Kinetic Mechanism for Formation of the Active, Dimeric UvrD Helicase-DNA Complex
2003; Elsevier BV; Volume: 278; Issue: 34 Linguagem: Inglês
10.1074/jbc.m304223200
ISSN1083-351X
AutoresNasib K. Maluf, Janid A. Ali, Timothy M. Lohman,
Tópico(s)RNA and protein synthesis mechanisms
ResumoEscherichia coli UvrD protein is a 3′ to 5′ SF1 helicase required for DNA repair as well as DNA replication of certain plasmids. We have shown previously that UvrD can self-associate to form dimers and tetramers in the absence of DNA, but that a UvrD dimer is required to form an active helicase-DNA complex in vitro. Here we have used pre-steady state, chemical quenched flow methods to examine the kinetic mechanism for formation of the active, dimeric helicase-DNA complex. Experiments were designed to examine the steps leading to formation of the active complex, separate from the subsequent DNA unwinding steps. The results show that the active dimeric complex can form via two pathways. The first, faster path involves direct binding to the DNA substrate of a pre-assembled UvrD dimer (dimer path), whereas the second, slower path proceeds via sequential binding to the DNA substrate of two UvrD monomers (monomer path), which then assemble on the DNA to form the dimeric helicase. The rate-limiting step within the monomer pathway involves dimer assembly on the DNA. These results show that UvrD dimers that pre-assemble in the absence of DNA are intermediates along the pathway to formation of the functional dimeric UvrD helicase. Escherichia coli UvrD protein is a 3′ to 5′ SF1 helicase required for DNA repair as well as DNA replication of certain plasmids. We have shown previously that UvrD can self-associate to form dimers and tetramers in the absence of DNA, but that a UvrD dimer is required to form an active helicase-DNA complex in vitro. Here we have used pre-steady state, chemical quenched flow methods to examine the kinetic mechanism for formation of the active, dimeric helicase-DNA complex. Experiments were designed to examine the steps leading to formation of the active complex, separate from the subsequent DNA unwinding steps. The results show that the active dimeric complex can form via two pathways. The first, faster path involves direct binding to the DNA substrate of a pre-assembled UvrD dimer (dimer path), whereas the second, slower path proceeds via sequential binding to the DNA substrate of two UvrD monomers (monomer path), which then assemble on the DNA to form the dimeric helicase. The rate-limiting step within the monomer pathway involves dimer assembly on the DNA. These results show that UvrD dimers that pre-assemble in the absence of DNA are intermediates along the pathway to formation of the functional dimeric UvrD helicase. DNA helicases are a ubiquitous class of enzymes that catalyze the separation of double-stranded (ds) 1The abbreviations used are: ds, double-stranded; ss, single-stranded; NLLS, non-linear least squares.1The abbreviations used are: ds, double-stranded; ss, single-stranded; NLLS, non-linear least squares. DNA to form the single-stranded (ss) DNA intermediates required for DNA replication, recombination, and repair in reactions that are coupled to the binding and hydrolysis of nucleoside triphosphates (1Matson S.W. Bean D.W. George J.W. Bioessays. 1994; 16: 13-22Crossref PubMed Scopus (269) Google Scholar, 2Lohman T.M. Bjornson K.P. Annu. Rev. Biochem. 1996; 65: 169-214Crossref PubMed Scopus (669) Google Scholar). The Escherichia coli UvrD helicase, also known as helicase II, plays essential roles in nucleotide excision repair and methyl-directed mismatch repair (3Sancar A. Science. 1994; 266: 1954-1956Crossref PubMed Scopus (506) Google Scholar, 4Modrich P. Lahue R. Annu. Rev. Biochem. 