has been characterized extensively (12-14). The primary structure of Gp32 (301 residues, 34 kDa) contains three distinct regions including the N-terminal "basic" (B-) domain (residues 1-22), the ssDNA-binding "core" domain (residues 22-253), and the C-terminal "acidic" (A-) domain (residues 254-301) (15). The x-ray structure of the core domain was determined upon co-crystallization with oligo(dT)6 (14). This domain contains an oligonucleotide/oligosaccharide binding-fold, and its structure is stabilized by a Zn2+ atom coordinated to residues His-64, Cys-77, Cys-87, and Cys-90. Molecular studies reveal that the B-domain is the specific region responsible for Gp32 homotypic associations and for the cooperativity of Gp32-ssDNA interactions (16, 17), whereas the A-domain is important for the heterotypic associations of Gp32 with other T4 DNA replication and recombination factors, including UvsX, UvsY, Gp59 (helicase loading/replication mediator protein), Dda (DNA helicase), Gp43 (DNA polymerase), Gp61 (primase), and others (18-21). Equilibrium and kinetic parameters of Gp32-ssDNA interactions, including binding site size n, intrinsic binding constant K, and cooperativity parameter 
, have been determined for a variety of polynucleotide lattices (22). Gp32-ssDNA interactions are characterized by high cooperativity, with generally 
1000 for all single-stranded lattices examined. Binding is sequence-nonspecific, but the observed affinity (K) varies with lattice composition according to the general hierarchy of: poly(dT) > ssDNA (random sequence) > ssRNA >> double-stranded DNA (22, 23).
Gp32 plays important pre- and post-synaptic roles in DNA strand exchange reactions catalyzed by the UvsX protein in concert with UvsY (6, 8-11, 24). During presynaptic filament formation, Gp32 denatures ssDNA secondary structure and provides an optimum lattice conformation for assembly of UvsX-ssDNA filaments. The net effect of Gp32 on UvsX-ssDNA filament formation is inhibitory, however, because the high affinity of Gp32-ssDNA interactions allows Gp32 to effectively compete with UvsX for binding sites on the ssDNA (25). Proper assembly of presynaptic filaments therefore requires the mediator function of UvsY protein in addition to UvsX and Gp32 (2, 3). UvsX exhibits a strict requirement for UvsY in strand exchange assays performed at salt concentrations approximating physiological ionic strength (11, 26, 27), consistent with the co-dependence of in vivo recombination processes on UvsX and UvsY (28-30). In the current model of T4 presynaptic filament assembly, UvsY weakens Gp32-ssDNA interactions and strengthens UvsX-ssDNA interactions, allowing UvsX filaments to nucleate onto pre-existing Gp32-ssDNA complexes, and ultimately to displace Gp32 from the lattice (27, 31-33). After transiently participating in presynapsis, Gp32 further stimulates DNA strand exchange by binding to the displaced single strand generated during UvsX-catalyzed D-loop formation (24).
The ultimate disruption of Gp32-ssDNA interactions during presynaptic filament assembly has been inferred from protein cross-linking and other data (31), but has never been observed directly. Understanding the dynamics of Gp32 during recombination and other DNA transactions requires a probe to selectively report on Gp32-ssDNA interactions in the presence of other DNA-binding proteins. Here we describe such a probe in the form of a covalent conjugate between Gp32 and 6-iodoacetamidofluorescein (Gp32F) (60, 61). Changes in the fluorescence signal of Gp32F allow for the detection and quantification of Gp32-ssDNA interactions in real time and in the presence of other proteins. Monitoring Gp32F fluorescence allows direct measurements of Gp32 displacement from the ssDNA during UvsX-ssDNA filament formation, and demonstrates the salt, nucleotide binding, and UvsY dependence of Gp32 displacement, in confirmation of the current model of T4 presynapsis. Results indicate that presynaptic filaments formed in the presence of Gp32 undergo assembly and collapse that is tightly linked to the ATP hydrolytic cycle, suggesting that in addition to the stabilizing effects of UvsY, ATP binding by UvsX is necessary for maximum occupancy of the ssDNA lattice by UvsX in the presence of Gp32. Competition for ssDNA binding sites between Gp32 and various nucleotide-liganded forms of UvsX may create opportunities for dynamic instability in presynaptic filaments, which could play important roles in T4 recombination metabolism.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Proteins and Nucleic Acids—Recombinant T4 Gp32, UvsX, wild-type UvsY, and two UvsY mutant proteins (UvsYK58A and UvsYK58A,R60A) were purified and stored as previously described (27, 34-36). All proteins were determined to be >98% pure based on SDS-polyacrylamide gels stained with Coomassie Blue. Furthermore, all protein stock solutions were tested and judged to be nuclease-free by previously published criteria (37). Extinction coefficients for Gp32, UvsX, and UvsY sp. at 280 nm were 41,306, 69,760, and 19,180 M-1 cm-1, respectively (38). Circular single-stranded DNA was extracted from purified bacteriophage M13mp18 particles as described (39, 40), and stored at -20 °C in TE buffer. The concentrations of ssDNA stock solutions were determined by the absorbance at 260 nm, using a conversion factor of 33 µg/ml/A260, and are expressed in units of micromoles of nucleotide residues per liter.
