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• W2078095931 abstract "RecBCD enzyme is a multifunctional nuclease that is essential for the major pathway of homologous genetic recombination in Escherichia coli. It has a potent helicase activity that uses ATP hydrolysis to unwind very long stretches of DNA. The functional form of RecBCD enzyme has been unclear, since Mr of 250,000-655,000 have been previously reported. We have isolated two oligomeric forms of the enzyme, one (monomeric) containing a single copy of the RecB, RecC, and RecD polypeptides, and the other (dimeric) containing two copies of each polypeptide. We show here that the monomeric form of the enzyme (Mr≈ 330,000) can form a stable initiation complex on the end of ds DNA. Depending on the nature of the ds end, KD estimates ranged from ≈ 0.1 nM to ≈ 0.7 nM in the presence of Mg2+ ions, which enhanced but was not required for binding. We further showed that the complex of monomeric RecBCD enzyme and a ds DNA end was competent to unwind DNA. A general model for the action of helicases has been proposed that uses repeated conformational changes between two states of a complex between DNA and a dimeric form of the enzyme. Our results make such a model unlikely for RecBCD enzyme. RecBCD enzyme is a multifunctional nuclease that is essential for the major pathway of homologous genetic recombination in Escherichia coli. It has a potent helicase activity that uses ATP hydrolysis to unwind very long stretches of DNA. The functional form of RecBCD enzyme has been unclear, since Mr of 250,000-655,000 have been previously reported. We have isolated two oligomeric forms of the enzyme, one (monomeric) containing a single copy of the RecB, RecC, and RecD polypeptides, and the other (dimeric) containing two copies of each polypeptide. We show here that the monomeric form of the enzyme (Mr≈ 330,000) can form a stable initiation complex on the end of ds DNA. Depending on the nature of the ds end, KD estimates ranged from ≈ 0.1 nM to ≈ 0.7 nM in the presence of Mg2+ ions, which enhanced but was not required for binding. We further showed that the complex of monomeric RecBCD enzyme and a ds DNA end was competent to unwind DNA. A general model for the action of helicases has been proposed that uses repeated conformational changes between two states of a complex between DNA and a dimeric form of the enzyme. Our results make such a model unlikely for RecBCD enzyme. INTRODUCTIONThe RecBCD enzyme (EC 3.1.11.5) is a large ATP-dependent enzyme that is involved in recombination and repair of DNA in Escherichia coli (reviewed in (1Taylor A.F. Kucherlapati R. Smith G.R. Genetic Recombination. American Society for Microbiology, Washington, DC1988: 231-263Google Scholar)). It is encoded by the recB, recC, and recD genes, whose gene products have Mr of 134,000, 129,000, and 67,000, respectively, as inferred from DNA sequence data and N-terminal peptide analysis(2Finch P.W. Storey A. Chapman K.E. Brown K. Hickson I.D. Emmerson P.T. Nucleic Acids Res. 1986; 14: 8573-8582Crossref PubMed Scopus (56) Google Scholar, 3Finch P.W. Storey A. Brown K. Hickson I.D. Emmerson P.T. Nucleic Acids Res. 1986; 14: 8583-8594Crossref PubMed Scopus (56) Google Scholar, 4Finch P.W. Wilson R.E. Brown K. Hickson I.D. Thompkinson A.E. Emmerson P.T. Nucleic Acids Res. 1986; 14: 4437-4451Crossref PubMed Scopus (44) Google Scholar, 5Masterson C. Boehmer P.E. McDonald F. Chaudhuri S. Hickson I.D. Emmerson P.T. J. Biol. Chem. 1992; 267: 13564-13572Abstract Full Text PDF PubMed Google Scholar). The enzyme has a potent ATP-dependent exonuclease that is active on either ds 1The abbreviations used are: dsdouble-strandedsssingle-strandedbpbase pair(s)ntnucleotide(s)SSBE. coli single-stranded DNA binding proteinMOPS4-morpholinepropanesufonic acid. or ss DNA and a weak ATP-stimulated endonuclease activity that acts only on ss DNA. It can use the energy of ATP hydrolysis to unwind ds DNA, either transiently or permanently, in a highly processive reaction(6Taylor A.F. Smith G.R. Cell. 1980; 22: 447-457Abstract Full Text PDF PubMed Scopus (156) Google Scholar, 7Roman L.J. Kowalczykowski S.C. Biochemistry. 