THE JOURNAL or BIOLOGICAL CHEMISTRY Vol. 260, No. 16, hue of August 10, pp. 6859-6885, 1976 Printed in U.S.A. Ten Proteins Required for Conversion of 4x174 Single- stranded DNA to Duplex Form in Vitro RESOLUTION AND RECONSTITUTION* (Received for publication, February 13, 1975) RANDY SCHEKMAN,~ JOEL H. WEINER,~ ALAN WEINER,~ AND ARTHUR KORNBERG From the Department of Biochemistry, Stanford University School of Medicine, Stanford, California 94305 SUMMARY Protein requirements for conversion of 4x174 single- stranded DNA to a double-stranded replicative form with a small gap (RF 11) have been determined by resolution and re- constitution of the multienzyme system from extracts of gently lysed Escherichia coli. Assays depended on: (a) com- plementation of extracts of thermosensitive mutants and (b) fractionation of extracts of wild type cells to divide essen- tial components into groups, each of which was further re- solved. These procedures have yielded eight proteins: dnaB protein, dnaC protein, proteins i and n (two novel proteins without a defined genetic locus), dnaG protein, DNA polymer- ase I11 holoenzyme (polymerase III* and copolymerase III*), and DNA unwinding protein; purification procedures for the first four are presented here. (Closure of RF 31 requires as with phage M13, DNA polymerase I and ligase.) Soluble extracts of gently lysed Escherichia coli convert 4x174 single-stranded DNA to the double-stranded replicative form (1). Enzymes involved in this reaction appear to participate in host chromosome replication. Conditionally lethal mutants of E. coli, defective in initiation at the chromosome origin (dnaA, dnaC) and ongoing DNA replication (dnaB, dnaE, and dnaG) all show in vitro defects in 4x174 RF* formation (2, 3). Proteins corresponding to these dna genes may be assayed and purified using complementation of extracts prepared from mutant cells. Partially resolved enzyme fractions have been added to deficient extracts and assayed at an elevated temperature at which the mutant protein is inactive. The dnaB, dnaC, dnaE, and dnaG gene products have been purified in this manner (4-7). However, * This work was supported in parts by grants from the National Institutes of Health and the National Science Foundation. Present address, Department of Biology, University of Cali- fornia at San Diego, La Jolla, California 92037. 5 Fellow of Darnon Runyon Memorial Fund for Cancer Re- search. 7 Fellow of Helen Hay Whitney Foundation. Present address, Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139. The abbreviations used are: RF, circular double-stranded DNA of replicative form; RFI, covalently closed RF, RF 11, RF with a discontinuity in one strand; NEM, N-ethylmaleimide. these four proteins were insufficient for RF synthesis from single- stranded DNA. In another approach extracts from wild type cells are subjected to fractionation. A procedure is adopted which generates two fractions, each of which is required; each fraction is then sub- divided further. This approach together with the complementa- tion assay has been employed for complete resolution of the components involved in the 9x174 reaction (8, 9). This report presents the strategy of the fractionation pattern, and the assays and purification procedures developed for four of the essential proteins. Information about the size, properties, abundance, and possible functions of these proteins are suggested from these studies. EXPERIMENTAL PROCEDURE Materials Materials were from sources previously described (1,2). Freshly dissolved calf thymus DNA (Calbiochem) was denatured and at- tached to cellulose by the procedure of Alberts (10) dried at 37" and stored