Proc. Natl. Acad. Sci. USA Vol. 93, pp. 10051-10056, September 1996 Biochemistry

ATPase activity of Escherichia coli Rep helicase crosslinked to single-stranded DNA: Implications for ATP driven helicase translocation (kinetics/mechanism/protein-DNA crosslinking/protein oligomerization/energy transduction) ISAAC WONG AND TIMOTHY M. LOHMANt Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, 660 South Euclid Avenue, Box 8231, St. Louis, MO 63110 Communicated by Carl Frieden, Washington University School of Medicine, St. Louis, MO, May 22, 1996 (received for review March 24, 1996)

ABSTRACT To examine the coupling ofATP hydrolysis to translocate unidirectionally along ssDNA, although direct evi- helicase translocation along DNA, we have purified and dence in support of this is lacking. On the other hand, we (14) characterized complexes of the Escherichia coli Rep protein, a have shown that unidirectional translocation along ssDNA is not dimeric DNA helicase, covalently crosslinked to a single- essential for DNA unwinding by the E. coli Rep helicase, a stranded hexadecameric oligodeoxynucleotide (S). Crosslinked 3' ->5' DNA helicase. Furthermore, we have proposed an active, Rep monomers (PS) as well as singly ligated (P2S) and doubly rolling model for Rep-catalyzed DNA unwinding (13) based on ligated (P2S2) Rep dimers were characterized. The equilibrium our finding that one subunit ofthe Rep dimer binds directly to the and kinetic constants for Rep dimerization as well as the duplex region to be unwound while the other subunit binds to steady-state ATPase activities of both PS and P2S crosslinked ssDNA (13, 14), and we noted that such a rolling model could also complexes were identical to the values determined for un- be envisioned as a mechanism for translocation of the Rep dimer crosslinked Rep complexes formed with dT16. Therefore, ATP along ssDNA. hydrolysis by both PS and P2S complexes are not coupled to DNA To distinguish between these mechanisms for helicase trans- dissociation. This also rules out a strictly unidirectional sliding location along ssDNA, we reasoned that any directional bias in mechanism for ATP-driven translocation along single-stranded translocation requires coupling of ATP hydrolysis to movement. DNA by either PS or the P2S dimer. However, ATP hydrolysis by In particular, a strictly unidirectional sliding mechanism (no the doubly ligated P2S2 Rep dimer is coupled to single-stranded "slippage"), in which the same protein subunit maintains contact DNA dissociation from one subunit ofthe dimer, although loosely with the ssDNA during translocation, would require tight cou- (low efficiency). These results suggest that ATP hydrolysis can pling to hydrolysis, while a "rolling" mechanism without direc- drive translocation of the dimeric Rep helicase along DNA by a tional bias would not. Toward this end, we characterized the "rolling" mechanism where the two DNA binding sites of the ATPase activities of covalently crosslinked Rep-single-stranded- dimer alternately bind and release DNA. Such a mechanism is oligodeoxynucleotide complexes, reasoning that severe inhibition biologically important when one subunit binds duplex DNA, of the ATPase activities of such complexes would result if followed by subsequent unwinding. ATP-driven translocation occurs by a strictly unidirectional slid- ing mechanism. Such crosslinked Rep-ssDNA complexes will DNA helicases are motor proteins that use the chemical energy also provide a useful means for studying putative intermediates of nucleoside 5'-triphosphate (NTP) hydrolysis to perform the in the DNA unwinding reaction. mechanical work of disrupting the base pairs between comple- mentary strands of duplex DNA to form single-stranded DNA MATERIALS AND METHODS (ssDNA) intermediates (1, 2). These molecular motors are es- Reagents and Buffers. [a-32P]ATP (3000 Ci/mmol; 1 Ci = 37 sential components of most DNA metabolic and processing GBq) was obtained from Amersham. Spectrophotometric grade machineries in all organisms (2), and mutations in helicases glycerol, HPLC grade methanol, and 99% triethylamine were involved in DNA repair processes have been linked to a number obtained from Aldrich. Sulfo-N-succinimidyl-6-(4'-azido-2'- of human skin cancers (3-5). Despite the importance of these nitrophenylamino)hexanoate (sulfo-SANPAH) was obtained enzymes and the large number of DNA helicases that have been from Pierce. Amino-modifier-C2-dT phosphoramidite was ob- identified, mechanistic studies on these enzymes have only begun tained from Glen Research (Sterling, VA). All solutions were recently (for reviews, see refs. 1, 6, and 7). made with reagent grade chemicals, except as noted above, using DNA helicases must translocate along DNA to unwind DNA Milli-Q H20-i.e., distilled H20 that was de-ionized using a processively and thus have features in common with motor Milli-Q Water Purification System (Millipore). All binding ex- proteins such as cytoplasmic kinesin (8-10). DNA helicases periments and ATPase assays were carried out in BBM buffer [20 appear generally to be oligomeric, primarily dimeric or hexameric mM Tris HCl, pH 7.5 at 4°C/6 mM NaCl/5 mM