1989; 28: 2863-2873Crossref PubMed Scopus (147) Google Scholar). The enzyme is active on linear, but not circular, ds DNA and thus requires a ds terminus for its unwinding or nuclease activity(8Taylor A.F. Smith G.R. J. Mol. Biol. 1985; 185: 431-443Crossref PubMed Scopus (125) Google Scholar, 9Goldmark P.J. Linn S. J. Biol. Chem. 1972; 247: 1849-1860Abstract Full Text PDF PubMed Google Scholar).Published reports suggest that RecBCD enzyme can exist in either a monomeric (B1C1D1) or a dimeric (B2C2D2) form. 2We have previously referred to these two forms as “trimer” and “hexamer” (17Ganesan S. Smith G.R. J. Mol. Biol. 1993; 229: 67-78Crossref PubMed Scopus (53) Google Scholar) but will henceforth refer to enzyme molecules containing one or two copies each of the RecB, RecC, and RecD polypeptides as monomers and dimers, respectively. The native Mr of the enzyme was initially reported to be about 250,000(9Goldmark P.J. Linn S. J. Biol. Chem. 1972; 247: 1849-1860Abstract Full Text PDF PubMed Google Scholar), as estimated by glycerol gradient centrifugation, consistent with the enzyme molecule containing one copy each of the RecB, RecC, and RecD polypeptides (with a predicted Mr of 330,000). A higher Mr form of the enzyme, apparently dimeric, was observed in sonicates of E. coli(10Telander-Muskavitch, K. M., 1981, Exonuclease V, the recBC Enzyme of Escherichia coli. Ph.D. dissertation, University of California, Berkeley.Google Scholar), together with the previously reported form, but was lost during subsequent purification steps. RecBCD enzyme purified from a strain that overproduced the enzyme had a native Mr of 655,000 but which decreased to about 270,000 in the presence of 0.5 M NH4Cl(11Dykstra C.C. Palas K.M. Kushner S.R. Cold Spring Harbor Symp. Quant. Biol. 1984; 49: 463-467Crossref PubMed Google Scholar). The higher Mr form was not observed in a subsequent purification(12Palas K.M. Kushner S.R. J. Biol. Chem. 1990; 265: 3447-3454Abstract Full Text PDF PubMed Google Scholar). More recently, RecBCD enzyme has been produced either by overproduction of the three subunits within E. coli or by mixing of purified subunits(5Masterson C. Boehmer P.E. McDonald F. Chaudhuri S. Hickson I.D. Emmerson P.T. J. Biol. Chem. 1992; 267: 13564-13572Abstract Full Text PDF PubMed Google Scholar). Mr estimations by gel filtration or native polyacrylamide gel electrophoresis are consistent with the enzyme from these sources being a monomer, but the specific activity of the reconstituted material was only a few percent of that of the native enzyme(5Masterson C. Boehmer P.E. McDonald F. Chaudhuri S. Hickson I.D. Emmerson P.T. J. Biol. Chem. 1992; 267: 13564-13572Abstract Full Text PDF PubMed Google Scholar).Interest in the subunit structure of RecBCD enzyme was rekindled by a “rolling dimer” model of helicase action which relies on a symmetric, dimeric enzyme structure(13Lohman T.M. Mol. Microbiol. 