E. coli Rep oligomers are required to initiate DNA unwinding in vitro.

E. coli Rep protein is a 3' to 5' SF1 superfamily DNA helicase which is monomeric in the absence of DNA, but can dimerize upon binding either single-stranded or duplex DNA. A variety of biochemical studies have led to proposals that Rep dimerization is important for its helicase activity; however, recent structural studies of Bacillus stearothermophilus PcrA have led to suggestions that SF1 helicases, such as E. coli Rep and E. coli UvrD, function as monomeric helicases. We have examined the question of whether Rep oligomerization is important for its DNA helicase activity using pre-steady state stopped-flow and chemical quenched-flow kinetic studies of Rep-catalyzed DNA unwinding. The results from four independent experiments demonstrate that Rep oligomerization is required for initiation of DNA helicase activity in vitro. No DNA unwinding is observed when only a Rep monomer is bound to the DNA substrate, even when fluorescent DNA substrates are used that can detect partial unwinding of the first few base-pairs at the ss-ds-DNA junction. In fact, under these conditions, ATP hydrolysis causes dissociation of the Rep monomer from the DNA, rather than DNA unwinding. These studies demonstrate that wild-type Rep monomers are unable to initiate DNA unwinding in vitro, and that oligomerization is required.

Large contributions of coupled protonation equilibria to the observed enthalpy and heat capacity changes for ssDNA binding to Escherichia coli SSB protein.

Many macromolecular interactions, including protein-nucleic acid interactions, are accompanied by a substantial negative heat capacity change, the molecular origins of which have generated substantial interest. We have shown previously that temperature-dependent unstacking of the bases within oligo(dA) upon binding to the Escherichia coli SSB tetramer dominates the binding enthalpy, DeltaH(obs), and accounts for as much as a half of the observed heat capacity change, DeltaC(p). However, there is still a substantial DeltaC(p) associated with SSB binding to ssDNA, such as oligo(dT), that does not undergo substantial base stacking. In an attempt to determine the origins of this heat capacity change, we have examined by isothermal titration calorimetry (ITC) the equilibrium binding of dT(pT)(34) to SSB over a broad pH range (pH 5. 0-10.0) at 0.02 M, 0.2 M NaCl and 1 M NaCl (25 degrees C), and as a function of temperature at pH 8.1. A net protonation of the SSB protein occurs upon dT(pT)(34) binding over this entire pH range, with contributions from at least three sets of protonation sites (pK(a1) = 5.9-6.6, pK(a2) = 8.2-8.4, and pK(a3) = 10.2-10.3) and these protonation equilibria contribute substantially to the observed DeltaH and DeltaC(p) for the SSB-dT(pT)(34) interaction. The contribution of this coupled protonation ( approximately -260 to -320 cal mol(-1) K(-1)) accounts for as much as half of the total DeltaC(p). The values of the "intrinsic" DeltaC(p,0) range from -210 +/- 33 cal mol(-1) degrees K(-1) to -237 +/- 36 cal mol(-1)K(-1), independent of [NaCl]. These results indicate that the coupling of a temperature-dependent protonation equilibria to a macromolecular interaction can result in a large negative DeltaC(p), and this finding needs to be considered in interpretations of the molecular origins of heat capacity changes associated with ligand-macromolecular interactions, as well as protein folding.

Structure of the DNA binding domain of E. coli SSB bound to ssDNA.

The structure of the homotetrameric DNA binding domain of the single stranded DNA binding protein from Escherichia coli (Eco SSB) bound to two 35-mer single stranded DNAs was determined to a resolution of 2.8 A. This structure describes the vast network of interactions that results in the extensive wrapping of single stranded DNA around the SSB tetramer and suggests a structural basis for its various binding modes.

An oligomeric form of E. coli UvrD is required for optimal helicase activity.