as a powder at -20". Buffer A is 50 mM Tris-HC1 (pH 7.5), 0.1 M NaCl, 20% glycerol (v/v), 1 mM EDTA, and 1 mM dithiothreitol. Buffer B is 50 mM imidazole-HC1 (pH 7.0), 20% glycerol, 1 mM EDTA, and 1 mM dithiothreitol. Buffer C is 50 mM Tris-HC1 (pH 7.5), 3oyO glycerol, 1 mM EDTA, and 10 mM dithiothreitol. Buffer D is 50 my Tris-HC1 (pH 7.5), 20% glycerol, and 1 mM EDTA. Buffer E is 20 mM potas- sium phosphate (pH 6.5), 20% glycerol, and 1 mM EDTA. Tris-HC1 and imidazole-HC1 stock solutions (2 M) were prepared at room temperature and diluted as required. DNA unwinding protein was Fraction 3b (0.38 mg/ml (11)); dnaG protein (Fraction V, 0.04 mg/ml)* was prepared according to Bouch6 et al.' DNA polymerase I11 holoenzyme was purified as before (12) except that Bio-Gel A-0.5m (200 to 400 mesh) replaced Bio-Gel A-5m. Fraction IV was precipitated with an equal volume of saturated ammonium sulfate (neutralized with ammonium hy- droxide). After 30 min at 0" the precipitate was centrifuged at 31,500 X g for 10 min and the supernatant was discarded. Am- monium sulfate precipitates stored at 0" were stable for at least 2 months; when redissolved in Buffer A (containing 10 mM dithio- threitol) they retained 50y0 of their activity after 1 week at 0'. Escherichia coli PC 79 (I?, his-, strR, malA, xyl-, mtl-, thi-, pol Al, sup-, dnaD7 (now referred to as dnaC)) was provided by Dr. P. Carl (University of Illinois, Urbana). E. coli BT1029 (dnaB, thy, pol Al, endo I-) waa provided by Dr. H. Hoffmann-Berling (Heidelberg). E. coli H560 was grown and soluble extracts were prepared as previously described (1,12) except that the 37" heating step was performed with 35-ml aliquots in polyethylene centrifuge J.-P. BouchB, K. Zechel, and A. Kornberg (1975) J. Biol. Ckm. 260. in press 5859 5860 tubes for 2 min followed by centrifugation at 48,000 X g for 30 min at 0". E. coli HMS-83, provided by Dr. R. L. McMacken, of this department, was grown as E. coliH560 (I). Methods General Procedures Ammonium Sulfate Precipitation-Solid ammonium sulfate was added at 0" over a 10-min interval, the solutions were stirred for another 20 min and centrifuged for 10 rnin at 23,000 X g. Solutions for extracting ammonium sulfate precipitates contained varying amounts of ammonium sulfate added to Buffer A (e.g. solution AS 0.20 was made up by adding 20 g of ammonium sulfate to 100 ml of Buffer A). A glass homogenizer was used manually for these extractions. Assay of dnaC Protein Preparation of Mutant Enzyme Fraclion-E. coli PC79 was grown at 30" in Hershey broth, harvested, and resuspeiided as described before for E. coli H560 (1). The soluble extract (Fraction I) was prepared as before (1, 12) except that spermidine was omitted and lysis was for 1 hour at 0". Fraction I was adjusted to 0.2 M NaCl and applied to a column of IIEAE-cellulose (equili- brated in Buffer 1) + 0.2 M NaCl) %th the volume of Fraction I. The flow-through was collected and solid ammonium sulfate was added to 40% saturation (0.242 g added/ml). The solution was stirred for 30 rnin at O", and then centrifuged at 39,000 X g for 10 rnin. The pellet (Fraction 11) was taken up in Buffer I), >Seth the volume of Fraction I (about 10 mg of protein/ml) and dialyzed against 100 volumes of Buffer I) + 0.05 M NaCl for 4 hours. The dialyzed fraction divided into aliquots was stored in liquid nitro- gen and remained stable for at least 3 months. Assay-Assays were performed in 25 rl at 30" for 20 min. Com- ponents were added in the following order at 0': 5 to 10 ~1 of dilu- tion buffer (loyo sucrose, 50 mM Tris-HC1 (pH 7.5), 20 mM dithio- threitol, 0.2 mg/ml of bovine serum albumin, and 50 mM NaCl), 40 