1996; 65: 101-133Crossref PubMed Scopus (1318) Google Scholar) and is also required for rolling circle replication of certain plasmids (5Bruand C. Ehrlich S.D. Mol. Microbiol. 2000; 35: 204-210Crossref PubMed Scopus (75) Google Scholar).The UvrD protein (720 amino acids, molecular mass of 81,989 Da) is a member of the SF1 helicase superfamily (6Gorbalenya A.E. Koonin E.V. Curr. Opin. Struct. Biol. 1993; 3: 419-429Crossref Scopus (1024) Google Scholar) and is operationally defined as a 3′ to 5′ helicase (7Matson S.W. J. Biol. Chem. 1986; 261: 10169-10175Abstract Full Text PDF PubMed Google Scholar), based on the fact that a 3′ ssDNA tail placed adjacent to the duplex DNA stimulates the unwinding reaction in vitro. However, UvrD can also initiate DNA unwinding, albeit less efficiently, from DNA nicks and blunt-ended DNA in multiple turnover experiments in vitro (8Runyon G.T. Lohman T.M. J. Biol. Chem. 1989; 264: 17502-17512Abstract Full Text PDF PubMed Google Scholar, 9Runyon G.T. Bear D.G. Lohman T.M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6383-6387Crossref PubMed Scopus (94) Google Scholar, 10Dao V. Modrich P. J. Biol. Chem. 1998; 273: 9202-9207Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 11Mechanic L.E. Frankel B.A. Matson S.W. J. Biol. Chem. 2000; 275: 38337-38346Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). In single turnover DNA unwinding experiments (i.e. single cycle with respect to the DNA), a 3′ ssDNA tail of at least 15 nucleotides is required to observe optimal unwinding by a UvrD dimer in vitro (12Ali J.A. Maluf N.K. Lohman T.M. J. Mol. Biol. 1999; 293: 815-834Crossref PubMed Scopus (93) Google Scholar, 13Maluf N.K. Fischer C.J. Lohman T.M. J. Mol. Biol. 2003; 325: 913-935Crossref PubMed Scopus (163) Google Scholar). Even though an 18-bp DNA substrate possessing a 3′ ssDNA tail of 4–10 nucleotides is sufficient to bind a single UvrD monomer with high affinity, no unwinding is observed (13Maluf N.K. Fischer C.J. Lohman T.M. J. Mol. Biol. 2003; 325: 913-935Crossref PubMed Scopus (163) Google Scholar). This reflects the fact that a UvrD dimer is required for helicase activity in vitro (13Maluf N.K. Fischer C.J. Lohman T.M. J. Mol. Biol. 2003; 325: 913-935Crossref PubMed Scopus (163) Google Scholar). In the active dimeric UvrD-DNA complex, one UvrD subunit binds tightly to the ss/dsDNA junction, whereas the second subunit binds to the 3′ ssDNA tail (13Maluf N.K. Fischer C.J. Lohman T.M. J. Mol. Biol. 2003; 325: 913-935Crossref PubMed Scopus (163) Google Scholar, 17Maluf N.K. Lohman T.M. J. Mol. Biol. 2003; 325: 889-912Crossref PubMed Scopus (40) Google Scholar). Neither the E. coli Rep helicase, which is also a 3′ to 5′ SF1 helicase (15Cheng W. Hsieh J. Brendza K.M. Lohman T.M. J. Mol. Biol. 2001; 310: 327-350Crossref PubMed Scopus (118) Google Scholar) with structural homology to UvrD (24Korolev S. Hsieh J. Gauss G.H. Lohman T.M. Waksman G. Cell. 1997; 90: 635-647Abstract Full Text Full Text PDF PubMed Scopus (432) Google Scholar), nor an ATPase-deficient UvrD mutant can substitute functionally for the second UvrD monomer (13Maluf N.K. Fischer C.J. Lohman T.M. J. Mol. Biol. 2003; 325: 913-935Crossref PubMed Scopus (163) Google Scholar), indicating that the two UvrD monomers interact to form a functional helicase.Whereas the structurally homologous E. coli Rep protein is monomeric in the absence of DNA (14Chao K.L. Lohman T.M. J. Mol. Biol. 1991; 221: 1165-1181Crossref PubMed Scopus (84) Google Scholar), but also must oligomerize to form an active helicase in vitro (15Cheng W. Hsieh J. Brendza K.M. Lohman T.M. J. Mol. Biol. 2001; 310: 327-350Crossref PubMed Scopus (118) Google Scholar, 16Ha T. Rasnik I. Cheng W. Babcock H.P. Gauss G.H. Lohman T.M. Chu S. Nature. 