1992; 6: 5-14Crossref PubMed Scopus (133) Google Scholar). In that model, monomer A of the dimeric helicase binds (already unwound) ss DNA behind the enzyme, and monomer B binds ds DNA immediately ahead. Monomer A releases its ss DNA and binds to ds DNA ahead of monomer B, which melts its bound ds DNA and remains bound to the ss DNA so produced. Translocation and unwinding thus result from the cycle of alternating binding and unbinding.Unwinding of ds DNA by RecBCD enzyme has been studied by electron microscopy(6Taylor A.F. Smith G.R. Cell. 1980; 22: 447-457Abstract Full Text PDF PubMed Scopus (156) Google Scholar, 14Muskavitch K.M.T. Linn S. J. Biol. Chem. 1982; 257: 2641-2648Abstract Full Text PDF PubMed Google Scholar). The enzyme unwinds DNA processively, in the presence of SSB, with the production of either asymmetric structures (a ss loop and two ss tails) or apparently symmetric structures (two ss loops). Both types of structure travel along the DNA at ≥300 bp/s, while the loops grow at about 100 nt/s. The relative abundance of the two structures is determined by the concentration of SSB(15Taylor A.F. Smith G.R. ICN-UCLA Symp. Mol. Cell. Biol. 1980; 19: 909-917Google Scholar), suggesting that they arise by a common mechanism(6Taylor A.F. Smith G.R. Cell. 1980; 22: 447-457Abstract Full Text PDF PubMed Scopus (156) Google Scholar).We proposed that, as a minimal model, RecBCD enzyme need contact only one strand of ds DNA and could produce the observed DNA structures by assimilating the DNA ahead of itself and releasing it behind itself at a slower rate(6Taylor A.F. Smith G.R. Cell. 1980; 22: 447-457Abstract Full Text PDF PubMed Scopus (156) Google Scholar). Roman and Kowalczykowski (16Roman L.J. Kowalczykowski S.C. Biochemistry. 1989; 28: 2873-2881Crossref PubMed Scopus (68) Google Scholar) proposed that unwinding by RecBCD enzyme results from the action of two helicases, acting at different rates on the two strands of the DNA, and suggested that the helicases may reside in the RecB and RecD subunits of the enzyme. Ganesan and Smith (17Ganesan S. Smith G.R. J. Mol. Biol. 1993; 229: 67-78Crossref PubMed Scopus (53) Google Scholar) combined the latter model with the “rolling dimer” model of Lohman (13Lohman T.M. Mol. Microbiol. 1992; 6: 5-14Crossref PubMed Scopus (133) Google Scholar) and suggested that a dimeric form of RecBCD enzyme unwinds DNA. Two copies of the RecB subunit were proposed to act in tandem to translocate along one strand of the DNA, while a pair of (RecC + RecD) complexes translocated along the other strand. The two complexes were postulated to travel at different rates and hence produce the ss loops observed by electron microscopy. The choice of subunits in this latter model was prompted by the UV-cross-linking patterns of the subunits on the ends of ds DNA(17Ganesan S. Smith G.R. J. Mol. Biol. 1993; 229: 67-78Crossref PubMed Scopus (53) Google Scholar), by the abilities of the RecB and RecD subunits to bind ATP(18Julin D.A. Lehman I.R. J. Biol. Chem. 1987; 262: 9044-9051Abstract Full Text PDF PubMed Google Scholar), and by the ATPase activity of isolated RecB protein(19Boehmer P.E. Emmerson P.T. J. Biol. Chem. 1992; 267: 4981-4987Abstract Full Text PDF PubMed Google Scholar).During the course of purifying RecBCD enzyme we observed, purified, and characterized both monomeric and dimeric forms of RecBCD enzyme. We report here that stable complexes were formed between ds DNA and the monomeric form of RecBCD enzyme and that such complexes were competent for unwinding. These results establish the active form of RecBCD enzyme as a monomer and question the physiological relevance of the dimer.EXPERIMENTAL PROCEDURESRecBCD EnzymeRecBCD enzyme was purified from strain V182(20Amundsen S.K. Neiman A.M. Thibodeaux S.M. Smith G.R. Genetics. 1990; 126: 25-40Crossref PubMed Google Scholar), which carries the recB, recC, and recD genes on an 18-kilobase fragment of E. coli DNA cloned in pBR322. Lysis and purification to produce Fraction IV were as described elsewhere(8Taylor A.F. Smith G.R. J. Mol. Biol. 1985; 185: 431-443Crossref PubMed Scopus (125) Google Scholar, 21Amundsen S.K. Taylor A.F. Chaudhury A.M. Smith G.R. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 5558-5562Crossref PubMed Scopus (183) Google Scholar). All experiments, except where noted and the dynamic light scattering determination, used the “first” purification described next. Fraction IV was applied to an Affi-Gel heparin column (Bio-Rad) in buffer C (22Eichler D.C. Lehman I.R. J. Biol. Chem. 1977; 252: 499-503Abstract Full Text PDF PubMed Google Scholar) containing 0.15 M NH4Cl. The column was washed with the same buffer and eluted with a 0.15-0.5 M NH4Cl gradient. Most of the enzyme did not bind to the column and was reapplied at 0.05 M NH4Cl and eluted with a 0.05-0.6 M NH4Cl gradient. Enzyme that eluted from the second Affi-Gel heparin column (at 0.1-0.2 M NH4Cl) was concentrated by binding to a 10-ml column of DEAE-Sepharose and eluting with a gradient of NH4Cl. Peak fractions were pooled and designated Fraction V-B, to distinguish it from Fraction V, the eluate of the heparin agarose column (Life Technologies, Inc.), employed both in previous purifications (21Amundsen S.K. Taylor A.F. Chaudhury A.M. Smith G.R. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 5558-5562Crossref PubMed Scopus (183) Google Scholar) and in the “second” purification described below. Fractions VI-M (the monomeric form of the enzyme) and VI-D (an apparent mixture of monomeric and dimeric forms) are described more fully under “Results.”In the “second” purification, Fraction V was purified as previously described (8Taylor A.F. Smith G.R. J. Mol. Biol. 1985; 185: 431-443Crossref PubMed Scopus (125) Google Scholar, 21Amundsen S.K. Taylor A.F. Chaudhury A.M. Smith G.R. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 5558-5562Crossref PubMed Scopus (183) Google Scholar) and concentrated and purified on a DEAE-Sepharose column, as above, to yield Fraction VII, which lacks detectable contaminants as judged by SDS-polyacrylamide gel electrophoresis.Units of activity are those of the ATP-dependent ds DNA exonuclease activity of the enzyme(22Eichler D.C. Lehman I.R. J. Biol. Chem. 1977; 252: 499-503Abstract Full Text PDF PubMed Google Scholar). RecBCD enzyme concentrations were derived from the A280 of enzyme fractions, using the ∈280 calculated by Roman and Kowalczykowski (7Roman L.J. Kowalczykowski S.C. Biochemistry. 1989; 28: 2863-2873Crossref PubMed Scopus (147) Google Scholar).Other ProteinsEnzymes were typically purchased from New England Biolabs. Mr standards, from gel filtration calibration kits (Pharmacia Biotech Inc.), were: thyroglobulin (669,000), ferritin (440,000), catalase (232,000), aldolase (158,000), bovine serum albumin (67,000 and multimers thereof), and ovalbumin (43,000).Glycerol Gradient CentrifugationSamples were loaded onto 5.5-ml 20-40% glycerol gradients in buffer C (22Eichler D.C. Lehman I.R. J. Biol. Chem. 1977; 252: 499-503Abstract Full Text PDF PubMed Google Scholar) containing 100 μg/ml polyvinylpyrrolidonone K-60 (Matheson, Colemen & Bell). Gradients were centrifuged for 15 h at 50,000 rpm at 5°C in a Beckman SW55Ti rotor. 43 or 44 three-drop fractions were collected, by bottom puncture, per gradient. Protein concentrations were measured by micro-Bradford (23Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (213347) Google Scholar) assay in microtiter trays.Preparation of Substrate DNAsHairpin-shaped “BamHI” OligonucleotideAn oligonucleotide with the sequence 5′-GATCCGTGCACATTAATCATATGCGCCCGGATTCCGCTAAGCGGAATCCGGGCGCATATGATTAATGTGCACG-3′ was obtained from Oligos Etc. (Wilsonville, OR). The sequence is self-complementary and forms a 33-bp ds region with a (presumed) 3-nt loop and a 4-nt 5′-overhang that is part of a BamHI site. The oligonucleotide