pg (4 pi) of mutant Fraction 11, 1 to 6 pl of the fraction to be assayed, and 10 p1 of a reaction mixture containing 0.45 nmol of [cx-~*P]~CTP (200 to 600 cpm/pmol), 1.25 nmol each of dATP, dGTP, and TTP, 125 nmol of MgC12,lOO nmol of spermidine-HCl, 20 nmol of ATP, 2.5 nmols each of GTP, UTP, and CTP, 0.33 nmol of +X174 DNA, and 100 ng of rifampicin. DNA synthesis was measured by incorporation of the labeled deoxynucleotide into an acid-insoluble form. One unit is defined as 1 pmol of total deoxy- nucleotide incorporated in 1 min; the value for dCMP incorpora- tion was multiplied by 4. Preparation of DNA-cellulose Binding and Nonbinding Fractions E. coli H560 (150 g of cell paste in 750 ml of 10% sucrose and 50 mM Tris-HC1 (pH 7.5)) was lysed and centrifuged to yield Fraction I (500 ml) (Table I) (1, 2). Ammonium sulfate (0.226 g/ml) was added and the precipitate was collected as described above. The precipitate was resuspended in 50 ml of AS 0.24 and the insoluble proteins were collected. This procedure was repeated with 10 ml of AS 0.20. The precipitate was resuspended in Buffer D to a final volume of 11 ml and applied to a Sephadex G-25 column (170 ml, 3-cm diameter) equilibrated in and eluted with Buffer D. The void volume fractions of column buffer conductivity (1.8 mmho) were pooled (42 ml, Fraction 11) and solid NaCl was added to a final concentration of 0.2 M. This fraction was then applied to a DEAE- cellulose (DE52) column (10 ml, 2-cm diameter) equilibrated in Buffer D + 0.20 M NaCl, followed by a 10-ml wash with the same buffer. Fractions containing the bulk of the protein were pooled (50 ml), ammonium sulfate was added (0.272 g/ml), and the pre- cipitate was collected and resuspended in 5 ml of Buffer B. This fraction was then applied to a Sephadex G-25 column (45 ml, 2-cm diameter) equilibrated in and eluted with Buffer B. The void volume fractions of column buffer conductivity (1.7 mmho) were pooled (15 ml, Fraction 111) and applied to a DNA-cellulose column (12 ml, 3.5-cm diameter) also equilibrated in Buffer B. The column was then washed with 100 ml of Buffer B. Nonbinding (flow-through) fractions (5 ml) were collected and assayed as described below. The DNA-cellulose binding proteins were eluted with 40 ml of Buffer B + 2 M NaCl, precipitated with ammonium sulfate (0.226 g/ml), resuspended in 2.5 ml of Buffer B, dialyzed against 500 ml of Buffer D + 0.2 M NaCI, and stored in aliquots in liquid nitrogen for at least 6 months with no loss of activity. Ammonium sulfate precipitates of Fractions I1 or I11 may be stored at -20" for several days and subsequently carried through the remaining steps. Resolution of DNA-cellulose Nonbinding Proteins The nonbinding fractions from the aboxe preparation were pooled (35 ml), precipitated with ammonium sulfate (0.272 g/ml), collected, and resuspended in 1.5 ml of Buffer A. Part of the sample (1.2 ml) was applied to a Sephadex G-150 column (42 ml, 2-cm diameter) equilibrated in Buffer A and eluted by this buffer. TWO essential proteins were separated (Fig. 1); one was in the void vol- ume (dnaB protein) and the other was included at K,, = 0.25 (protein i). Peak fractions of each were pooled and aliquots stored in liquid nitrogen. Quantitative determination of the preparation through this stage is presented in Table I. Assay of DNA Binding and Nonbinding Fractions Activity of the binding fraction was determined by assaying varying amounts in the presence of dnaC protein and the nonbind- ing fraction. Assays were in 25 pl at 30" for 20 min. Components were mixed as follows: 10pl of dilution fraction, 2 pl of nonbinding fraction, 0.2 to 2 pl (1 to 10 pg) of binding