2002; 419: 638-641Crossref PubMed Scopus (389) Google Scholar), UvrD protein can self-assemble to form dimers and tetramers in the absence of DNA (17Maluf N.K. Lohman T.M. J. Mol. Biol. 2003; 325: 889-912Crossref PubMed Scopus (40) Google Scholar, 18Runyon G.T. Wong I. Lohman T.M. Biochemistry. 1993; 32: 602-612Crossref PubMed Scopus (83) Google Scholar). These UvrD self-assembly equilibria have been characterized quantitatively by analytical ultracentrifugation methods over a range of UvrD concentrations, solution conditions, and temperatures (17Maluf N.K. Lohman T.M. J. Mol. Biol. 2003; 325: 889-912Crossref PubMed Scopus (40) Google Scholar), including those conditions used to study UvrD-catalyzed DNA unwinding in vitro (13Maluf N.K. Fischer C.J. Lohman T.M. J. Mol. Biol. 2003; 325: 913-935Crossref PubMed Scopus (163) Google Scholar). Considering that a UvrD dimer is required for helicase activity in vitro, the question arises as to whether UvrD dimers that can assemble in the absence of DNA are "on the pathway" to formation of the active dimeric helicase. Alternatively, it is possible that pre-assembled UvrD dimers are not functional and thus would need to first dissociate in order to form an active dimeric helicase on the DNA.To address this question, we performed a series of pre-steady state kinetic studies, using chemical quenched flow methods, to examine the kinetic mechanism by which pre-assembled UvrD dimers form an active dimeric helicase on the DNA. Experiments were performed over a range of UvrD concentrations such that the initial population of UvrD monomers and pre-assembled dimers could be varied significantly. Based on the measured equilibrium constants for UvrD dimerization and tetramerization (17Maluf N.K. Lohman T.M. J. Mol. Biol. 2003; 325: 889-912Crossref PubMed Scopus (40) Google Scholar), we could then correlate the kinetics of formation of the active UvrD dimeric helicase-DNA complex with the population of free UvrD monomers and dimers in solution. The results of these studies show that the active, dimeric helicase can be formed either by rapid binding to the DNA substrate of pre-assembled UvrD dimers (dimer path) or by the sequential binding of two UvrD monomers followed by dimer assembly on the DNA (monomer path). The data further indicate that the rate-limiting step in the monomer pathway involves assembly of the active, dimeric UvrD complex from two UvrD monomers bound to the DNA. This rate-limiting step is not observed along the dimer pathway, suggesting that the pre-assembled UvrD dimer interface is similar to the interface involved in the active helicase dimer on the DNA. The "double-mixing" quenched flow methods described here and elsewhere (21Pang P.S. Jankowsky E. Planet P.J. Pyle A.M. EMBO J. 2002; 21: 1168-1176Crossref PubMed Scopus (203) Google Scholar) enable one to examine the kinetics and mechanism of formation of the active helicase complex on the DNA and should prove useful in studies of helicase-DNA assembly reactions in general.EXPERIMENTAL PROCEDURESBuffers—Buffers were made with reagent grade chemicals using distilled water that was further deionized using a Milli-Q system (Millipore Corp., Bedford, MA). Buffer T20 is 10 mm Tris, pH 8.3, at 25 °C, 20 mm NaCl, and 20%(v/v) glycerol. Storage buffer is 20 mm Tris, pH 8.3, at 25 °C, 200 mm NaCl, 50%(v/v) glycerol, 1 mm EDTA, 0.5 mm EGTA, and 25 mm 2-mercaptoethanol. Storage minimal buffer is 20 mm Tris, pH 8.3, at 25 °C, 200 mm NaCl, and 50%(v/v) glycerol.UvrD Protein and DNA Substrates—UvrD protein was purified to greater than 99% homogeneity, and the protein concentration was determined spectrophotometrically using an extinction coefficient of ϵ280 = 1.06 × 105m–1 cm–1, as described (18Runyon G.T. Wong I. Lohman T.M. Biochemistry. 