was labeled at its 5′-end, using [g-32P]ATP (DuPont NEN, 3000 Ci/mmol or ICN, 7000 Ci/mmol) and polynucleotide kinase. A portion of the labeled oligonucleotide was reacted with ATP and T4 DNA ligase, and the dimerized oligonucleotide was purified by polyacrylamide gel electrophoresis, as was the unligated labeled oligonucleotide. Labeled oligonucleotides with 5′ ss overhangs of 3, 2, 1, or 0 nt were prepared by incubation of 5′-end-labeled oligonucleotide with the Klenow fragment of DNA polymerase I in the presence of appropriate mixtures of dATP, dCTP, dGTP, and TTP, and purified by polyacrylamide gel electrophoresis.“Nicked hairpin DNA”The 1652-bp DdeI fragment of plasmid pBR322 x+F(24Smith G.R. Kunes S.M. Schultz D.W. Taylor A. Triman K.L. Cell. 1981; 24: 429-436Abstract Full Text PDF PubMed Scopus (212) Google Scholar) was gel-purified and labeled at its 3′-ends by incubation with the Klenow fragment of DNA polymerase I in the presence of [α-32P]TTP (New England Nuclear; 800 Ci/mmol). After digestion with AvaI and heat inactivation of the restriction enzyme, the DNA (two labeled AvaI-DdeI fragments, of 157 and 1495 bp) was ligated with a self-complementary hairpin-shaped oligonucleotide terminated with an AvaI site (5′-CCGAGTCTAGAGGGCCTATGGCCCTCTAGAC-3′; Oligos, Etc.). The oligonucleotide bore a 5′-hydroxyl and hence formed a covalent bond on only one strand. The mixture of ligation products was purified on an 8% polyacrylamide gel in Tris acetate-EDTA buffer(25Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Three closely spaced radioactive bands of about 170 bp were observed. The desired product, the middle band, was a 157-bp fragment of pBR322 bearing a 3′-32P label on the DdeI end and a hairpin-shaped oligonucleotide cap on the other end, with a nick in one strand adjacent to the cap. Fragments were extracted from gel slices by electroelution, recovered by ethanol precipitation, and repurified by the same method.DNA concentrations are given as molarities of DNA molecules, unless otherwise stated, and were calculated from the A260 of the plasmid DNA solutions and the specific activities of the labeled DNA species, as measured by trichloroacetic acid precipitation(22Eichler D.C. Lehman I.R. J. Biol. Chem. 1977; 252: 499-503Abstract Full Text PDF PubMed Google Scholar).Nondenaturing Polyacrylamide Gel ElectrophoresisGels were run in Hoeffer Mighty Small slab gel electrophoresis units, using alumina backplates and 0.75-mm spacers. Gels used an acrylamide:bisacrylamide ratio of 37.5:1 and were run at 4°C. Glycerol (10%, v/v, final concentration) and bromophenol blue (0.04%) were added to samples prior to loading. The continuous buffer system gels were run at 4°C for 1 h at 100 V, prior to changing the buffer and loading the samples, and then run with periodic replacement of the electrophoresis buffer. After electrophoresis, gels were fixed in 40% methanol, 10% acetic acid, the proteins were stained with Fast Stain (Zoion Research, Allston, MA), and the gels were dried between acetate sheets, or on Whatmann DEAE paper, prior to autoradiography.Determination of Dissociation ConstantsDNA substrates were the 5′-end-labeled BamHI hairpin-shaped oligonucleotide or its derivatives, as described above. For some experiments, dilutions of the DNA were mixed with constant amounts of RecBCD enzyme (Fraction VI-M) in 10 μl of a solution containing 50 mM MOPS-KOH (pH 7.0), 1 mM EDTA, 100 μg/ml each of bovine serum albumin and polyvinylpyrrolidonone K-60. After 10 min at 20°C, samples were run on minigels (4-15% polyacrylamide gradient gels in 50 mM MOPS-KOH (pH 7.0), 1 mM EDTA) as described above. For other experiments, reaction mixtures were prepared as above, but with 25 pM DNA and varying