Labeling of Gp32 with 6-IAF—All steps were carried out in the dark and at 4 °C. 2 mg of Gp32 protein were dialyzed overnight against 1 liter of Buffer A. Meanwhile a 10 mM stock of 6-IAF in Me2SO was prepared. A 3-fold molar excess of 6-IAF was added to the dialyzed Gp32 via drop by drop additions with gentle mixing. The solution was incubated overnight, after which 100 mM 2-mercaptoethanol was added to consume excess 6-IAF. After 1 h of further incubation, the mixture was dialyzed overnight against 3 x 1 liters of Gp32 storage buffer. SDS-PAGE of the resulting fluorescein-Gp32 conjugate (Gp32F) revealed a single yellow band under visible light prior to Coomassie Blue staining, which upon staining appeared to run at approximately the same molecular weight as unmodified Gp32. The labeling stoichiometry of Gp32F was calculated from the absorbance at 492 and 280 nm, respectively, according to manufacturer's instructions.
ATPase Assays—The ssDNA-dependent ATPase activity of UvsX protein in the presence and absence of Gp32 and Gp32F proteins was measured by a modification of the coupled spectrophotometric assay described (37). All reactions contained 20 mM Tris acetate, pH 7.4, 10 mM magnesium acetate, 90 mM potassium acetate, 2 mM ATP, 6 units/ml pyruvate kinase, 6 units/ml lactate dehydrogenase, 2.3 mM phosphoenolpyruvate, 0.23 mM NADH, 4.5 µM M13mp18 ssDNA, 0.5 µM UvsX, and 0-1.4 µM of either Gp32 or Gp32F. Reactions at 37 °C were started by the addition of UvsX protein to a preincubated mixture of the other components. Reaction velocities were calculated from the change in absorbance at 380 nm using the linear portions of time courses.
ATP concentrations present at different times during fluorescence time course experiments were determined using the ATP determination kit from Molecular Probes. Ten-microliter aliquots were removed from reaction mixtures in fluorescence cuvettes at different times and immediately quenched with 10 mM EDTA. Quenched samples then were boiled for 5 min before analysis. Luminescence was read on a FLX-800 Plate Reader (Sunnyvale, CA) in 96-well assay microplates. Values were calculated based on an ATP standard curve. All ATP concentrations were determined in triplicate. The generation of ADP versus AMP products was estimated based on experimentally measured ATP concentrations and by assuming a 2:1 ratio of ADP:AMP produced by UvsX protein (6).4
Fluorescence Measurements—All fluorescence data were collected on a Quantamaster QM-4 fluorometer (Photon Technology International, South Brunswick, NJ). This fluorometer was equipped with a 75-watt xenon arc lamp as an excitation source, and excitation/emission monochrometers to measure steady-state fluorescence. All data were collected in the steadystate mode and corrected for effects of dilution, solution change, photobleaching, inner filter effects, and intrinsic protein fluorescence as described (25, 34, 41, 42). The slit widths for excitation and emission were 1 and 5 nm, respectively. The excitation wavelength was 460 nm. A long pass cutoff glass filter at 495 nm was used for all of the emission data collection. Data represent the averages of three independent multiwavelength scans (fluorescence emission spectra) or three independent readings at a fixed emission wavelength of 519 nm (fluorescence titrations and time courses), respectively. Sample volumes were generally 0.6-1.0 ml. Photobleaching was minimized by keeping shutters closed although not acquiring data, and was negligible in all experiments.