Pre-steady-state chemical quenched-flow techniques were used to study DNA unwinding catalyzed by Escherichia coli UvrD helicase (helicase II), a member of the SF1 helicase superfamily. Single turnover experiments, with respect to unwinding of a DNA oligonucleotide, were used to examine the DNA substrate and UvrD concentration requirements for rapid DNA unwinding by pre-bound UvrD helicase. In excess UvrD at low DNA concentrations (1 nM), the bulk of the DNA is unwound rapidly by pre-bound UvrD complexes upon addition of ATP, but with time-courses that display a distinct lag phase for formation of fully unwound DNA, indicating that unwinding occurs in discrete steps, with a "step size" of four to five base-pairs as previously reported. Optimum unwinding by pre-bound UvrD-DNA complexes requires a 3' single-stranded (ss) DNA tail of 36-40 nt, whereas productive complexes do not form readily on DNA with 3'-tail lengths

Adenine base unstacking dominates the observed enthalpy and heat capacity changes for the Escherichia coli SSB tetramer binding to single-stranded oligoadenylates.

Isothermal titration calorimetry (ITC) was used to test the hypothesis that the relatively small enthalpy change (DeltaHobs) and large negative heat capacity change (DeltaCp,obs) observed for the binding of the Escherichia coli SSB protein to single-stranded (ss) oligodeoxyadenylates result from the temperature-dependent adenine base unstacking equilibrium that is thermodynamically coupled to binding. We have determined DeltaH1,obs for the binding of 1 mole of each of dT(pT)34, dC(pC)34, and dA(pA)34 to the SSB tetramer (20 mM NaCl at pH 8.1). For dT(pT)34 and dC(pC)34, we found large, negative values for DeltaH1,obs of -75 +/- 1 and -85 +/- 2 kcal/mol at 25 degrees C, with DeltaCp,obs values of -540 +/- 20 and -570 +/- 30 cal mol-1 K-1 (7-50 degrees C), respectively. However, for SSB-dA(pA)34 binding, DeltaH1,obs is considerably less negative (-14 +/- 1 kcal/mol at 25 degrees C), even becoming positive at temperatures below 13 degrees C, and DeltaCp,obs is nearly twice as large in magnitude (-1180 +/- 40 cal mol-1 K-1). These very different thermodynamic properties for SSB-dA(pA)34 binding appear to result from the fact that the bases in dA(pA)34 are more stacked at any temperature than are the bases in dC(pC)34 or dT(pT)34 and that the bases become unstacked within the SSB-ssDNA complexes. Therefore, the DeltaCp,obs for SSB-ssDNA binding has multiple contributions, a major one being the coupling to binding of a temperature-dependent conformational change in the ssDNA, although SSB binding to unstacked ssDNA still has an "intrinsic" negative DeltaCp,0. In general, such temperature-dependent changes in the conformational "end states" of interacting macromolecules can contribute significantly to both DeltaCp,obs and DeltaHobs.

A two-site kinetic mechanism for ATP binding and hydrolysis by E. coli Rep helicase dimer bound to a single-stranded oligodeoxynucleotide.

Escherichia coli Rep helicase catalyzes the unwinding of duplex DNA in reactions that are coupled to ATP binding and hydrolysis. We have investigated the kinetic mechanism of ATP binding and hydrolysis by a proposed intermediate in Rep-catalyzed DNA unwinding, the Rep "P2S" dimer (formed with the single-stranded (ss) oligodeoxynucleotide, (dT)16), in which only one subunit of a Rep homo-dimer is bound to ssDNA. Pre-steady-state quenched-flow studies under both single turnover and multiple turnover conditions as well as fluorescence stopped-flow studies were used (4 degrees C, pH 7.5, 6 mM NaCl, 5 mM MgCl2, 10 % (v/v) glycerol). Although steady-state studies indicate that a single ATPase site dominates the kinetics (kcat=17(+/-2) s-1; KM=3 microM), pre-steady-state studies provide evidence for a two-ATP site mechanism in which both sites of the dimer are catalytically active and communicate allosterically. Single turnover ATPase studies indicate that ATP hydrolysis does not require the simultaneous binding of two ATP molecules, and under these conditions release of product (ADP-Pi) is preceded by a slow rate-limiting isomerization ( approximately 0.2 s-1). However, product (ADP or Pi) release is not rate-limiting under multiple turnover conditions, indicating the involvement of a second ATP site under conditions of excess ATP. Stopped-flow fluorescence studies monitoring ATP-induced changes in Rep's tryptophan fluorescence displayed biphasic time courses. The binding of the first ATP occurs by a two-step mechanism in which binding (k+1=1.5(+/-0.2)x10(7) M-1 s-1, k-1=29(+/-2) s-1) is followed by a protein conformational change (k+2=23(+/-3) s-1), monitored by an enhancement of Trp fluorescence. The second Trp fluorescence quenching phase is associated with binding of a second ATP. The first ATP appears to bind to the DNA-free subunit and hydrolysis induces a global conformational change to form a high energy intermediate state with tightly bound (ADP-Pi). Binding of the second ATP then leads to the steady-state ATP cycle. As proposed previously, the role of steady-state ATP hydrolysis by the DNA-bound Rep subunit may be to maintain the DNA-free subunit in an activated state in preparation for binding a second fragment of DNA as needed for translocation and/or DNA unwinding. We propose that the roles of the two ATP sites may alternate upon binding DNA to the second subunit of the Rep dimer during unwinding and translocation using a subunit switching mechanism.