MgCl2/10% (1, 6, 7); for example, the Escherichia coli Rep helicase functions (vol/vol) glycerol]. Triethyl-ammonium bicarbonate buffer as a dimer (11), with both subunits able to bind DNA and ATP (TEAB, 1 M) was made by bubbling CO2 (g) derived from (10, 12, 13). Of particular mechanistic interest is how helicases subliming dry ice into an aqueous solution containing >1 M translocate along DNA and how this is coupled to NTP hydro- triethylamine at 0°C for 4 hr or until a pH of 7.5 was achieved; lysis. In this regard, most helicases display a macroscopic "polar- Milli-Q H20 was then added to achieve a final concentration of ity" in DNA unwinding assays in vitro-i.e., unwinding by most 1 M. Kinase buffer was 50 mM Tris HCl (pH 7.5 at 25°C), 10 mM helicases is stimulated if the DNA duplex possesses a 5' ssDNA MgCl2, and 10 mM 2-mercaptoethanol. "tail" flanking the duplex (a 5' ->3' helicase) or a 3' ssDNA tail Proteins, Enzymes, and Oligodeoxynucleotides. E. coli Rep (a 3' ->5' helicase). This observation has led to the suggestion protein was purified to >99% homogeneity from E. coli MZ-1/ that helicases couple the energy derived from ATP hydrolysis to Abbreviations: ssDNA, single-stranded DNA; sulfo-SANPAH, sulfo- The publication costs of this article were defrayed in part by page charge N-succinimidyl-6-(4'-azido-2'-nitro-phenylamino)hexanoate. payment. This article must therefore be hereby marked "advertisement" in tTo whom reprint requests should be addressed. e-mail: accordance with 18 U.S.C. §1734 solely to indicate this fact. [email protected]. 10051 Downloaded by guest on September 27, 2021 10052 Biochemistry: Wong and Lohman Proc. Natl. Acad. Sci. USA 93 (1996) pRepO (15) as described (16). Rep concentration was deter- sample to 25 ml and the sample was dialyzed for 6 h against mined spectrophotometrically using s20 = 7.68 x 104 M-l cm-I 1 liter of the same buffer containing 100 mM NaCl. The for Rep monomer (14). An ATPase-deficient mutant of Rep, dialysate was reloaded onto the Macro-Prep Hi-Q column K281, in which Lys-28 was replaced with Ile, was constructed and that had been reequilibrated with 100 mM NaCl. The column purified to >99% purity (unpublished data). T4 polynucleotide was rinsed with 10 ml buffer containing 100 mM NaCl and kinase was purchased from United States Biochemical. Oligode- eluted with 1 M NaCl as before. The fractions containing oxynucleotides dT16, d(T3C2T12), d(T7C2T8), d(T12C2T3), where protein and DNA were pooled and dialyzed against 5x C2 denotes amino-modifier-C2-dT, were synthesized using an binding buffer. Due to the thermodynamic stability of P2S Applied Biosystems model PCR-mate 391 DNA synthesizer and and the negative cooperativity of binding DNA to P2S to were purified to >99% homogeneity as described (17) and form P2S2, the purified crosslinked mixture contained a signif- dialyzed (Spectra/Por 7 MWCO 1000) into Milli-Q H20 for icant fraction of uncrosslinked Rep subunits in the form of P2S. storage. Oligodeoxynucleotide concentrations were determined However, all uncrosslinked DNA has been removed. spectrophotometrically in 10 mM Tris-HCl (pH 7.5), 1 mM ATPase Assay. ATPase activity was determined at 4°C by EDTA, and 150 mM NaCl at 25°C using 8260 = 1.29x105 measuring the initial rate of conversion ofATP to ADP using M-l cm-1 per molecule. [a-32P]ATP (Amersham) in BBM buffer. Reactions were Synthesis and Purification ofd(T7S2T8). Solid sulfo-SANPAH typically initiated by addition of 10 ,ul of 10 mM ATP to 90 was added to d(T7C2T8) (350 ,uM) in 0.5 M Na-carbonate ,ul protein. At regular time intervals, 10 ,lI aliquots were (pH 9.0) buffer to a final concentration of 25 mM and removed and quenched into 10 ,ul 0.5 M EDTA (pH 8.5). allowed to react in the dark at 25°C for .4 h. Three volumes Intervals between time points ranged from S to 20 s depend- of ice-cold ethanol was added and the sample chilled at ing on the anticipated rate of hydrolysis. Eight to 10 time -20°C for 1 h to precipitate the DNA. The large bright points were taken per assay, but only the linear portion of orange pellet was collected by centrifugation at 4°C and each time course, <60% product formation, was used to rinsed with 1 volume of 1,4-dioxane, followed by 1 volume of determine the initial velocity. The extent of ADP formation 100% ethanol, then dried and resuspended in Milli-Q H20 was monitored by spotting 1 ,lI onto polyethyleneimine- at 55°C for 5 min. The sample was chilled on ice for 30 min cellulose TLC plates (Merck). TLC plates were developed and centrifuged for 15 min at 4°C. The supernatant was using 0.3 M potassium phosphate (pH 7.0) as the mobile transferred to a fresh microfuge tube, being careful to avoid phase, dried, and imaged on a Betascope 603 direct ,3-emis- an orange oily residue that clings to the old microfuge tube. sion imager (Betagen). Spots corresponding to radiolabeled The solution was then purified through a 25 ml Bio-Gel P2 supplied by column in 50 mM Na-carbonate buffer (pH 9.0). The faintly ATP and ADP were quantitated using software pink band eluting in the void volume was collected and the manufacturer. contained d(T7S2T8). An equal volume