1993; 32: 602-612Crossref PubMed Scopus (83) Google Scholar). For all experiments performed here, aliquots of UvrD were dialyzed, as needed, versus storage minimal buffer, and stored for periods up to ∼5 months at –20 °C without any loss of helicase activity. Any further dilutions of UvrD were made into storage minimal buffer for experiments performed on that same day; unused protein was discarded.Oligodeoxynucleotides were synthesized using an ABI model 391 DNA synthesizer (Applied Biosystems, Foster City, CA) and purified as described (19Wong I. Chao K.L. Bujalowski W. Lohman T.M. J. Biol. Chem. 1992; 267: 7596-7610Abstract Full Text PDF PubMed Google Scholar). The DNA substrate used here, referred to as 3′-(dT20)-ds18, consists of a 3′ ssDNA region (dT20) attached to an 18-bp duplex DNA and is the same DNA used in previous studies (13Maluf N.K. Fischer C.J. Lohman T.M. J. Mol. Biol. 2003; 325: 913-935Crossref PubMed Scopus (163) Google Scholar, 17Maluf N.K. Lohman T.M. J. Mol. Biol. 2003; 325: 889-912Crossref PubMed Scopus (40) Google Scholar). The sequence of the DNA strand without the 3′-(dT20) ssDNA tail (the top strand) is 5′-GCCTCGCTGCCGTCGCCA-3′. This top strand was radiolabeled with 32P at the 5′ end using T4 polynucleotide kinase (U. S. Biochemical Corp.) and purified as described (19Wong I. Chao K.L. Bujalowski W. Lohman T.M. J. Biol. Chem. 1992; 267: 7596-7610Abstract Full Text PDF PubMed Google Scholar). The radiolabeled strand was mixed with a 1.25-fold excess of bottom strand in 10 mm Tris, pH 8.3 (at 25 °C), and 50 mm NaCl, heated to 95 °C for 5 min, and then cooled slowly to room temperature. The "protein trap" was a 10-bp DNA hairpin with a 3′-(dT40) ssDNA tail (5′-GCCTCGCTGCT5GCAGCGAGGCT40-3′) and was used to prevent additional binding of free UvrD to the DNA substrate after DNA unwinding has been initiated (see below). The "DNA trap" was an 18-nucleotide ssDNA complementary to the 18-nucleotide top strand of the DNA substrate and was included to ensure that no re-annealing of the radiolabeled top strand occurred with the unwound 3′-(dT20)-ssDNA bottom strand either during unwinding or after quenching. The DNA trap anneals with the unwound 18-nucleotide top strand, but this species (a blunt ended 18-bp dsDNA) can be easily separated from the 3′-(dT20)-ds18 radiolabeled DNA substrate by non-denaturing gel electrophoresis (see below).Double-mixing Quenched Flow Experiments—Chemical quenched flow experiments were carried out using a three-pulsed quenched flow apparatus (KinTek RQF-3, University Park, PA) in the "double-mixing mode" maintained at 25 °C using a circulating water bath. UvrD, in Buffer T20 containing 0.2 mg/ml bovine serum albumin, was preincubated at 4 °C (on ice) for at least 20 min and then incubated in a 25 °C heat block for 15 min, after which it was loaded into one loop of the quenched flow apparatus and allowed to incubate for an additional 5 min at 25 °C before the experiment was started. Further incubation times (at 25 °C) up to 1 h had no effect on the results. The other loop was loaded with the radiolabeled 3′-(dT20)-ds18 DNA substrate in Buffer T20. The first push of the quenched flow apparatus rapidly mixed the contents of these two loops together, and the binding reaction (in the absence of ATP) was allowed to proceed for Δt 1 seconds. The second push then rapidly mixed these reaction contents with Buffer T20 containing 1 mm ATP:Mg2+, 2 μm protein trap, and 1 μm DNA trap. This second mixing event initiates unwinding of any DNA on which