The importance of coulombic end effects: experimental characterization of the effects of oligonucleotide flanking charges on the strength and salt dependence of oligocation (L8+) binding to single-stranded DNA oligomers.

Binding constants Kobs, expressed per site and evaluated in the limit of zero binding density, are quantified as functions of salt (sodium acetate) concentration for the interactions of the oligopeptide ligand KWK6NH2 (designated L8+, with ZL = 8 charges) with three single-stranded DNA oligomers (ss dT-mers, with |ZD| = 15, 39, and 69 charges). These results provide the first systematic experimental information about the effect of changing |ZD| on the strength and salt dependence of oligocation-oligonucleotide binding interactions. In a comparative study of L8+ binding to poly dT and to a short dT oligomer (|ZD| = 10),. Proc. Natl. Acad. Sci. USA. 93:2511-2516) demonstrated the profound thermodynamic effects of phosphate charges that flank isolated nonspecific L8+ binding sites on DNA. Here we find that both Kobs and the magnitude of its power dependence on salt activity (|SaKobs|) increase monotonically with increasing |ZD|. The dependences of Kobs and SaKobs on |ZD| are interpreted by introducing a simple two-state thermodynamic model for Coulombic end effects, which accounts for our finding that when L8+ binds to sufficiently long dT-mers, both DeltaGobso = -RT ln Kobs and SaKobs approach the values characteristic of binding to poly-dT as linear functions of the reciprocal of the number of potential oligocation binding sites on the DNA lattice. Analysis of our L8+-dT-mer binding data in terms of this model indicates that the axial range of the Coulombic end effect for ss DNA extends over approximately 10 phosphate charges. We conclude that Coulombic interactions cause an oligocation (with ZL < |ZD|) to bind preferentially to interior rather than terminal binding sites on oligoanionic or polyanionic DNA, and we quantify the strong increase of this preference with decreasing salt concentration. Coulombic end effects must be considered when oligonucleotides are used as models for polyanionic DNA in thermodynamic studies of the binding of charged ligands, including proteins.

Calorimetric studies of E. coli SSB protein-single-stranded DNA interactions. Effects of monovalent salts on binding enthalpy.

Isothermal titration calorimetry (ITC) was used to examine the effects of monovalent salts (NaCl, NaBr, NaF and ChCl) on the binding enthalpy (DeltaHobs) for E. coli SSB tetramer binding to the single-stranded oligodeoxythymidylates, dT(pT)69 and dT(pT)34 over a wide range of salt concentrations from 10 mM to 2.0 M (25 degrees C, pH 8.1), and when possible, the binding free energy and entropy (DeltaG degrees obs, DeltaS degrees obs). At low monovalent salt concentrations (<0.1 M), the total DeltaHobs for saturating all sites on the SSB tetramer with ssDNA shows little dependence on salt concentration, but is extremely large and exothermic (DeltaHobs=-150(+/-5) kcal/mol). This is much larger than any DeltaHobs previously reported for a protein-nucleic acid interaction. However, at salt concentrations above 0.1 M, DeltaHobs is quite sensitive to NaCl and NaBr concentration, becoming less negative with increasing salt concentration (DeltaHobs=-70(+/-1)-kcal/mol in 2 M NaBr). These salt effects on DeltaHobs were mainly a function of anion type and concentration, with the largest effects observed in NaBr, and then NaCl, with little effect of [NaF]. These large effects of salt on DeltaHobs appear to be coupled to a net release of weakly bound anions (Br- and Cl-) from the SSB protein upon DNA binding. However, at lower salt concentrations (

Comparisons between the structures of HCV and Rep helicases reveal structural similarities between SF1 and SF2 super-families of helicases.