of 1 M TEAB buffer Concentration Dependence of the ATPase Activity of (pH 7.5) was added to the pooled fractions and the sample Crosslinked Rep-d(T7S2T8). Crosslinked Rep-d(T7S2T8) was se- loaded onto a 900 mg Maxi-Clean C18 cartridge (Alltech rially diluted to span a range of concentrations from 2 ,uM to 27 Associates) activated with 1 ml methanol then equilibrated nM. The solutions were allowed to equilibrate at 4°C in the cold with 5 ml 10 mM TEAB (pH 7.5). Following washes of 5 ml room for 30 min and then assayed for ATPase activity as 10 mM TEAB (pH 7.5) and 1 ml of Milli-Q H20, d(T7S2T8) described above. The resulting isotherm was analyzed by nonlin- was eluted with 50% (vol/vol) methanol and dried in the ear least-squares methods (18) and the following three equilibria: Speed-Vac. The purified DNA was >97% derivatized as K2 determined spectroscopically using an extinction coefficient at 260 nm, 8260 = 1.4 x 105 M-1cm-1. d(T3S2T12) and K3 d(T12S2T3) were prepared in the same manner. Care was PS* + P'=±P2S taken to avoid buffers that contained ammonia or primary K5 amines-e.g., Tris or EDTA-and to minimize exposure of 2PS*;=±P2S2 samples to light. However, the compound is stable in ambient light for at least 20 min. For a mixture of native and crosslinked Rep monomers, the Synthesis and Purification of Crosslinked Rep-d(T7S2T8. concentration of PS at any concentration of total Rep pro- Rep protein was dialyzed extensively against 5x binding tomer, PT, is given by the real nonnegative root of Eq. 1: buffer (100 mM Tris, pH 7.5/30 mM NaCl/5 mM EDTA/ K2(K2K3 + [p1 2K2K5)[pS12 50% glycerol). Crosslinking was carried out at 1 ,uM Rep [PS]3 + 2K2K3K5+3K3 monomers and S ,tM d(T7S2T8) in 25 ml 2x binding buffer mM mM mM EDTA/20% (40 Tris, pH 7.5/12 NaCl/2 +K2[1 - K3PT(2r - 1)] + 1[PS] glycerol) in a shallow 3-inch-diameter dish in the cold room. 2K2K3,K5 [S The sample was UV irradiated for 4-6 h. Buffer containing 50 mM Tris (pH 7.5) and 20% glycerol without salt was rPT added to replace volume loss due to evaporation, and 5 M 2K32K,K5° [1] NaCl added to a final concentration of 100 mM. This was = loaded onto a 2 ml Macro-Prep Hi-Q anion exchange column where r 0.61 is the fraction of crosslinked subunits. The (Bio-Rad) equilibrated in 50 mM Tris (pH 7.5), 100 mM concentrations of the other species are given by Eqs. 2a-d. NaCl, and 20% glycerol, rinsed with 4 ml of the same buffer, - (1 r)(2K2K4[PS] + K2 + 1)[PS] [2a] and eluted with buffer containing 1 M NaCl. Fractions of six -P]= (2r - 1)K2K3[PS] + r drops each were collected. Pink DNA containing fractions were pooled (-3 ml) and carefully layered onto a 30 ml [PS*] = K2[PS] [2b] Bio-Gel P-6 column equilibrated with buffer containing 50 = [2c] mM Tris (pH 7.5), 1 M NaCl, and 10% glycerol running at [P2S] K2K3[PS][P] a 5 ml/h. Fractions of 1.5 ml were collected while monitoring [P2S2] = K22K[PS]2 [2d] the absorbance of the eluate at both 254 nm and 280 nm. Crosslinked complexes (A280 > A260) eluted in the void The fit is not sensitive to the isomerization between PS and volume which was collected and pooled. Buffer containing PS*; however, the equilibrium was included, constrainingK2 = 50 mM Tris (pH 7.5) and 20% glycerol was added to the 13 as determined by Bjornson et al. (19). The specific ATPase Downloaded by guest on September 27, 2021 Biochemistry: Wong and Lohman Proc. Natl. Acad. Sci. USA 93 (1996) 10053

activity of PS and PS* in the fit was constrained at 2 s-1 as A determined for native uncrosslinked Rep (20). Kinetics of Transient Formation of P2S2. For the experi- ments measuring the transient formation of P2S2, 75 ,ul of a Rep-d(T7S2T8) solution (2 ,AM or 1.66 ,uM) preincubated with 0.34 ,uM Rep was mixed with 75 Al of 2x [dT16] at t = 0. At 20-s intervals, 10 Al aliquots were removed and mixed with 10 ,ul 2x [ATP] and allowed to react for 10 s. Ten microliters was then transferred into 10 p,l of '0.5 M EDTA to quench the reaction. In this experiment, the time courses were not linear beyond 10 s. However, within the first 10 s of the reaction, the rate of ATP hydrolysis was constant as determined by rapid k, k2 ki k2 2P+2Sw- 'PS+P+S tPS*+P+S rPS*+PS ' 2PS* ki k2 k2 'ji

B __ Rep-DNA M4 P2S+S of P2S2 k-4 _4 free DNA Scheme I 1 2 3 4 quench-flow experiments (data not shown). The data were FIG. 1. Synthesis of photoreactive crosslinkable single-stranded oli- analyzed to obtain the rate constants for Scheme I and kcat for godeoxynucleotides. Three analogues of dT16, d(T3C2T12), d(T7C2T8), P2S2 using FITSIM (21). and d(T12C2T3), were synthesized (A) with amino-C2-modifier-dT phos- All Rep species bound to dT16 were treated as being phoramidite (denoted C2) containing a primary amine attached via a equivalent to the crosslinked Rep-d(T7S2T8) species with the linker arm to the C5 of thymidine incorporated at positions 4, 8, and 13 exception that the rate of dissociation of dT16 from P2S2 when from the 5' end. These were then chemically coupled to the hetero- bifunctional crosslinker sulfo-SANPAH in the dark via its amino-reactive the other subunit was crosslinked to was halved Rep d(T7S2T8) succinimidyl functionality to yield the