Determination of Gp32F ssDNA-binding Parameters—Forward, reverse, and salt-back titrations were performed as described (43), in Buffer B (with NaCl concentration as indicated) at 25 °C. The binding site size, n, of Gp32F on ssDNA was calculated from the end point of reverse titrations of Gp32F with ssDNA, monitored by the increase in Gp32F fluorescence, under tight binding conditions (Buffer B + 50 mM NaCl). Intrinsic affinity (K) and cooperativity () parameters for Gp32F-ssDNA interactions were derived from forward titrations of 3.5 µM ssDNA with Gp32F under weak binding conditions (Buffer B + 260 to 340 mM NaCl). Binding was monitored by the increase in Gp32F fluorescence at 519 nm. Control titrations lacking M13mp18 ssDNA were conducted at each NaCl concentration, and were subtracted from their corresponding experimental data sets. Binding data in terms of fractional saturation of the ssDNA lattice (
) with Gp32F ligand were derived from corrected fluorescence data. The observed affinity K was determined at each salt concentration from the midpoint of the titration according to Equation 1,
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was determined by curve fitting to a sigmoidal function using DynaFit (BioKin, Ltd.). Holding K constant, the data then were compared by inspection to theoretical isotherms derived from the equations of McGhee and von Hippel (44, 45), while selecting different pairs of and K values to best describe the lower half of the binding data (43). Due to the highly cooperative nature of Gp32F-ssDNA interactions and the interdependence of and K, the "optimal" is best given as a range of values that can adequately describe the data. Therefore the error in the corresponding K results from assuming values at the high, middle, and low ends of the range. The resulting K values were corrected for magnesium ion effects using Equations 3 and 4 of Record et al. (47), assuming 
= 0.71, where is the fraction of counterions thermodynamically bound to ssDNA, and assuming m'= 3, where m' is the number of charge interactions formed between Gp32 and ssDNA (22, 46, 47). Ionic effects on Gp32F-ssDNA interactions were estimated according to Equation 2,
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Gp32F-ssDNA Disruption Assays—Competition titrations of pre-formed Gp32F-ssDNA with UvsX protein were performed under steady-state conditions at 25 °C in Buffer B containing 200 mM NaCl, 1 mM ATP, ATP-regenerating system (6 units/ml pyruvate kinase + 2.3 mM phosphoenolpyruvate), 3.5 µM M13mp18 ssDNA, 3.5 µM Gp32F, variable UvsX, and 0 or 1 µM UvsY depending on experiment. Control titrations lacking ssDNA or UvsY contained a volume of TE or UvsY storage buffer, respectively, equal to the volume of the missing component.
Time Resolved ssDNA Binding Competition Assays—The competition between Gp32F and UvsX proteins for a limiting number of ssDNA binding sites was measured as a function of time by adding M13mp18 ssDNA (3.5 µM) to a solution containing both proteins (0.5 µM Gp32F, 0.875 µM UvsX) and monitoring subsequent changes in fluorescein fluorescence. The ssDNA concentration used is stoichiometric with respect to Gp32F alone (binding site size, n = 7 nucleotide residues) or to UvsX alone (n = 4) but cannot saturate both proteins simultaneously (22, 25). Reactions were carried out at 25 °C in Buffer B containing 200 mM NaCl. To compare the effects of different nucleotides on Gp32F/UvsX competition, some reaction mixtures contained 1 mM AMP, 1 mM ADP, 1 mM ATP