Three helicase structures have been determined recently: that of the DNA helicase PcrA, that of the hepatitis C virus RNA helicase, and that of the Escherichia coli DNA helicase Rep. PcrA and Rep belong to the same super-family of helicases (SF1) and are structurally very similar. In contrast, the HCV helicase belongs to a different super-family of helicases, SF2, and shows little sequence homology with the PcrA/Rep helicases. Yet, the HCV helicase is structurally similar to Rep/PcrA, suggesting preservation of structural scaffolds and relationships between helicase motifs across these two super-families. The comparison study presented here also reveals the existence of a new helicase motif in the SF1 family of helicases.

Kinetic mechanism for the sequential binding of two single-stranded oligodeoxynucleotides to the Escherichia coli Rep helicase dimer.

Escherichia coli Rep helicase is a DNA motor protein that unwinds duplex DNA as a dimeric enzyme. Using fluorescence probes positioned asymmetrically within a series of single-stranded (ss) oligodeoxynucleotides, we show that ss-DNA binds with a defined polarity to Rep monomers and to individual subunits of the Rep dimer. Using fluorescence resonance energy transfer and stopped-flow techniques, we have examined the mechanism of ss-oligodeoxynucleotide binding to preformed Rep dimers in which one binding site is occupied by a single-stranded oligodeoxynucleotide, while the other site is free (P2S dimer). We show that ss-DNA binding to the P2S Rep dimer to form the doubly ligated P2S2 dimer occurs by a multistep process with the initial binding step occurring relatively rapidly with a bimolecular rate constant of k1 = approximately 2 x 10(6) M-1 s-1 [20 mM Tris (pH 7.5), 6 mM NaCl, 5 mM MgCl2, 5 mM 2-mercaptoethanol, and 10% (v/v) glycerol, 4 degrees C]. A minimal kinetic mechanism is proposed which suggests that the two strands of ss-DNA bound to the Rep homodimer are kinetically distinct even within the P2S2 Rep dimer, indicating that this dimer is functionally asymmetric. The implications of these results for the mechanisms of DNA unwinding and translocation by the functional Rep dimer are discussed.

Major domain swiveling revealed by the crystal structures of complexes of E. coli Rep helicase bound to single-stranded DNA and ADP.

Crystal structures of binary and ternary complexes of the E. coli Rep helicase bound to single-stranded (ss) DNA or ssDNA and ADP were determined to a resolution of 3.0 A and 3.2 A, respectively. The asymmetric unit in the crystals contains two Rep monomers differing from each other by a large reorientation of one of the domains, corresponding to a swiveling of 130 degrees about a hinge region. Such domain movements are sufficiently large to suggest that these may be coupled to translocation of the Rep dimer along DNA. The ssDNA binding site involves the helicase motifs Ia, III, and V, whereas the ADP binding site involves helicase motifs I and IV. Residues in motifs II and VI may function to transduce the allosteric effects of nucleotides on DNA binding. These structures represent the first view of a DNA helicase bound to DNA.

Thermodynamics of oligoarginines binding to RNA and DNA.