concentrations of RecBCD enzyme, and analyzed on 5% polyacrylamide gels. Incubation and gel electrophoresis were in the presence of 3 mM Mg(OAc)2 or 2 mM EDTA. The free and retarded radioactive DNA bands were detected and quantitated by PhosphorImager analysis. Results were analyzed by nonlinear regression for varying DNA concentration or by Hill plots for varying enzyme concentration (26Stone S.R. Hughes M.J. Jost J.P. Jost J.P. Saluz H.P. A Laboratory Guide to in Vitro Studies of Protein-DNA Interactions. Birkhäuser Verlag, Basel1991: 164-194Google Scholar).Gel Assay for Unwinding by RecBCD EnzymeRecBCD enzyme was incubated with the nicked hairpin DNA described above, with ATP omitted, for 10 min at 20°C to allow binding to the end of the DNA. Reactions were started by the addition of ATP (which was mixed, in some reactions, with an agent to prevent multiple rounds of reaction), incubated for 10 s at 20°C in a final volume of 20 μl, and stopped by addition of EDTA (10 mM final concentration), SDS (0.1%), sucrose (10%), and tracking dyes (0.04%). Final concentrations, after addition of enzyme, ATP, and DNA, were: 20 mM MOPS-KOH (pH 7.0), 4 mM Mg(OAc)2, 100 μg/ml polyvinylpyrrolidonone K-60, 5 mM ATP, and 1 nM labeled DNA substrate. Samples were analyzed on an 8% polyacrylamide gel in Tris acetate-EDTA buffer, and the gel was dried prior to exposure to film(27Taylor A.F. Smith G.R. J. Mol. Biol. 1990; 211: 117-134Crossref PubMed Scopus (32) Google Scholar). Final concentrations of inhibitory agents were: 0.13% Sarkosyl (ICN), 0.13% heparin (Sigma), or 40 nM ds DNA molecules (a DdeI digest of phage l DNA).Dynamic Light ScatteringThe translational diffusion coefficient of RecBCD enzyme was measured by dynamic light scattering, using a DynaPro-801 instrument (Protein Solutions Inc., Charlottesville, VA). The enzyme was dialyzed into 20 mM potassium phosphate (pH 6.8), 0.1 mM EDTA, 0.1 mM dithiothreitol, immediately before measurement. The AutoPro software of the machine calculated the hydrodynamic radius of the protein and its predicted Mr, based on data for typical globular proteins.RESULTSTwo Forms of RecBCD EnzymeAs a final step in its purification, RecBCD enzyme was sedimented through a glycerol gradient (Fig. 1A). The enzyme loaded onto the gradient was >95% pure, as judged by Coomassie staining of SDS-polyacrylamide gels (not shown), yet the protein sedimented as two peaks on the glycerol gradient. The Mrs of the two species were estimated by comparison with the sedimentation of known proteins in a parallel tube(28Freifelder D. Physical Biochemistry. Applications to Biochemistry and Molecular Biology. 2nd Ed. W. H. Freedman, New York1982Google Scholar). The estimated Mr of the slower sedimenting material was 313,000-345,000 (Table 1), consistent with it comprising one copy each of RecB, RecC, and RecD (expected Mr of 330,000). The slower sedimenting peak fractions from this and other gradients were pooled as Fraction VI-M (for “monomer”). The faster sedimenting material, with an estimated Mr of 560,000-690,000, was similarly pooled, as Fraction VI-D (for “dimer”), as its Mr suggested that it was a dimer of the slower sedimenting material.Table 1 Open table in a new tab Material equivalent to Fractions VI-M and VI-D, obtained from a previous glycerol gradient purification, was analyzed in the same experiment. The monomeric fraction from that gradient contained very little of the “dimer” species (Fig. 1B), while the “dimer” material contained approximately equal weights of the two forms of the enzyme (Fig. 1C). The origin of the monomer-sized material in this “dimer” fraction is unclear: it may have arisen from inefficient purification in the previous glycerol gradient, or it may