S, or 1 mM ATP (with or without ATP regenerating system consisting of 2.3 mM phosphoenolpyruvate, and 6 units/ml pyruvate kinase). To study UvsY effects, some reactions contained variable amounts of wild-type UvsY protein (0-0.875 µM), or UvsYK58A (0.875 µM) or UvsYK58A,R60A (0.875 µM) mutant proteins. UvsY proteins, when present, were preincubated along with Gp32F and UvsX in a buffer/nucleotide mixture prior to the addition of ssDNA. In all reactions, a baseline fluorescence signal was obtained by collecting a 100-s time course (
ex = 460 nm, em = 519 nm) prior to ssDNA addition. ssDNA was added with rapid mixing, signal was re-acquired, and a 300-700-s time course (depending on experiment) was collected. For reaction series containing variable protein components, solvent composition was kept constant by adding appropriate amounts of protein storage buffers.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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ssDNA-induced Changes in Gp32F Fluorescence—Results demonstrate that Gp32F fluorescence is enhanced upon binding to ssDNA, but is not affected by UvsX and UvsY proteins. Fluorescence emission spectra of Gp32F were measured at 200 mM NaCl in the presence/absence of ssDNA, UvsX, or UvsY (Fig. 1). Excited at 460 nm, Gp32F reaches its peak emission at 518 nm in the absence of ssDNA. The addition of M13mp18 ssDNA to the solution causes a 
2-fold enhancement in the intensity of Gp32F fluorescence, with the emission peak slightly red-shifted to 519 nm (Fig. 1). Under identical solution conditions, the addition of double-stranded DNA has no effect on Gp32F fluorescence (data not shown). Therefore Gp32F retains preferential ssDNA binding activity, and the binding reaction alters the environment of the conjugated fluorescein molecule in a way that favors enhanced fluorescence.
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Gp32F Binds to ssDNA with High Affinity and Cooperativity— The ssDNA-induced enhancement of Gp32F fluorescence was used to measure the ssDNA binding parameters of Gp32F, for purposes of comparison with unmodified Gp32. Salt-back titrations (data not shown) demonstrated that Gp32F forms tight, stoichiometric complexes with ssDNA at NaCl concentrations 
200 mM, whereas higher NaCl concentrations destabilize Gp32F-ssDNA interactions with a dissociation midpoint at 350 mM NaCl. Reverse titrations of Gp32F with ssDNA (data not shown) under tight binding conditions (50 mM NaCl) demonstrated that Gp32F has a binding site size of 7.6 ± 0.2 nucleotide residues, which is similar to values reported previously for unmodified Gp32 (22). This finding suggests that Gp32F and Gp32 are equally effective in eliminating ssDNA secondary structure, allowing both to saturate random sequence ssDNA lattices.
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for Gp32F-ssDNA interactions were estimated by performing forward titrations of a fixed amount of M13mp18 lattice with protein under salt conditions that permit relatively weak, non-stoichiometric binding (260, 300, or 340 mM NaCl) (Fig. 2). ssDNA binding parameters were calculated as described under "Experimental Procedures" and are listed in Table 2. All titration curves revealed obvious sigmoidicity, indicative of highly cooperative binding of Gp32F to ssDNA. The results indicate that with increasing NaCl concentration, the magnesium-corrected intrinsic binding constant, KMg-corr, decreases markedly, whereas the cooperativity parameter remains on the order of 103. A log-log plot of KMg-corr versus [NaCl] (data not shown) was linear with a least-squares slope of dlogKMg-corr/dlog[NaCl] = -5.7. Then from Equation 2 and assuming m' = 3 (22), the value of a = 3.6 indicating 3-4 anions displaced per Gp32F bound to ssDNA.