We have examined the equilibrium binding of a series of synthetic oligoarginines (net charge z = +2 to +6) containing tryptophan to poly(U), poly(A), poly(C), poly(I), and double-stranded (ds) DNA. Equilibrium association constants, K(obs), measured by monitoring tryptophan fluorescence quenching, were examined as functions of monovalent salt (MX) concentration and type, as well as temperature, from which deltaG(standard)obs, deltaH(obs), and deltaS(standard)obs were determined. For each peptide, K(obs) decreases with increasing [K+], and the magnitude of the dependence of K(obs) on [K+], delta log K(obs)/delta log[K+], increases with increasing net peptide charge. In fact, the values of delta log K(obs)/delta log[K+] are equivalent for oligolysines and oligoarginines possessing the same net positive charge. However, the values of K(obs) are systematically greater for oligoarginines binding to all polynucleotides, when compared to oligolysines with the same net charge. The origin of this difference is entirely enthalpic, with deltaH(obs), determined from van't Hoff analysis, being more exothermic for oligoarginine binding. The values of deltaH(obs) are also independent of [K+]; therefore, the salt concentration dependence of deltaG(standard)obs is entirely entropic in origin, reflecting the release of cations from the nucleic acid upon complex formation. These results suggest that hydrogen bonding of arginine to the phosphate backbone of the nucleic acids contributes to the increased stability of these complexes.

Crystal structure of the homo-tetrameric DNA binding domain of Escherichia coli single-stranded DNA-binding protein determined by multiwavelength x-ray diffraction on the selenomethionyl protein at 2.9-A resolution.

The crystal structure of the tetrameric DNA-binding domain of the single-stranded DNA binding protein from Escherichia coli was determined at a resolution of 2.9 A using multiwavelength anomalous dispersion. Each monomer in the tetramer is topologically similar to an oligomer-binding fold. Two monomers each contribute three beta-strands to a single six-stranded beta-sheet to form a dimer. Two dimer-dimer interfaces are observed within the crystal. One of these stabilizes the tetramer in solution. The other interface promotes a superhelical structure within the crystal that may reflect tetramer-tetramer interactions involved in the positive cooperative binding of the single-stranded DNA-binding protein to single-stranded DNA.

A two-site mechanism for ATP hydrolysis by the asymmetric Rep dimer P2S as revealed by site-specific inhibition with ADP-A1F4.

The Escherichia coli Rep helicase is a dimeric motor protein that catalyzes the transient unwinding of duplex DNA to form single-stranded (ss) DNA using energy derived from the binding and hydrolysis of ATP. In an effort to understand this mechanism of energy transduction, we have used pre-steady-state methods to study the kinetics of ATP binding and hydrolysis by an important intermediate in the DNA unwinding reaction--the asymmetric Rep dimer state, P2S, where ss DNA [dT(pT)15] is bound to only one subunit of the Rep dimer. To differentiate between the two potential ATPase active sites inherent in the dimer, we constructed dimers with one subunit covalently cross-linked to ss DNA and where one or the other of the ATPase sites was selectively complexed to the tightly bound transition state analog ADP-A1F4. We found that when ADP-A1F4 is bound to the Rep subunit in trans from the subunit bound to ss DNA, steady-state ATPase activity of 18 s(-1) per dimer (equivalent to wild-type P2S) was recovered. However, when the ADP-A1F4 and ss DNA are both bound to the same subunit (cis), then a titratable burst of ATP hydrolysis is observed corresponding to a single turnover of ATP. Rapid chemical quenched-flow techniques were used to resolve the following minimal mechanism for ATP hydrolysis by the unligated Rep subunit of the cis dimer: E + ATP <==> E-ATP <==> E'-ATP <==> E'-ADP-Pi <==> E-ADP-Pi <==> E-ADP + Pi <==> E + ADP + Pi, with K1 = (2.0 +/- 0.85) x 10(5) M(-1), k2 = 22 +/- 3.5 s(-1), k(-2) < 0.12 s(-1), K3 = 4.0 +/- 0.4 (k3 > 200 s(-1)), k4 = 1.2 +/- 0.14 s(-1), k(-4) << 1.2 s(-1), K5 = 1.0 +/- 0.2 mM, and K6 = 80 +/- 8 microM. A salient feature of this mechanism is the presence of a kinetically trapped long-lived tight nucleotide binding state, E'-ADP-Pi. In the context of our "subunit switching" model for Rep dimer translocation during processive DNA unwinding [Bjornson, K. B., Wong, I., & Lohman, T. M. (1996) J. Mol. Biol. 263, 411-422], this state may serve an energy storage function, allowing the energy from the binding and hydrolysis of ATP to be harnessed and held in reserve for DNA unwinding.