have resulted from instability of the dimer form of the enzyme. Observations with nondenaturing gels (see below) suggest that the dimeric material also returns to the monomeric state during electrophoresis, suggesting that the dimer may indeed be unstable.To estimate the relative numbers of copies of each polypeptide in the two forms of RecBCD enzyme, fractions VI-M and VI-D were analyzed by SDS-polyacrylamide gel electrophoresis, and the Coomassie-stained gels were quantitated by densitometry. The observed relative intensities for the three polypeptides were within 15% of those predicted for a protein with one subunit each of 134,000, 129,000, and 67,000 (Table 2), as previously reported (7Roman L.J. Kowalczykowski S.C. Biochemistry. 1989; 28: 2863-2873Crossref PubMed Scopus (147) Google Scholar, 29Murphy K.C. J. Biol. Chem. 1994; 269: 22507-22516Abstract Full Text PDF PubMed Google Scholar) for purified enzyme. While the relative Coomassie staining abilities of the RecB, RecC, and RecD polypeptides have not been measured, the relative staining abilities of several proteins are proportional to the number of positively charged amino acids they contain(30Tal M. Silberstein A. Nusser E. J. Biol. Chem. 1985; 260: 9976-9980Abstract Full Text PDF PubMed Google Scholar). As shown in Table 2, the three polypeptides of RecBCD enzyme contain similar densities of positively charged amino acids. Hence, the equimolar staining of the three polypeptide bands is consistent with RecBCD enzyme containing equal numbers of copies of the RecB, RecC, and RecD polypeptides. This result, together with the estimated Mr of monomeric enzyme, shows that monomeric enzyme contains one copy of each polypeptide.Table 2 Open table in a new tab Quantitation of a Coomassie-stained native polyacrylamide gel showed that >60% of the protein in Fraction VI-D was in the dimeric form and that 95% of Fraction VI-M was in the monomeric form. Hence, the observation of equal ratios of polypeptides in the two forms (Table 2) implies that the dimeric form in Fraction VI-D also contained equal numbers of each subunit. The faster sedimenting form of RecBCD enzyme must thus be a simple dimer of the monomeric form, with two copies each of the RecB, RecC, and RecD polypeptides.Fig. 1 also shows the ds DNA exonuclease activity and specific activity of each gradient fraction. The specific activity of the dimeric enzyme was less than 25% of that of the monomeric enzyme. Exonuclease activity of the dimeric enzyme was apparently not due to contamination with monomeric enzyme, as the specific activity of the dimeric enzyme was constant across the peak of dimer material (Fig. 1, A and C). One explanation for a lower specific activity, a dimer with the active site of one monomer occluded, would predict a specific activity half that of the monomer, greater than that observed. Alternatively, the dimeric form may be devoid of ds DNA exonuclease activity and the observed nuclease activity may be that of monomeric enzyme that has arisen from the dimer before or during the assay.Estimation of Mr by Dynamic Light ScatteringThe hydrodynamic radius of the protein in Fraction VII from the second purification of RecBCD enzyme was measured by dynamic light scattering. Fraction VII was >95% pure, as assayed by SDS-polyacrylamide gel electrophoresis and contained ~90% monomer as assayed on a native polyacrylamide gel. Monomeric RecBCD enzyme had a hydrodynamic radius of 6.6 (± 0.2) nm, with an inferred Mr of 282,000 ± 18,000. Fraction VI-D had an apparent hydrodynamic radius of 11.2 nm; the instrument was unable to resolve the contributions from the monomeric and dimeric species in the sample and hence could not estimate their separate Mrs.The Mr of the