fraction, 2 to 5 pg of Fraction 111 dnaC protein, and 10 pl of the reaction mixture de- scribed for assay of the dnaC protein. DNA synthesis was meas- ured as above. When the resolved nonbinding fractions were puri- fied and became available, protein i (Fraction V, 4 to 40 ng/assay) and dnaB protein (Fraction V, 20 ng/assay) were used (see "Re- sults"). Assay for Protein n, NEM-sensitive DNA-cellulose Binding Protein The binding fraction contains two essential components which are inactivated by the sulfhydryl-blocking agent, NEM : DNA polymerase I11 holoenzyme and another component, called protein n. The binding fraction was treated with 10 mM NEM (freshly dis- solved in water) at 30" for 15 min and excess NEM was removed with 20 mM dithiothreitol at 0". Assays for protein n were mixed as follows: 10 p1 of dilution buffer, 0.1 to 2 p1 of the fraction to be assayed, 5pl of a mixture of NEM-treated binding fraction (1 to 10 pg), protein i (Fraction V, 40 ng), dnaB protein (Fraction V, 20 ng), DNA polymerase I11 holoenzyme (Fraction IV, 0.33 pg), and dnaC protein (Fraction 111, 2 to 5 pg), followed by 10 pl of the reaction mixture used for assay of the dnaC protein. Alternatively, purified DNA unwinding protein (700 ng) plus dnaG protein (20 ng) can replace the NEM-treated binding fraction. 10 1 I I I EXCLUDED INCLUDED FRACTION FRACTION N 8- 0, 0 X z I- o LL v) z 3 2 . t 3 FRACTION FIQ. 1. Sephadex G-150 chromatography of the DNA-cellulose nonbinding fraction. The DNA-cellulose flow-through fraction (see "Methods") (1.2 ml, 13 mg, 470 unit.s/mg) waa applied to a Sephadex G-150 column (42 ml, 2-cm diameter) equilibrated in and eluted with Buffer A. Fractions of 1.25 ml were collected at a flow rate of 20 ml/hour. Aliquots (2 pl) of the void volume protein com- bined with portions (2 pl) of the column fractions were used to assay for the included fraction (see "Methods"). The excluded fraction was then assayed by combining 2-pl aliquots of the in- cluded peak with 2-pl portions of the void volume region. 5861 TABLE I1 Stages in reconstitution Assays were performed as described under "Methods." Where indicated, reactions contained 2 to 5 pg of dnaC protein (Fraction III), 20 pg of Fraction I1 (depleted of dnaC protein), 10 pg of DNA-cellulose binding proteins, 10 pg of DNA-cellulose non- binding proteins, 40 ng of protein i (Fraction V), 20 ng of dnaB protein (Fraction IV), 10 pg of NEM-treated DNA.cellulose binding proteins, 0.3 pg of DNA polymerase I11 holoenzyme (Fraction IV), 0.13 pg of protein n (Fraction V), 40 ng of dnaG protein (Fraction V), and 0.73 pg of DNA unwinding protein (Fraction 3b). TABLE I Resolution of DNA-cellulose binding fractions, dnaB protein, and protein i Assays were performed as under "Methods." Activity was measured with 2- to 4-pg additions of dnaC protein (Fraction 111). Stage I1 was assayed with dnaC protein and an excess of binding or nonbinding fractions. Stage I11 was assayed with dnaC protein, DNA-cellulose binding fraction, and either an excess of Sephadex G-150 excluded fraction or included fraction to permit titration of the component to be assayed. Stages in fractionation Extract. .......................... Stage I Ammonium sulfate. ............... DEAE-cellulose ................... DNA-cellulose binding fraction.. .. Nonbinding fraction. ............. Sephadex G-150 excluded fraction (dnaB protein). ................... Included fraction (protein i) ...... Stage I1 Stage 111 Protein mglml 19.7 2.7 6.1 5.3 11.7 1.5 0.6 Total Units x lo-' 90 124 121 119 8.6 40.2 Specific activity unils/mg 790 1,360 9,100 6,800 2,300 13,400 Glycerol Gradient Sedimentations These were performed in an SW 