A mutation in E. coli SSB protein (W54S) alters intra-tetramer negative cooperativity and inter-tetramer positive cooperativity for single-stranded DNA binding.

E. coli SSB tetramer binds with high affinity and cooperatively to single-stranded (ss) DNA and functions in replication, recombination and repair. Curth et al. (Biochemistry, 32 (1993) 2585-2591) have shown that a mutant SSB protein, in which Trp-54 has been replaced by Ser (W54S) in each subunit, binds preferentially to ss-polynucleotides in the (SSB)35 mode in which only 35 nucleotides are occluded per tetramer under conditions in which wild-type (wt) SSB binds in its (SSB)65 mode. The W54S mutant also displays increased UV sensitivity and slow growth phenotypes, suggesting defects in vivo in both repair and replication (Carlini et al. (Molecular Microbiology, 10 (1993) 1067)). We have characterized the energetics of SSBW54S binding to poly(dT) as well as short oligodeoxyribonucleotides (dA(pA)69, dT(pT)34, dC(pC)34) to determine the basis for this dramatic change in binding mode preference. We find that the W54S mutant remains a stable tetramer; however, its affinity for ss-DNA as well as both the intra-tetramer negative cooperativity and its inter-tetramer positive cooperativity in the (SSB)35 mode (omega 35) are altered significantly compared to wtSSB. The increased intra-tetramer negative cooperativity makes it more difficult for ss-DNA to bind the third and fourth subunits of the W54S tetramer, explaining the increased stability of the (SSB)35 mode in complexes with poly(dT). When bound to dA(pA)69 in the (SSB)35 mode, W54S tetramer also displays a dramatically lower inter-tetramer positive cooperativity (omega 35 = 77(+/-20)) than wtSSB (omega 35 > or = 10(5)) as well as a significantly lower affinity for ss-DNA. These results indicate that a single amino acid change can dramatically influence the ability of SSB tetramers to bind in the different SSB binding modes. The altered ss-DNA properties of the W54S SSB mutant are probably responsible for the observed defects in replication and repair and support the proposal that the different SSB binding modes may function selectively in replication, recombination and/or repair.

Kinetic measurement of the step size of DNA unwinding by Escherichia coli UvrD helicase.

The kinetic mechanism by which the DNA repair helicase UvrD of Escherichia coli unwinds duplex DNA was examined with the use of a series of oligodeoxynucleotides with duplex regions ranging from 10 to 40 base pairs. Single-turnover unwinding experiments showed distinct lag phases that increased with duplex length because partially unwound DNA intermediate states are highly populated during unwinding. Analysis of these kinetics indicates that UvrD unwinds duplex DNA in discrete steps, with an average "step size" of 4 to 5 base pairs (approximately one-half turn of the DNA helix). This suggests an unwinding mechanism in which alternating subunits of the dimeric helicase interact directly with duplex DNA.

ATP hydrolysis stimulates binding and release of single stranded DNA from alternating subunits of the dimeric E. coli Rep helicase: implications for ATP-driven helicase translocation.

DNA helicases are motor proteins that unwind duplex DNA during DNA replication, recombination and repair in reactions that are coupled to ATP binding and hydrolysis. In the process of unwinding duplex DNA processively, DNA helicases must also translocate along the DNA filament. To probe the mechanism of ATP-driven translocation by the dimeric E. coli Rep helicase along single stranded (ss) DNA, we examined the effects of ATP on the dissociation kinetics of ssDNA from the Rep dimer. Stopped-flow experiments show that the dissociation rate of a fluorescent ss oligodeoxynucleotide bound to one subunit of the dimeric Rep helicase is stimulated by ssDNA binding to the other subunit, and that the rate of this ssDNA exchange reaction is further stimulated approximately 60-fold upon ATP hydrolysis. This ssDNA exchange process occurs via an intermediate in which ssDNA is transiently bound to both subunits of the Rep dimer. These results suggest a rolling or subunit switching mechanism for processive ATP-driven translocation of the dimeric Rep helicase along ssDNA. Such a mechanism requires the extreme negative cooperativity for DNA binding to the second subunit of the Rep dimer, which insures that the doubly DNA-ligated Rep (P2S2) dimer is formed only transiently and relaxes back to the singly ligated Rep (P2S) dimer. The fact that other oligomeric DNA helicases share many functional features with the dimeric Rep helicase suggests that similar mechanisms for translocation and DNA unwinding may apply to other dimeric as well as hexameric DNA helicases.