corresponding photoreactive relative to when neither subunit was crosslinked. Because our crosslinking hexadecamers, d(T3S2Tu2), d(T7S2T8), and d(T12S2T3). In- version of FITSIM does not allow output factors to be floated, cubation with Rep in the presence of UV irradiation at 366 nm yields the best fit value of kcat for P2S2 was obtained by executing fits covalently crosslinked Rep-DNA complexes as shown by SDS/PAGE using different values of kcat for P2S2 and manually minimizing (B). Lanes: 1, control without irradiation; 2-4, irradiation products with the sums of squares of differences. d(T3S2T12), d(T7S2T8), and d(T12S2T3), respectively. Data Analysis. FITSIM (21) was executed on an IBM PS/2 Model 76. NONLIN (18) was executed on a Hewlett-Packard unaffected by the crosslinking. Under these reaction conditions, 715. All other data analyses were performed using KALEIDA- the only species present are P2S, PS (including PS*), and P. The GRAPH (Synergy Software, Reading, PA) on an Apple Macin- addition of 42 nM P converted all of the PS to P2S, which led to tosh Quadra 700. the increased ATPase activity. Therefore, 121 nM (one half of 200 nM + 42 nM), or 61% of the original 200 nM Rep protomers RESULTS were crosslinked to d(T7S2T8). Because only one Rep monomer or one subunit of a dimer can Rep dimerization is induced upon DNA binding (11) such that bind to a 16-base oligonucleotide (11, 12), we synthesized three the dimerization equilibrium constant increases by at least 4-5 analogs of dT16: d(T3S2T12), d(T7S2T8), and d(T12S2T3), each orders of magnitude on binding DNA (12, 13) and dimerization containing a single thymidine with a photoreactive azido-moiety increases Rep's ATPase activity dramatically (19, 20, 22). Con- attached to its C5 position (designated S2) as described in Fig. LA. sequently, Rep can form multiple complexes with a 16-base Pilot crosslinking studies were performed, the reaction products oligodeoxynucleotide, depending on the DNA and Rep concen- were separated by denaturing SDS/PAGE, and the radioactive trations. Rep monomers can be free (P) or bound to ssDNA (PS), bands to free and crosslinked DNA were corresponding quanti- and two types of Rep dimers can form, P2S and P2S2, where one tated (Fig. 1B). These studies indicated that the three oligode- or both subunits are bound to ssDNA, respectively (12, 13). These oxynucleotides can be crosslinked to Rep; however, they showed different crosslinking efficiencies, with d(T3S2T12) being the least 10.0 effective (-35% Rep crosslinked) and d(T7S2T8) the most ef- (- fective (80-90%). We therefore purified mg quantities of uo

crosslinked Rep-d(T7S2T8). - Although all of the DNA in the "purified" samples of Rep- 2 7.5 d(T7S2T8) was crosslinked to Rep subunits, the sample also

contained free Rep protein dimerized (noncovalently) to 6- crosslinked Rep, because DNA binding induces Rep dimeriza- tion. We determined the fraction of crosslinked Rep subunits by 5.0 directly titrating an aliquot of the purified sample, containing 200 50 100 150 nM total Rep protomers, with free Rep monomers (containing (Rep] added no DNA) while monitoring the ATPase activity (Fig. 2). The (nM monomers) ATPase activity increased linearly on addition of free Rep FIG. 2. Direct titration to determine fraction of crosslinked Rep monomers to a concentration of 42 nM and then plateaued at an subunits. The "purified" Rep-d(T7S2T8) sample contains a mixture of activity of 9.5 s-1 per total Rep monomer, corresponding to an crosslinked and uncrosslinked Rep subunits. To determine the fraction of activity of 19 s-1 per crosslinked P2S. This plateau value is crosslinked Rep protomers in this mixture, Rep-d(T7S2T8) (200 AM total identical within error to the rate of ATP hydrolysis by the protomers) was mixed with increasing amounts of free Rep monomers uncrosslinked P25, indicating that the ATPase activity of P2S was (BBM buffer at 4°C) and the ATPase activity was monitored. Downloaded by guest on September 27, 2021 10054 Biochemistry: Wong and Lohman Proc. Natl. Acad. Sci. USA 93 (1996)

are shown schematically in Table 1. Under the conditions of our Table 2. Kinetic and equilibrium constants for protein experiments (BBM buffer at 4°C), Bjornson etal. (19) have shown dimerization of crosslinked versus native Rep that ssDNA-induced Rep dimerization proceeds according to the Crosslinked Native mechanism in Scheme I with kinetic and equilibrium constants shown in Table 2 (19, 22). PS* + P 4 P2S To determine if DNA crosslinking influenced the energetics of k3, M-'s-' 4.3 ± 0.3 x 105 4.5 + 0.3 x 105* k-3, s-1 2.5 ± 0.04 x 10-3 2.7 + 0.08 x 10-3* Rep dimerization, we first examined the kinetics of dissociation 1.7 0.1 x 108 1.7 0.5 x 108* of the crosslinked P2S dimer to form P and PS by monitoring the K3, M-1 loss of ATPase activity following the addition of P2S + S 4 P2S2 time-dependent k4, M-'s-I 394 ± 6 387 ± 3t a mutant Rep protein, K281, to trap dissociated crosslinked Rep k-4, 51 <1.1 x 10-3 <1.1 X 10-3t monomers (PS). K281 is defective in ATPase activity due to a 2PS* 4 P2S2 Lys-28 to Ile substitution in the conserved GX4GKT sequence of k5, M1 s 1.5 + 0.1 x 103 1.3 ± 