an active dimeric UvrD helicase has assembled within the time, Δt 1, while also preventing any further binding of UvrD to the DNA substrate after the first incubation time (Δt 1). The DNA unwinding reactions initiated after the second push of the quenched flow were allowed to proceed for Δt 2 = 20 s, after which the reaction contents were expelled into an Eppendorf tube containing 100 μlof0.4 m EDTA + 10% (v/v) glycerol to quench the reaction. A Δt 2 of 20 s is sufficient to allow any DNA unwinding reactions that were initiated by UvrD to be completed (13Maluf N.K. Fischer C.J. Lohman T.M. J. Mol. Biol. 2003; 325: 913-935Crossref PubMed Scopus (163) Google Scholar). The background fraction of ssDNA in the sample at t = 0 was determined by mixing the DNA substrate with Buffer T20 + 0.2 mg/ml bovine serum albumin, in the absence of UvrD. The DNA samples were analyzed by non-denaturing PAGE (10% PAGE) to separate the duplex DNA from the ssDNA, and the fraction of DNA unwound was quantitated as described (13Maluf N.K. Fischer C.J. Lohman T.M. J. Mol. Biol. 2003; 325: 913-935Crossref PubMed Scopus (163) Google Scholar).The kinetics of dissociation of active UvrD helicase-DNA complexes were also performed using the KinTek quenched flow in the double-mixing mode. One syringe contained a mixture of 50 nm UvrD and 3 nm radiolabeled 3′-(dT20)-ds18 DNA substrate, which had been incubated for at least 5 min at 25 °C in one loop of the quenched flow. The other loop contained varying concentrations of the unlabeled 3′-(dT20)-ds18 DNA substrate. The contents of these two loops were then rapidly mixed and allowed to incubate for Δt 1 seconds, after which a second push mixed the solution with 1 mm ATP:Mg2+ to start the DNA unwinding reaction, along with 2 μm protein trap and 1 μm DNA trap, as described above.Analysis of Kinetic Data—The biphasic kinetic time courses for formation of the active UvrD helicase-DNA complexes were fit initially using non-linear least squares (NLLS) to the double-exponential function shown in Equation 1 to estimate the observed rates (k obs,1 and k obs,2) and amplitudes (A 1 and A 2) of both phases. F(t)=A1(1-e-kobs,1t)+A2(1-e-kobs,2t)(Eq. 1) For experiments performed at total initial UvrD concentrations ≥120 nm, both the monomer and pre-assembled UvrD dimer concentrations were in sufficiently large excess over the DNA substrate concentration (2 nm initial concentration) so that the reactions were pseudo-first order with respect to both the UvrD monomer and dimer concentrations.The software package SCIENTIST (MicroMath Software, St. Louis, MO) was used to analyze the kinetic data by numerical integration methods. The pre-steady state quenched flow experiments performed at sufficiently low total UvrD concentrations such that no UvrD dimer was present at the start of the reaction were analyzed according to Scheme 1, using the differential equations in Equations 2, 3, 4, 5, 6. d[Uf]dt=-k1[Uf][Df]-k2[Uf][UD]+k-1[UD]+k-2[U2D](Eq. 2) d[Df]dt=-k1[Uf][Df]+k-1[UD](Eq. 3) d[UD]dt=k1[Uf][Df]+k-2[U2D]-(k-1+k2)[UD](Eq. 4) d[U2D]dt=k2[UD][Uf]+k-3[U2D*]-(k-2+k3)[U2D](Eq. 5) d[U2D*]dt=k3[U2D]-k-3[U2D*](Eq. 6) At the start of the reaction (t = 0), we set [Ut] = [Uf], [Dt] = [Df], and [UD] = [U2D] = [U2D*] = 0, where [Ut] is the total UvrD monomer concentration, and [Dt] is the total DNA substrate concentration. The fraction of DNA molecules unwound as a function of time, F(t), is related to the concentration of the active helicase species (U2D* in Scheme 1), according to Equation 7, F(t)=AU2D*[U2D*][Dt](Eq. 7) where we note that [U2D*] is itself a function of time. A U2D* is the probability that the [U2D*] complex will proceed to form fully unwound ssDNA