ATPase activity of Escherichia coli Rep helicase crosslinked to single-stranded DNA: implications for ATP driven helicase translocation.

To examine the coupling of ATP hydrolysis to helicase translocation along DNA, we have purified and characterized complexes of the Escherichia coli Rep protein, a dimeric DNA helicase, covalently crosslinked to a single-stranded hexadecameric oligodeoxynucleotide (S). Crosslinked Rep monomers (PS) as well as singly ligated (P2S) and doubly ligated (P2S2) Rep dimers were characterized. The equilibrium and kinetic constants for Rep dimerization as well as the steady-state ATPase activities of both PS and P2S crosslinked complexes were identical to the values determined for un-crosslinked Rep complexes formed with dT16. Therefore, ATP hydrolysis by both PS and P2S complexes are not coupled to DNA dissociation. This also rules out a strictly unidirectional sliding mechanism for ATP-driven translocation along single-stranded DNA by either PS or the P2S dimer. However, ATP hydrolysis by the doubly ligated P2S2 Rep dimer is coupled to single-stranded DNA dissociation from one subunit of the dimer, although loosely (low efficiency). These results suggest that ATP hydrolysis can drive translocation of the dimeric Rep helicase along DNA by a "rolling" mechanism where the two DNA binding sites of the dimer alternately bind and release DNA. Such a mechanism is biologically important when one subunit binds duplex DNA, followed by subsequent unwinding.

ATPase activity of Escherichia coli Rep helicase is dramatically dependent on DNA ligation and protein oligomeric states.

The Escherichia coli Rep helicase catalyzes the unwinding of duplex DNA using the energy derived from ATP binding and hydrolysis. Rep functions as a dimer but assembles to its active dimeric form only on binding DNA. Each promoter of a dimer contains a DNA binding site that can bind either single-stranded (S) or duplex (D) DNA. The dimer can bind up to two oligodeoxynucleotides in five DNA-ligation states: two half-ligated states, P2S and P2D, and three fully-ligated states, P2S2, P2D2, and P2SD. We have previously shown that the relative stabilities of these ligation states are allosterically regulated by the binding and hydrolysis of ATP and have proposed an "active rolling" model for DNA unwinding where the enzyme cycles through a series of these ligation states in a process that is coupled to the catalytic cycle of ATP hydrolysis [Wong, I., & Lohman, T.M., (1992), Science 256, 350-355]. THe basal ATPase activity of Rep protein is stimulated by ss DNA binding and by protein dimerization. We have measured the steady-state ATPase activities of Rep bound to dT(pT)15 in each distinct ss DNA ligation state (PS, P2S, and P2S2) to compare with our previous measurements with unligated Rep monomer (P) [Moore, K.J.M., & Lohman, T.M. (1994) Biochemistry 33, 14550]. We find the ATPase activity of Rep is influenced dramatically by both dimerization and ss DNA ligation state, with the following kcat values for ATP hydrolysis increasing by over 4 orders of magnitude: 2.1 x 10(-3) s(-1) for P, 2.17 +/- 0.04 s(-1) for PS, 16.5 +/- 0.2 s(-1) for P2S, and 71 +/- 2.5 s(-1) for P2S2 (20 mM Tris-HCl, pH 7.5, 6mM NaCl, 5 mM MgCl2, 10% glycerol, 4 degrees C). The apparent KM's for ATP hydrolysis are 2.05 +/- 0.1 microM for PS and 2.7 +/- 0.2 microM for P2S. These widely different ATPase activities reflect the allosteric effects of DNA ligation and demonstrate that cooperative communication occurs between the ATP and DNA site of both subunits of the Rep dimer. These results further emphasize the need to explicitly consider the population distribution of oligomerization and DNA ligation states of the helicase when attempting to infer information about elementary processes such as helicase translocation based solely on macroscopic steady-state ATPase measurements.