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) of Gp32F is 12-fold lower than that of unmodified Gp32. Other aspects of ssDNA binding, including binding site size, cooperativity, and salt dependence of KMg-corr, are highly similar between Gp32F and Gp32. The high extrapolated KMg-corr values of Gp32F and Gp32 are consistent with the experimentally observed stoichiometric binding of both proteins to ssDNA in 200 mM NaCl. The moderately diminished affinity of Gp32F for ssDNA, compared with unmodified Gp32, is confirmed by the observation that a slight excess of Gp32 can slowly but completely displace Gp32F from ssDNA in 200 mM NaCl.5
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In the absence of UvsY, the addition of limiting ssDNA to a Gp32F/UvsX mixture in 200 mM NaCl, 1 mM ATP (+ ATP regenerating system) causes a rapid jump in Gp32F fluorescence followed by a slower increase to a plateau signal reflective of a saturated Gp32F-ssDNA complex (Fig. 4). Therefore Gp32F wins the kinetic competition with UvsX for ssDNA binding sites under these conditions. Under otherwise identical conditions, inclusion of UvsY in the protein mixture affords a distinct curve upon ssDNA addition: an immediate fluorescence jump followed by an exponential fluorescence decay (Fig. 4). The initial, acute signal enhancement represents the rapid binding of Gp32F onto ssDNA, whereas the subsequent exponential decay curve represents the slower nucleation and/or propagation of UvsX filaments onto this Gp32F-covered ssDNA in the presence of UvsY, with concomitant displacement of Gp32F. The addition of excess ssDNA at the end of this experiment restored Gp32F fluorescence back to its high ssDNA-enhanced level (data not shown), suggesting that the Gp32F displaced during filament assembly is free in solution and competent to bind free lattice. Thus the pre-steady state kinetic competition data (Fig. 4) are consistent with the steadystate titration data in Fig. 3, both demonstrating that UvsY is required for UvsX to out-compete Gp32F for ssDNA in the presence of 200 mM NaCl and 1 mM ATP. The complete reaction in Fig. 4 reflects the sequential filament assembly process that is thought to occur in vivo, details of which will be presented under "Discussion."
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S for ATP plus regenerating system generates a qualitatively similar time course (Fig. 5A). UvsY is still required for optimal Gp32F displacement in reactions containing ATPS.6
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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An important outcome of this study is the observation that UvsX protein requires both ATP and UvsY to displace Gp32 from ssDNA under stringent salt conditions. The existence of transiently ATP-stabilized UvsX-ssDNA interactions was previously inferred from differential effects of ATPS versus ATP on binding isotherms and from the high catalytic turnover of UvsX ATPase activity (25). In this study, direct evidence shows that both ATPS and ATP are capable of inducing a form of UvsX that can displace Gp32 from ssDNA. With ATP as substrate, the high-affinity form is maintained under steady-state conditions for ATP hydrolysis, as long as the ATP:product ratio remains high. However, the depletion of ATP substrate and accumulation of ADP/AMP products leads to loss of the high affinity form and ultimately to re-occupation of ssDNA by Gp32 at the expense of UvsX. This is the first direct evidence for modulation of the ssDNA binding state of UvsX during the ATPase cycle, a process that was previously identified for the Escherichia coli RecA protein (46). Note that although ATP binding by UvsX is necessary for Gp32 displacement, it is not sufficient because all displacement reactions in 200 mM NaCl are co-dependent on UvsY and ATP (ATPS). We recently showed that UvsY globally stabilizes UvsX-ssDNA interactions, with the most stable complex formed in the presence of both UvsY and ATPS (33). The implication is that Gp32-ssDNA binding energy is surmounted through the synergistic stabilizing effects of UvsY and nucleoside triphosphate ligands on UvsX-ssDNA interactions.
Based on previous studies, a model of UvsY-mediated presynaptic filament assembly was proposed in which a tripartite UvsY-Gp32-ssDNA complex acts as a key intermediate (2, 3, 32). UvsY introduces structural changes in ssDNA that weaken Gp32-ssDNA interactions within this complex, thus "priming" it to accept UvsX-ssDNA filament nucleation events and to initiate the displacement of Gp32. Results of the current study are consistent with this model, reflected in Fig. 9. The data demonstrate that UvsY itself does not displace Gp32 from ssDNA because the Gp32F-ssDNA fluorescence signal is not perturbed by UvsY (Fig. 1) under salt conditions in which both proteins are known to co-occupy the lattice (32, 33). This observation supports previous conclusions that both proteins retain intimate contacts with ssDNA within the UvsY-Gp32-ssDNA intermediate (19, 32).