0.2 x 103t the putative ATP binding site (I.W., unpublished experiments). k-5, 51 6.9 ± 0.1 x 10-3 7.9 ± 0.1 x 10-3t This mutant serves as an efficient trap for dissociated PS species K5, M-1 2.2 ± 0.2 x 105 1.65 ± 0.25 x 105t because the heterodimer formed between a wild-type and a K281 protomer shows no steady-state ATPase activity (I.W., unpub- Data are for BBM buffer at 4°C. lished experiments). Since P2S first undergoes rate-limiting dis- *From Bjornson et al. (19). tFrom Wong et al. (20). sociation to form PS and P before PS can then dimerize with K281, the time-dependent loss of activity reflects the rate of the ATPase activity of unligated Rep monomer, P, is 1000-fold dissociation of the dimer. The time course of the ATPase activity lower than that of PS, its ATPase activity is not detectable (20). of Rep-d(T7S2T8) (0.4 ,uM total Rep protomer) mixed with excess The ATP concentration dependence of the ATPase activity K281 (2.0 ,uM monomer) is shown in Fig. 34 fitted to a single (BBM buffer at 4°C) of crosslinked PS normalized with respect exponential with an observed rate constant of 2.5 + 0.04 x to total PS concentration (Fig. 44, 0) is well described by a 10-3s- . This rate constant is identical to that measured for rectangular hyperbola with best fit values ofkcat = 2.29 + 0.07 s- I dissociation of native P2S dimer (2.7 + 0.8 x 10-3s-1) by and KM = 2.3 + 0.19 ,uM, and is virtually indistinguishable from fluorescence stopped-flow experiments under identical solution data obtained with uncrosslinked PS (a) (kcat = 2.17 + 0.04 s- conditions (19). and KM = 2.05 + 0.1 ,uM) (20). The change in the ATPase activity of the crosslinked Rep- The steady-state ATPase activity of the crosslinked P2S dimer, d(T7S2T8) mixture as a function of concentration (27 nM to 2.0 normalized to the total P2S concentration is shown in Fig. 4B (0). ,uM total protomer) (Fig. 3B) showed further that the equilibrium The data was fitted to a rectangular hyperbola, with best fit values constants for Rep dimerization were unaffected by ssDNA of kcat = 18.3 ± 0.3 s-I and KM = 3.1 ± 0.2 ,_tM. Also shown are crosslinking. The increase in ATPase activity with increasing data previously obtained for uncrosslinked P2S (0) fitted to kcat = protein concentration was biphasic with an intermediate plateau 16.5 ± 0.2 s-I and KM = 2.7 ± 0.2 ,uM (20). Therefore, region between 100 to 300 nM. Consistent with the negative crosslinked and uncrosslinked P2S also have identical ATPase cooperativity for DNA binding (12, 13, 22), the intermediate activities within experimental uncertainty. plateau region represents formation of P2S while the second Lastly, we measured kcat for ATP hydrolysis by the fully transition reflects formation of the P2S2 dimer. Due to the low ligated Rep dimer, P2S2, with one or both of the Rep subunits solubility limits of Rep, it was not possible to saturate this second crosslinked to ssDNA. Due to the negative cooperativity for phase. From nonlinear analysis of the data, we determined a DNA binding to the Rep dimer, it is difficult to populate a dimerization constant, K3 = 1.7 + 0.1 x 108 M- 1, for the sufficiently large fraction of P2S2 at equilibrium. However, we formation of P2S from PS* and P and an apparent kcat = 18 + 1.2 have previously shown that the ATPase activity of P2S2 can be s-1 for ATP hydrolysis by P2S. We were not able to resolve the accurately determined by measuring the ATPase activity as a dimerization constant, K5, for forming P2S2 from 2 PS*, due to the function of time following the addition of excess dT16 to a lack of an end-point for the second transition. We were, however, solution of P2S (20) since this results in a transient accumu- able to obtain a best fit value of kcat = 77 + 9 s-I for ATP lation of P2S2 prior to re-equilibration to PS (Fig. 5). The hydrolysis by P2S2 by constraining K5 to 1.6 x 105 M-1 as results from a series of such experiments performed at 0.5 ,uM measured for native Rep (20). The population distribution of PS, P2S and 6.25, 12.5, 25, 50, and 100 ,uM dT16 are shown in Fig. PS*, P2S, and P2S2 calculated from these values of K2, K3, and K5 5A. The observed transient increase in ATPase activity was are shown overlaid on the fit to the data. dependent on [dTI6] and correlates well with transient forma- We then measured the ATPase activities of each ssDNA tion of P2S2. The subsequent loss of activity corresponds to ligation state of the crosslinked Rep to compare with those dissociation of the dimer to yield an equilibrium mixture of determined for uncrosslinked complexes (20). The monomer PS*, PS, and P2S2 as shown previously (20). The [dTI6] species, PS, was formed at 1 nM total subunit concentration; dependence of the rate of the transient increase gives the based on our previous studies of uncrosslinked Rep-dT16 com- apparent association rate constant, k4, for binding ssDNA to plexes as well as the value ofK3 determined in the above titration, P2S. The amplitude of this transient is directly proportional to only monomers, P and PS, are present at this concentration. Since the difference between the kcat values of P2S and P2S2. The rate Table 1. ATPase activities of ssDNA ligation states of crosslinked versus native Rep kcat, S- KM, AM Crosslinked Native Crosslinked Native O P 2 x 10-3* 7 x 10-3* o~- PS 2.30 + 0.07 2.17 ± 0.04t 2.30 + 0.19 2.05 +0.1t 8 P2S 18.3 + 0.3 16.5 ± 0.2t 3.1 ± 0.2 2.7 + 0.2t P2S2 68 2 71 2.5t Data are for BBM buffer at 4°C. *From Moore and Lohman (23). tFrom Wong et al. (20). Downloaded by guest on September 27, 2021 Biochemistry: Wong and Lohman Proc. Natl. Acad. Sci. USA 93 (1996) 10055