upon initiation of DNA unwinding. The second step in Scheme 1 was considered to be in rapid equilibrium relative to the third step. This was constrained in the NLLS fitting by setting k –2 ≫ k 3 in Equations 2, 3, 4, 5, 6 (in general, fixing k –2 = 100 s–1 was sufficient to satisfy this constraint) and then allowing k 2 to float. The equilibrium constant, K 2, was then calculated from K 2 = k 2/k –2.Global NLLS analysis of the pre-steady state quenched flow experiments performed over the entire UvrD concentration range used, where both dimers and monomers can be present at the start of the reaction, were analyzed using Scheme 2. The differential equations describing Scheme 2 are given in Equations 8, 9, 10, 11, 12, 13. d[Uf]dt=-k1[Uf][Df]-k2[Uf][UD]+k-1[UD]+k-2[U2D](Eq. 8) d[Df]dt=-k1[Uf][Df]+k-1[UD]-k5[U2][Df]+k-5[U2D*](Eq. 9) d[U2]dt=-k5[U2][Df]+k-5[U2D*](Eq. 10) d[UD]dt=k1[Uf][Df]+k-2[U2D]-k-1[UD]+k2[UD][Uf](Eq. 11) d[U2D]dt=k2[UD][Uf]+k-3[U2D*]-(k-2+k3)[U2D](Eq. 12) d[U2D*]dt=k3[U2D]+k5[U2][Df]-(k-3+k-5)[U2D*](Eq. 13) At t = 0, the initial concentrations of UvrD monomer and dimer, immediately after mixing of the reactants, were calculated using Equations 14 and 15. [Uf]=0.5-1+1+8L20[Ut]4L20(Eq. 14) [U2]=0.5L20-1+1+8L20[Ut]4L202(Eq. 15) where [Ut] is the total UvrD monomer concentration before mixing. Each of these concentrations is multiplied by 0.5 because the equilibrium between monomer and dimer is established at the concentrations that exist before the reactants are mixed (diluted 2-fold) to start the reaction. We have assumed that the rates of association and dissociation of the dimer are slow relative to all other rate constants in Scheme 2. Furthermore, at t = 0, [UD] = [U2D] = [U2D*] = 0. The rapid equilibrium condition for formation of the U2D species was constrained in the analysis as described above, and F(t) is defined as in Equation 7 above.Scheme 2View Large Image Figure ViewerDownload Hi-res image Download (PPT)Uncertainties are reported at the 68% confidence limit (±1 S.D.) and were calculated using SCIENTIST. In some NLLS analyses performed here, some of the fitting parameters were fixed at their experimentally determined values, whereas other parameters were allowed to float. In order to estimate the extent to which the uncertainty associated with the fixed parameters propagates to the calculated uncertainty of the floated parameters, a Monte Carlo method was employed as described previously (17Maluf N.K. Lohman T.M. J. Mol. Biol. 2003; 325: 889-912Crossref PubMed Scopus (40) Google Scholar). The UvrD species fraction distributions were calculated as described (17Maluf N.K. Lohman T.M. J. Mol. Biol. 2003; 325: 889-912Crossref PubMed Scopus (40) Google Scholar).RESULTSKinetics of Formation of the Active UvrD Helicase-DNA Complex under Conditions Where Both UvrD Monomers and Dimers Are Populated in the Absence of DNA—Chemical quenched flow experiments were performed to study the kinetics of formation of the active UvrD helicase-DNA complex, starting from free DNA substrate and free UvrD protein. All experiments were carried out in Buffer T20 (10 mm Tris, pH 8.3, 20 mm NaCl, 20%(v/v) glycerol) at 25 °C, which are the identical conditions used to determine the equilibrium constants describing UvrD self-association, as well as the stoichiometries of UvrD binding to the DNA substrate (17Maluf N.K. Lohman T.M. J. Mol. Biol. 2003; 325: 889-912Crossref PubMed Scopus (40) Google Scholar). We have shown that under these solution conditions, UvrD can self-assemble to form dimers and tetramers in the absence of DNA, with dimerization constant, L 20 = [U2]/[U]2 = (2.33 ± 0.30) μm–1 and overall tetramerization constant, L 40 = [U4]/[U]4 = (5.11 ± 0.80) μm–3 (17Maluf N.K. Lohman T.M. J. Mol. Biol. 2003; 325: 889-912Crossref PubMed Scopus (40) Google Scholar). By using these equilibrium constants, we show in Fig. 1 the predicted distributions of UvrD monomers, dimers, and tetramers as a function of total UvrD concentration. Under these conditions, at least 90% of the UvrD protein exists in either the monomeric or dimeric state at total UvrD monomer concentrations of ≤500 nm.Fig. 1UvrD oligomeric species fraction distributions. Fraction of total UvrD (monomer units) found as free monomers (M), dimers (D), and tetramers (T), as a function of total UvrD concentration. The species fractions were calculated using the dimerization and tetramerization self-association equilibrium constants, L 20 = (2.33 ± 0.30) μm–1, and L 40 = (5.11 ± 0.80) μm–3 determined in Buffer T20, at 25 °C, as described by Maluf and Lohman (17Maluf N.K. Lohman T.M. J. Mol. Biol. 2003; 325: 889-912Crossref PubMed Scopus (40) Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT)In previous studies (13Maluf N.K. Fischer C.J. Lohman T.M. J. Mol. Biol. 2003; 325: 913-935Crossref PubMed Scopus (163) Google Scholar) we have shown that a dimer of UvrD is required to observe helicase activity in single turnover DNA unwinding experiments in vitro. As designed, these experiments are single turnover (single round) with respect to the DNA substrate, whereas multiple ATP turnovers are required for DNA unwinding of even an 18-bp DNA substrate. However, these studies did not address whether UvrD dimers that can pre-assemble in the absence of DNA (hereafter referred to as the "free dimer") occur on the pathway to formation of the active UvrD helicase dimer on the DNA substrate. If formation of the free UvrD dimer occurs off-pathway, it would compete with formation of the active helicase dimer and thus inhibit formation of the active helicase-DNA complex. On the other hand, if formation of the free UvrD dimer occurs on the pathway to forming the active helicase dimer, then an increase in total UvrD concentration would increase the population of the free UvrD dimer, which would increase the rate of assembly of the active helicase dimer on the DNA substrate. We therefore examined the kinetic mechanism of formation of the active dimeric helicase-DNA complex, starting with free UvrD and free DNA, over a range of UvrD concentrations to determine whether the pre-assembled UvrD dimer occurs "on" or "off" the pathway to formation of the active helicase-DNA complex. Quantitative analysis of these kinetic studies was possible only because we have determined the equilibrium constants for assembly of the free UvrD dimer and tetramer from UvrD monomers under the identical solution conditions used in the kinetic studies (17Maluf N.K. Lohman T.M. J. Mol. Biol. 2003; 325: 889-912Crossref PubMed Scopus (40) Google Scholar). Such information is required in order to correlate the formation of active helicase-DNA complexes with the free concentrations of UvrD monomer, free UvrD dimer, or free UvrD tetramer.To study the kinetics of formation of the active dimeric UvrD helicase-DNA complex, we performed chemical quenched flow experiments, using the "double-mixing" mode of the KinTek quenched flow apparatus (see "Experimental Procedures"). As diagrammed in Fig. 2, one syringe contained UvrD in Buffer T20, whereas the other syringe contained 2 nm of a radiolabeled DNA unwinding substrate, also in Buffer T20. The DNA substrate used is the same substrate that we have used in previous studies (13Maluf N.K. Fischer C.J. Lohman T.M. J. Mol. Biol. 2003; 325: 913-935Crossref PubMed Scopus (163) Google Scholar, 17Maluf N.K. Lohman T.M. J. Mol. Biol. 2003; 325: 889-912Crossref