Results of kinetic competition assays (Figs. 4, 5, 6, 7, 8) suggest that there is a highly ordered sequence of protein-ssDNA association and dissociation events during presynaptic filament assembly. A model for this process is shown in Fig. 9. We propose that Gp32 coats the lattice first, removing ssDNA secondary structure (Fig. 9A). UvsY adds next, forming the UvsY-Gp32-ssDNA intermediate (Fig. 9B). UvsY next recruits ATP-bound UvsX subunits to the ssDNA, forming a presynaptic filament nucleation complex with local displacement of Gp32 (Fig. 9C). Further addition of ATP-bound UvsX to an ATP-capped filament end (presumably the 3' end) leads to directional propagation while progressively displacing more Gp32 (Fig. 9D). This sequence of events is logical given the rapid on-rates observed for the formation of Gp32-ssDNA cooperative clusters (53), the strong affinity of UvsY for Gp32-ssDNA (32), and given the prevailing conditions during T4 infection of E. coli, wherein sufficiently high concentrations of Gp32 are maintained to rapidly saturate any ssDNA that is generated during DNA replication, recombination, and repair transactions (12, 13). Therefore the kinetic competition assay appears to provide a reasonable biochemical model for the kinetics of the presynapsis process as it occurs in vivo. We note that filament assembly in vitro is a relatively slow process, requiring 2-3 min for the ensemble to approach steady-state under the conditions of our experiments (Figs. 4 and 5). Nevertheless, this rate appears to be kinetically competent with respect to rates of UvsX-catalyzed DNA strand exchange (6, 26, 54). The filament assembly rates we observe are also consistent with the slow approach to steady-state rates of ssDNA-dependent ATP hydrolysis observed for UvsX protein in the presence/absence of Gp32 and UvsY (8).7
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Our results indicate that there is a stoichiometric requirement for UvsY protein for optimal assembly of UvsX onto Gp32-saturated DNA. The rate of Gp32F displacement by UvsX/UvsY appears to approach a maximum value as the UvsY concentration approaches 1:1 stoichiometry with respect to UvsX and/or ssDNA binding sites (Fig. 7). The requirement for UvsY clearly is not all-or-none, however, because UvsY:UvsX molar ratios as low as 1:11, monomer:monomer (1:66 hexamer: monomer), are sufficient to promote low rates of Gp32F displacement. These findings are consistent with previous observations that the DNA strand exchange, ssDNA-dependent ATPase, and recombination-dependent DNA synthesis promoting activities of UvsX are optimal in the presence of stoichiometric UvsY, but are also significantly stimulated at lower UvsY:UvsX ratios (8-10). It is unclear from our data whether the low rates of Gp32F displacement observed at low UvsY: UvsX ratios represent the assembly of a few long filaments as opposed to many shorter ones, or if a critical UvsY:UvsX stoichiometry exists within each filament. More detailed kinetic studies (in progress) are needed to distinguish whether UvsY exerts its most important effects on filament nucleation, propagation, or both.
Experiments with UvsY missense mutants defective in protein-ssDNA interactions also demonstrate the key role of these interactions in mediating presynaptic filament assembly. The UvsY K58A and R60A mutations dramatically decrease, but do not totally eliminate, mediated Gp32F-ssDNA disruption by UvsX. Thus our data are consistent with previous observations that both missense mutants weakly stabilize UvsX-ssDNA interactions and retain residual recombination mediator activities in UvsX-catalyzed DNA strand exchange and ssDNA-dependent ATPase reactions (27, 33). The fact that both Gp32-ssDNA destabilization and UvsX-ssDNA stabilization functions depend largely on UvsY-ssDNA interactions (32, 33) suggests that there is linkage between the two processes. It is possible to envision a seamless progression from a pre-existing Gp32-ssDNA complex to a UvsY-Gp32-ssDNA intermediate to a UvsY- and nucleoside triphosphate-stabilized UvsX-ssDNA nucleation complex, with ssDNA structural change as the common denominator. The residual mediator activities of UvsYK58A and UvsYK58A,R60A mutants presumably result from a relatively weak stabilization of UvsX-ssDNA via protein-protein interactions.