6.0 2.5 . ', 2.0 ...... 0 0 -3

1 3.0 cn 1.0 ...... 0 0.5

0.0

0.0 20

.. 15 I PS r- 10 10.0 0.8 Cw 0 B -- ~P2S~ I l. 5 0 a *PS* / ri, > 1 0 p2 IN. 10-' 1U 103 5.0o 0.4 a I P S S (ATPJ (11M) &- cr FIG. 4. Steady-state ATPase activity of crosslinked Rep monomer PS (PS) and singly ligated Rep dimer (P2S). (A) Crosslinked PS was formed o.o 0.00o by performing experiments at 1 nM total protomers and ATPase activity 10-3 10-1 101 103 was assayed as a function of [ATP]. The solid line represents best fit of (Rep"t*, (txM) the data (0) to a rectangular hyperbola with kcat = 2.3 ± 0.07 s-1 and KM = 2.3 ± 0.19 ,uM. 0, Data for uncrosslinked PS obtained at 1 nM Rep and FIG. 3. An Kinetics and thermodynamics of crosslinked dimers. (A) 50 nM dT16. The dotted line represents best fit of the uncrosslinked data excess (2.0 of Rep mutant, K28I, was added to (0.4 AM) Rep-d(T7S2T8) to kcat = 2.17 ± 0.04 S-1 and KM = 2.05 ± 0.1 ,uM. (B) Free Rep to ,uM). PS formed from the dissociation of P2S PS and P is trapped by monomers (0.07 ,uM) were preincubated with 0.33 ,M crosslinked rapid rebinding to this mutant to form heterodimers which are defective mixture to form 0.2 uM crosslinked P2S and assayed for ATPase activity The time of the loss of activity therefore in ATPase activity. dependence as a function of [ATP]. The solid line represents best fit of the data (-) corresponds to the rate of P2S dimer dissociation and fits to a single to a rectangular hyperbola with kcat = 18.3 ± 0.08 S-1 and KM = 3.1 ± exponential with a rate constant of 2.5 ± 0.04 X 10-3-S1. (B) The 0.2 ,uM. 0, Data for uncrosslinked P2S obtained at 0.5 ,uM Rep monomers ATPase activity of Rep-d(T7S2T8) is very sensitive to protein concentra- and of 3 nM dT16 (>99% Rep is P2S under these concentrations). The tion due to the linkage to Rep dimerization. The solid line represents the dotted line represents the best fit of the uncrosslinked data to kt = nonlinear least-square best fit of the ATPase activities at different protein 16.5 ± 0.2 s-1 and KM = 2.7 + 0.2 ,uM. concentrations using kcat values of 2 s-1, 18 s-1, and 77 + 9 s-1 for the ATPase activities of PS + PS*, P2S, and P2S2, respectively. Equilibrium ciation or translocation, thus ruling out any mechanisms for dimerization constants K3 and K5 for formation of P2S from PS* + P and helicase translocation that require tight coupling. P2S2 from 2PS* were 1.7 ± 0.1 x 108 M-1 and 1.5 ± 0.1 x 105 M-1, respectively. The isomerization equilibrium for PS to PS* was fixed at K2 We also have shown that the time courses of ATP hydrolysis = 13. The species distributions are also shown. by both crosslinked and uncrosslinked P2S dimers remain linear until >60% of the starting ATP has been converted to of decay of the transient gives the rate of dissociation of P2S2 ADP. This result is consistent with the hypothesis that the to form 2PS*. These data were globally fitted together with ssDNA does not dissociate from the P2S dimer during steady- data obtained at a different protein concentration (not shown) state ATP hydrolysis. Net dissociation of ssDNA from P2S by FITSIM (21) according to Scheme I. Solid lines represent best would result in dimer dissociation and a resulting loss of fits using k4 = 3.94 0.06 x 102 M-1's-s, k5 = 1.5 + 0.1 x 103 linearity in the time course of ATP hydrolysis because the rate M-1-s'1, k-5 = 6.9 0.1 X 10-3s-1, and kcat = 68 2 s-1. The of dimerization to reform P2S would be rate-limiting under dissociation rate of DNA from P2S2, k-4, was not resolved beyond an upper limit of 1.1 x 10-3 s-'. The rate constant for 25 the dissociation of P2S to form PS* and P, k-3, was fixed at 0.0025 s-1 as determined in the mutant trapping experiment, 20 and the association rate constant, k3, was fixed at 4.3 x 105 I - 15 = M-1Ls-, calculated as k_3K3 using a value of K3 1.7 x 108 0 M-1 as determined in the titration described above. Fig. SB 10 shows the predicted species population distribution during the time course of the reaction shown in the top curve of Fig. SA 5 based on these rate constants, which confirms that the tran- 25 1.0 CD 0., a sient rise in ATPase activity results from the transient accu- 20 't ''.P2S -%_PS 0.8 C. o mulation of P2S2 which then dissociates to form 2PS. _. 