56 rotor of the Beckman L2-65B ultracentrifuge. Fractions were collected through a puncture at the bottom of the tube. Hemoglobin and myoglobin markers were located by measuring A120. RESULTS Strategy of Fractiondion The fractionation pattern of an extract of wild type cells can be divided into five stages (Table 11, Fig. 2). Separation of dnaC Protein by Ammonium Sulfate-Fraction I1 (see "Methods" for preparation of DNA-cellulose binding and nonbinding fraction) was deficient in dnaC protein (Table 11, Stage I). The latter could be assayed with the dnaC-depleted ammonium sulfate fraction from wild type cells or from dnaC mutant cells (Table 11, Footnote a). Separation of DNA-cellulose Binding and Nonbinding Proleins -Fraction I1 (depleted of dnaC protein) was applied to a column of DNA-cellulose and the nonbinding (flow-through) and binding fractions were collected. At this stage (Table 11, Stage 11), assays required dnaC protein and both the DNA-cellulose binding and nonbinding fractions. Resolution of DNA-cellulose Nonbinding Proteins on Sephadex G-150-Filtration resolved this fraction into excluded and in- cluded activities (Fig. 1). Both were needed in addition to the DNA-cellulose binding proteins and dnaC protein (Table I ; Table 11, Stage 111). The excluded activity was identified as the dnaB gene product by further purification (see below) and com- plementation assays (Table 111) ; the included activity, called protein i, also appeared to be a single component upon purifica- tion (see below), but no genetic locus has yet been found for it. NEM-sensitive DNA-cellulose Binding Proteins-Reconstitu- tion at Stage I11 (Table 11) was inactive when the DNA-cellulose binding fractions were treated with NEM (Table 11, Stage IV). The NEM-sensitive components could be resolved by Sephadex G-150 filtration into excluded and included fractions. The ex- Component omitted None (compiete mixture). ..... dnaC protein.. ................ Fraction I1 (depleted of dnaC protein) .................... DNA-cellulose binding pro- teins.. ..................... DNA-cellulose nonbinding proteins .................... Protein i.. .................... dnaB protein. ................. NEM -treated DNA-cellulose binding proteins. ......... DNA polymerase 111 holoen- zyme. ...................... Protein n.. ................... dnaG protein. ................ DNA unwinding protein.. ..... Stages $mol DNA synthesis 88 4.0 2.0 - 52 9.1 2.0 1.9 - 47 0.4 1.1 6.7 4.0 - 37 6.4 8.7 7.6 6.7 1.8 6.6 - 120 7.9 13.9 24.9 2.2 5.4 8.0 1.9 - a A complementation assay of the dnaC protein (Fraction 111) gave a value of 60 pmol with the dnaC mutant Fraction I1 (40 pg), but only 4 pmol without it; the value for Fraction I1 by itself was 2.0 pmol. SOLUBLE EXTRACT I AMMONIUM SULFATE cluded fraction was identified as DNA polymerase I11 holoen- zyme (12). The other NEM-sensitive activity, called protein n, could then be assayed upon the addition of NEM-treated DNA- cellulose binding proteins, DNA polymerase 111 holoenzyme, DNA-cellulose nonbinding proteins, and dnaC protein. Protein n has been purified to near homogeneity (see below) but has not been identified with any of the known genetic loci. NEM-resistant DNA-cellulose Binding Proteins-Complete re- constitution was achieved by replacing the NEM-treated DNA- cellulose binding fraction by the E. coli DKA unwinding protein (11) and dnaG protein (Table 11, Stage V). Purification of 5862 TABLE I11 dnaB Complementation assay Escherichia coli BT1029 was grown and soluble extracts were prepared as described for dnaC mutant enzyme fraction. Am- monium sulfate was added to Fraction I (0.240 g/ml) at 0". The suspension was centrifuged at 10,OOO X g for 20 min. The pellet was dissolved in ?