The enhancement of Gp32F fluorescence upon binding to ssDNA reflects an environmental change of the fluorescein moiety. The environmental change could be due to the proximity of bound ssDNA to the fluorophore, contiguous binding of Gp32F molecules in cooperative clusters, an ssDNA-induced conformational change in Gp32F, or some combination of these effects. The first of these possibilities seems unlikely based on structural considerations. Cysteine 166, the residue to which 6-IAF is covalently conjugated, is not expected to be involved in direct ssDNA contacts. In the x-ray crystallographic structure of the ssDNA-binding core domain of Gp32 (14), Cys-166 is located on the surface of sub-domain II of the core, on a distal turn of the oligonucleotide/oligosaccharide-binding fold motif of the protein and oriented away from the ssDNA binding surface. Cys-166 is 25 Å away from the nearest residue of the KRKTS sequence (a so-called "LAST" motif) found at positions 110-114 of Gp32, which is the major ssDNA binding surface identified in both x-ray and NMR studies (56, 57). Therefore the fluorescein moiety of Gp32F is probably distant from the bound ssDNA, making it unlikely that interactions with ssDNA directly enhance its fluorescence signal. Instead, evidence suggests that ssDNA-dependent conformational changes in the core domain may be driving the observed changes in Gp32F fluorescence. Cys-166 of Gp32 is located near one of the binding surfaces for Gp59, a T4-encoded helicase loading protein known to modulate the ssDNA binding affinity of Gp32 (21, 58), and can be chemically cross-linked to Gp59 (36). Also, Cys-166 of Gp32 is situated in a hydrogen-bonded turn with residue Asp-163, which is the target for the bacteriophage P2 Tin protein, a molecule that poisons Gp32 function (59). These observations suggest that Cys-166 resides in a region of Gp32 that shifts conformation in response to ssDNA as well as to regulators of ssDNA binding activity, which could also explain why the attachment of 6-IAF at Cys-166 has a moderate weakening effect on Gp32-ssDNA interactions. Other factors including the A- and B-domains of Gp32 may also influence the environment of the fluorescein moiety of Gp32F. These domains are predicted to be distant from Cys-166 in monomeric Gp32 due to chain topology (14), however, in cooperative clusters of Gp32-ssDNA the A- and B-domains could pack against the core domains of neighboring molecules in ways that change the chemical environment surrounding Cys-166. Currently no structural data are available on the inter- or intramolecular interactions of Gp32 core, A-, and B-domains.
The fluorescence properties and strong, cooperative ssDNA binding activity of Gp32F make it a useful reagent for studying a number of DNA transactions. Potential applications include: 1) detection and quantification of ssDNA; 2) measurement of ssDNA-binding parameters for other proteins via competitive binding assays; 3) real-time assays for ssDNA 
dsDNA interconversions involved in DNA synthesis, exonuclease, helicase unwinding, and other reactions; and 4) rapid kinetics and single-molecule studies of protein-ssDNA displacement reactions and ssDNA dsDNA interconversions. Future studies employing Gp32F are likely to yield important new insights on the dynamics of SSB proteins during homologous recombination, DNA replication, and DNA repair processes.
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FOOTNOTES |
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1 Supported by graduate fellowships in structural biology from the Department of Energy-Experimental Program to Stimulate Competitive Research.
2 To whom correspondence should be addressed: B407 Given Bldg., University of Vermont College of Medicine, Burlington, VT 05405. Tel.: 802-656-8260; Fax: 802-656-8220; E-mail: smorrica{at}zoo.uvm.edu.
3 The abbreviations used are: ssDNA, single-stranded DNA; SSB, single-stranded DNA binding protein; Gp32, bacteriophage T4 gene 32 protein; 6-IAF, 6-iodoacetamidofluorescein; Gp32F, T4 gene 32 protein labeled with 6-iodoacetamidofluorescein; ATPS, adenosine 5'-O-(thiotriphosphate).
4 J. Farb and S. Morrical, unpublished results.
5 N. Qian, J. Chen, and S. Morrical, unpublished experiments.
6 J. Liu and S. Morrical, unpublished results.
7 S. Morrical et al., unpublished observations.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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3 s). Protein amounts are at 1:1 stoichiometry with respect to ssDNA binding sites assuming a binding site size of n = 7 nucleotide residues per monomer of Gp32F, and of n = 4 nucleotide residues per monomer of UvsX or UvsY.
3' direction as ATP-bound UvsX subunits slowly add to the 3' filament end, displacing more Gp32 subunits in the process. E, the first UvsX subunits to bind are the first to hydrolyze ATP, generating a relatively aged, ADP-capped 5' filament end. The ADP-bound UvsX subunits are now vulnerable to displacement by Gp32. Differential competitive effects between Gp32 and the ATP-versus ADP-capped filament ends creates dynamic instability in the complex, which could lead to filament treadmilling.