0 0.6 o 15 DISCUSSION 0.4 0~ gr Ct Tables 1 and 2 summarize the kinetic and equilibrium con- 10 0.2 stants for Rep dimerization and the kcat values for ATP 0 hydrolysis by the crosslinked Rep-d(T7S2T8) complexes, PS, 5 B. i 0.0 0 50 100 150 200 250 P2S, and P2S2, and compares them with those measured for the Time (s) uncrosslinked complexes formed with dT16 (19, 22). All pa- rameters measured for the crosslinked and the uncrosslinked FIG. 5. Transient kinetic formation of P2S2 and determination of its species are identical within experimental error. Neither the ATPase activity. (A) Crosslinked P2S (0.5 ,uM) was mixed with 6.25 (A), Rep dimerization energetics nor the initial velocity steady- 12.5 (v), 25 (v), 50 (0), and 100 AM (0) dT16 to form transiently crosslinked at t = 0. At 20-s the ATPase activity was state ATPase activities of any of the ssDNA ligation states are P2S2 intervals, measured and was observed to increase transiently followed by a slower perturbed by DNA crosslinking. The ATPase activity of Rep decay. Solid lines represent best fits to all data sets using the rate constants was also not changed upon crosslinking to either d(T3S2T12) or shown in Tables 1 and 2 according to Scheme I. (B) The simulated d(T12S2T3) (data not shown). These results demonstrate that population distribution of species is displayed for the top curve in A ATP hydrolysis by Rep is not tightly coupled to DNA disso- showing transient formation of P2S2 from P2S and its dissociation to PS. Downloaded by guest on September 27, 2021 10056 Biochemistry: Wong and Lohman Proc. Natl. Acad. Sci. USA 93 (1996) these reaction conditions. However, because of the steady- (13), the macroscopic 3' -*5' "polarity" of DNA unwinding by state nature of the ATPase assay, this observation of linearity the Rep helicase may originate from the asymmetry inherent at alone does not constitute proof that dissociation does not the unwinding junction. This is supported by the results reported occur as there could be other compensating effects such as here because a random diffusion process would not require a tight from changes in the protein oligomeric state (20). The coupling of energy derived from ATP hydrolysis. Such a rolling crosslinking results, however, provide strong corroborating mechanism also provides a means for processive translocation of evidence that the ATPase activity of P2S is not coupled to the dimeric helicase. Whereas the increase in the rate of disso- DNA dissociation. The results are therefore most consistent ciation of ssDNA from one subunit of the P2S2 Rep dimer upon with the ssDNA remaining bound to Rep in both its PS and P2S ATP hydrolysis would result directly in an increased rate of complexes during multiple rounds of ATP hydrolysis. translocation, the lack of an ATP-stimulated DNA dissociation Because we have ruled out ATP coupled dissociation of from the P2S dimer would ensure that one subunit of the dimer ssDNA from both P2S and PS, the only remaining possible (although not always the same subunit) remains tightly bound to mode of translocation in these ligation states, if it occurs at all, the ssDNA lattice throughout the translocation cycle. would be via sliding (i.e., translocation while the same protein There is currently no direct evidence to support the common subunit maintains contact with the DNA). A related question assumption that unidirectional sliding of helicases along ssDNA is whether ATP hydrolysis fuels unidirectional sliding as a is crucial to their mechanism of DNA unwinding. Interestingly, mechanism for helicase translocation along ssDNA (24-26). observations that an increase in the length of the ssDNA lattice The extent of any directional bias during translocation must be results in a net increase in the ATPase activity of DNA helicases directly related to the degree of coupling between ATP have been cited as support for ATP-driven unidirectional trans- hydrolysis and movement. In one extreme, strictly unidirec- location (25). In this context, the crosslinked Rep-ssDNA com- tional translocation without "slippage" would require a high plexes described here would approximate the limit of Rep bound coupling efficiency between ATP hydrolysis and translocation to an infinitely long ssDNA lattice, yet we observe no net increase such that tethering of the DNA to the helicase by crosslinking in ATPase activity in these crosslinked complexes relative to would be expected to severely inhibit its ATPase activity. In the uncrosslinked complexes of Rep bound to dT16, a single-stranded other extreme, if ATP hydrolysis is independent of DNA oligodeoxynucleotide approximately equal in length to the site movement, then it could not possibly fuel a directionally biased size for the Rep monomer (12). Furthermore, we have also shown translocation. Therefore, our results indicate that transloca- that the #2-fold increase in ATPase activity of Rep bound to tion does not occur by a strictly unidirectional sliding mech- poly(dT) versus dT16 is due to an increase in the transient anism since the tethered single-stranded oligodeoxynucleotide population of the P2S2 species (20). would restrict translocation and thus inhibit ATP hydrolysis. We have previously proposed an active "rolling" model for Although we cannot completely rule out a biased directional Rep helicase-catalyzed DNA unwinding in which the dimer sliding mechanism, this would have to occur with low effi- simultaneously binds to both duplex and ssDNA (13). Our ciency. Furthermore, the fact that the same ssDNA stimulated current findings also rule out a strict unidirectional sliding ATPase