PubMed Scopus (40) Google Scholar) and consists of a 3′ (dT)20 ssDNA tail attached to an 18-bp duplex DNA (3′-dT20-ds18). The contents of these two syringes were rapidly mixed in the first "push" of the quenched flow and allowed to incubate for a period of time (Δt 1), after which the resulting sample was then rapidly mixed with a third solution of 1 mm ATP:Mg2+, along with a large excess of a "protein trap" (a DNA molecule that consists of a 3′ (dT)40 ssDNA tail attached to a 10-bp hairpin). This second mixing event serves to initiate unwinding of any DNA substrates on which an active dimeric UvrD complex has assembled during the time of the first incubation period (Δt 1), whereas the large excess of trap for free protein prevents any binding or rebinding of free UvrD to the DNA substrate. The resulting DNA unwinding reaction was allowed to proceed for a constant time of 20 s (Δt 2), which is sufficient time to allow any productively bound UvrD to complete the unwinding of the 18-bp duplex, as determined in our previous studies (13Maluf N.K. Fischer C.J. Lohman T.M. J. Mol. Biol. 2003; 325: 913-935Crossref PubMed Scopus (163) Google Scholar). After 20 s, the solution was then expelled into a solution containing 0.4 m EDTA which quenches the unwinding reaction. By performing a series of experiments in which we varied the time, Δt 1, between the first and second push, we obtain the kinetic time course that monitors selectively the formation of UvrD-DNA complexes that are active in DNA unwinding.Fig. 2Design of double-mixing quenched flow kinetics experiment. Schematic representation of the double-mixing quenched flow experiment for measuring the kinetics of formation of the active UvrD helicase-DNA substrate complex is shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The total UvrD monomer concentration that exists before mixing with the DNA substrate, i.e. before the first push of the quenched flow apparatus, was used to calculate the free UvrD monomer and dimer concentrations before the start of the UvrD-DNA binding reaction, using the known UvrD monomerdimer-tetramer equilibrium constants (17Maluf N.K. Lohman T.M. J. Mol. Biol. 2003; 325: 889-912Crossref PubMed Scopus (40) Google Scholar). However, since the first mixing event dilutes each sample 2-fold, the concentrations of free UvrD monomer and dimer used for analysis of the kinetics of assembly of the active helicase-DNA complex have been reduced by a factor of 2. 2In this analysis we have assumed that the monomer-dimer equilibrium relaxes slowly with respect to the rate of binding of UvrD monomers and dimers to the DNA, which is supported by simulations (see "Discussion"). However, if the concentrations of free UvrD monomer and dimer are calculated using the 2-fold lower total UvrD concentration that exists after the first push, the quantitative results are not affected significantly. The second mixing event, whereby DNA unwinding is initiated by any UvrD that has assembled on the DNA as an active helicase, occurs under single turnover DNA unwinding conditions (due to the inclusion of the protein trap), and thus this second dilution of the sample does not need to be considered in the analysis. Based on these considerations, we refer to the concentration of UvrD that exists in the syringe prior to the first mixing event (first push) as the "pre-mix" concentration, and we refer to the concentration of UvrD that exists subsequent to the first mixing event as the "post-mix" concentration.Fig. 3, A and B, shows the results of experiments in which radiolabeled 3′-(dT20)-ds18 DNA substrate (1 nm post-mix concentration) was mixed with U
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