monomer form estimated by this method is consistent with those measured by glycerol gradient centrifugation (preceding section) and by native gel electrophoresis (next section). These results confirm that the major form of the enzyme is indeed a monomer.Estimation of Mr by Native Polyacrylamide Gel ElectrophoresisProteins migrate in nondenaturing polyacrylamide gels as a function of both their size and their charge. We investigated gel conditions which would separate the monomeric and dimeric forms of RecBCD enzyme, both to confirm the estimates of their Mrs and to allow easier analysis of multiple samples, including those in the “gel shift” experiments described below. In a high pH nondenaturing discontinuous 5-20% gradient gel system(31Blackshear P.J. Methods Enzymol. 1984; 104: 237-255Crossref PubMed Scopus (180) Google Scholar), all of the protein standards migrated as a smooth function of their Mrs (Fig. 2). Monomeric and dimeric RecBCD enzyme migrated with apparent Mrs of 305,000 and 650,000, respectively (Fig. 2, lanes 2 and 3). In this system, with a resolving gel at pH 9.5, all the proteins should be highly charged, and hence their migration should be principally a function of their Mrs. RecBCD enzyme was unstable in this gel system: faster migrating species were seen, most of which did not appear in the other gel systems examined (Fig. 3 and data not shown). Their apparent Mrs were 220,000, 170,000, 160,000, 140,000, 130,000, and 120,000; we suppose that they arose from various combinations of the subunits of the enzyme.Figure 2:Molecular weight estimates and composition of RecBCD enzyme fractions determined by nondenaturing polyacrylamide gel electrophoresis. A high pH nondenaturing discontinuous 5-20% gradient gel (31Blackshear P.J. Methods Enzymol. 1984; 104: 237-255Crossref PubMed Scopus (180) Google Scholar) was run for 2 h in the cold room; the current was adjusted to maintain the upper buffer chamber temperature below 10°C. Lane 1 contained 0.5 μg each of thyroglobulin, ferritin, catalase, bovine serum albumin (Mr 67,000 and 134,000), and ovalbumin. The other lanes contain fractions from the first RecBCD enzyme purification. Lane 2, Fraction VI-M (790 units, 2.2 μg); lane 3, Fraction VI-D (1 μg); lane 4, Fraction IV (1700 units, 11 μg). Proteins were detected by Zoion Fast Stain. Linear regression of log(Mr) versus migration distance for the 669,000, 440,000, and 232,000 markers (r = −0.999) was used to estimate the Mrs of monomer (310,000) and dimer (630,000) in lanes 2 and 3.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3:Nondenaturing polyacrylamide gel electrophoresis of RecBCD enzyme from two enzyme preparations. A 5% polyacrylamide gel in 50 mM MOPS-KOH (pH 7.0), 2 mM EDTA was run for 2 h at 100 V. Lane 1 contained 1 μg of bovine serum albumin, providing Mr markers of 67,000, 134,000, and 200,000. Lane 15 contained 2 μg each of thyroglobulin (669,000) and ferritin (440,000). Lanes 2, 5, 7, 9, and 11 contained Fractions VI-D (0.7 μg) from the first purification. Lane 3 contained Fraction IV (500 units, 3.3 μg), and lane 4 contained Fraction VI-M (550 units, 1.5 μg), both from the first purification. The remaining lanes contained fractions from the second purification. Lane 6, Fraction VII (490 units, 4.1 μg); lane 8, Fraction V (110 units, 0.6 μg); lane 10, Fraction IV (85 units, 1.4 μg); lane 12, Fra" @default.
• W2078095931 created "2016-06-24" @default.
• W2078095931 creator A5017146305 @default.
• W2078095931 creator A5027526419 @default.
• W2078095931 date "1995-10-01" @default.
• W2078095931 modified "2023-10-18" @default.
• W2078095931 title "Monomeric RecBCD Enzyme Binds and Unwinds DNA" @default.
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