activity can be obtained with either dT16 or poly(dT) along ssDNA as a mechanism for a targeted search by the Rep (20) also indicates that biased directional translocation is not helicase for duplex DNA. We now propose, based on these required for maximal ATPase activity. findings, that the Rep helicase can translocate along ssDNA If ATP hydrolysis by a P2S Rep dimer does not stimulate via a similar rolling model without need for an explicit DNA dissociation or translocation by sliding, then how might directional bias, although this may occur by an as yet unknown a Rep dimer translocate along DNA? Unlike the P2S dimer, mechanism. The same rolling or subunit switching mechanism ATP hydrolysis by the doubly ligated P2S2 Rep dimer is linear can be readily extended to account for translocation of the for only the first "10 s, after which it decreases significantly, ring-like hexameric helicases with each subunit providing a indicating that continued ATP hydrolysis by the P2S2 dimer potential DNA binding site (1). leads to changes in its DNA ligation and/or dimerization states (20, 27). In fact, recent stopped-flow fluorescence measure- We thank Bill Van Zante for oligodeoxynucleotide synthesis and purification, and Keith Bjornson and Janid Ali for critical discussions ments show that dissociation of a single-stranded oligode- and Carl Frieden and Peter Burgers for comments on the manuscript. oxynucleotide from one subunit of the P2S2 Rep dimer is This work was supported by grants from the National Institutes of enhanced -60-fold during the course of ATP hydrolysis (27). Health (GM 45948) and the American Cancer Society (NP-756B). I.W. Therefore, the rate of DNA dissociation from one subunit of received partial support from an American Cancer Society Postdoc- P2S2, but not P2S or PS, is enhanced during ATP hydrolysis, toral Fellowship (PF-3671). indicating that ATP hydrolysis is coupled to DNA dissociation 1. Lohman, T. M. & Bjornson, K P. (1996) Annu. Rev. Biochem. 65, 169-214. from P2S2. However, even for this reaction, an average of 150 2. Matson, S. W. & Kaiser-Rogers, K. A. (1990) Annu. Rev. Biochem. 59, 289-329. 3. Friedberg, E. C. (1992) Cell 71, 887-889. ATPs are turned over for each net dissociation of DNA 4. Sancar, A. (1994) Science 266, 1954-1956. indicating a low coupling efficiency (<1%) under our condi- 5. Modrich, P. (1994) Science 266, 1959-1960. 6. Lohman, T. M. (1992) Mol. Microbiol. 6, 5-14. tions (BBM buffer at 4°C). Therefore, DNA dissociation from 7. Lohman, T. M. (1993) J. Biol. Chem. 268, 2269-2272. P2S2 is not obligatory during each ATPase cycle. This is 8. Hackney, D. D. (1994) Proc. Natl. Acad. Sci. USA 91, 6865-6869. 9. Gilbert, S., Webb, M. R., Brune, M. & Johnson, K. A. (1995)Nature (London) 373,671-676. consistent with our result that the initial steady-state ATPase 10. Moore, K. J. M. & Lohman, T. M. (1995) Biophys. J. 68, 180s-185s. activity of the P2S2 dimer is unaffected by DNA crosslinking. 11. Chao, K. & Lohman, T. M. (1991) J. Mol. Biol. 221, 1165-1181. 12. Wong, I., Chao, K. L., Bujalowski, W. & Lohman, T. M. (1992) J. Biol. Chem. 267, Based on these results we propose a "rolling" model of 7596-7610. translocation for the dimeric Rep helicase that takes advantage of 13. Wong, I. & Lohman, T. M. (1992) Science 256, 350-355. 14. Amaratunga, M. & Lohman, T. M. (1993) Biochemistry 32, 6815-6820. the two distinct DNA binding sites of the dimer (12, 13). In this 15. Colasanti, J. & Denhardt, D. T. (1987) Mol. Gen. Genet. 209, 382-390. model, net movement results from transient binding of ssDNA to 16. Lohman, T. M., Chao, K., Green, J. M., Sage, S. & Runyon, G. (1989)J. Biol. Chem. 264, 10139-10147. the unligated subunit of P2S followed by release of ssDNA from 17. Lohman, T. M. & Bujalowski, W. (1988) Biochemistry 27, 2260-2265. the first subunit. This is the same type oftranslocation mechanism 18. Johnson, M. L. & Frasier, S. G. (1993) Methods Enzymol. 117, 301-342. 19. Bjornson, K. P., Moore, K. J. M. & Lohman T. M. (1996) Biochemistry 35, 2268-2282. that we previously proposed for the dimeric Rep helicase during 20. Wong, I., Moore, K. J. M., Bjomson, K. P., Hsieh, J. & Lohman, T. M. (1996) Biochem- its DNA istry 35, 5726-5734. unwinding reaction (13), the only difference being that 21. Zimmerle, C. T. & Frieden, C. (1989) Biochem. J. 258, 381-387. translocation during unwinding occurs upon binding duplex 22. Wong, I., Amaratunga, M. & Lohman, T. M. (1993) J. Biol. ChenL 268, 20386-20393. 23. Moore, K. J. M. & Lohman, T. M. (1994) Biochemistry 33, 14550-14564. DNA, D, into the free Rep subunit to form a P2SD intermediate; 24. Brown, W. C. & Romano, L. J. (1989) J. Biol. Chem. 264, 6748-6754. this duplex DNA is then unwound to transiently form a P2S2 25. Young, M. C., Schultz, D. E., Ring, D. & von Hippel, P. H. (1994) J. Mol. Biol. 235, 1447-1458. complex. Translocation along ssDNAby this mechanism need not 26. Raney, K. D. & Benkovic, S. J. (1995) J. Biol. Chem. 270, 22236-22242. occurwith any net directionality. As we have previously proposed 27. Bjornson, K. P., Wong, I., & Lohman, T. M. (1996) J. MoL. Biol., in press. Downloaded by guest on September 27, 2021