& volume of Buffer A without NaC1. Assays were performed as described for dnaC complementation. Addi- tions of dnaC protein were 9 units of Fraction 111. Protein added to dnaB mutant fraction None. ............................ dnaB protein. ................... DNA unwinding protein. ......... Protein i.. ...................... Protein n ......................... dnaG protein ..................... DNA polymerase I11 holoenzyme. . dnaC protein None I Present )mol DNA synthesis 8.0 11.8 13.5 54.6 12.2 11.4 11.3 17.3 12.3 12.3 12.2 9.0 12.7 9.4 these proteins based on their role in the conversion of phage G4 single-stranded DNA to RF are presented elsewhere (1 1) .* The product at Stage V was RF I1 with a nearly full length linear synthetic strand (8). The procedures described above and outlined in Fig. 2 were guides to the existence and general properties of the several pro- teins and provided assays for them. The endogenous (over-all) replication activity of the extract was 800 to 1000 units/g of cell paste. On this basis, the activity of the individual components, expressed as units/g of cell paste, was: dnaU protein, 7000; dnaC protein, 3000; dnaG protein, 8000; DNA polymerase I11 holo- enzyme, 5000; protein n, 1400; protein i, 1100 units. In recent experiments with protein i activity levels of 5000 units/g of cell paste have been observed. Thus, each of the activities was present at a level equal to or greater than the endogenous activity. Inde- pendent purification procedures had to be developed to obtain each protein in optimal yields. The purification procedures for dnaC protein, protein i, dnaU protein, and protein n are given below. Protein mg/ml 17 78 4.5 Purification oj dnaC Protein This protein is the activity most readily identified as specific for 4x174 and also the most labile. Although dnaC activity can be isolated from E. coli H560, only 165 units/g of cell paste were recovered in extracts compared to 2600 to 3000 units/g for E. coli HMS-83. Beyond the enrichment of about 70-fold (Table IV) further attempts at purification have been frustrated by loss of activity. Glycerol gradient sedimentation of dnaC protein (Fig. 3) gave an S value of 2.0 corresponding to a molecular weight of about 20,000. Purijication of dnaB Protein The purification of dnaB protein is summarized in Table V. This protein was initially identified as the excluded factor on Sephadex G-150 gel filtration and subsequently shown to comple- ment a crude fraction prepared from a dnaB temperature-sensi- tive mutant (Table 111). In order to complement this mutant it was necessary to add dnaC protein in addition to dnal3 protein. Similar pleiotropic requirements seen with other mutant extracts Yield --__ unW 40 mg % 170 75 2650 55 TABLE IV Purification of dnaC protein Escherichia coli HMS83 (335 ml, 65 g) prepared as for H560 (see "Methods") was thawed at 10" and dilut.ed with 150 ml of 0.05 M Tris-HC1 (pH 7.5)/1070 sucrose. To the suspension was added 1.2 ml of 0.4 M dithiothreitol, 18.5 ml of 4 M KCl, 4.8 ml of lysozyme (20 mg/ml), 4.8 ml of lOyo Brij 58, 19.5 ml of 0.5 M EDTA, and 9.7 ml of 1 M spermidine-HCl. The pH was adjusted to 8.5 with 1.5 g of solid Tris base. The suspension was incubated for 25 min at 4O, then centrifuged at 48,000 X g for 30 min to produce Fraction I (415 ml). Ammonium sulfate (0.240 g/ml) was added; the precipitate collected aa described under "Meth- ods" was resuspended in 33.5 ml of AS 0.24. The undissolved material was collected and dissolved in Buffer B containing 0.2 M KCl (Fraction 11, 16 ml). Fraction I1 (5.5 ml) was dialyzed against two 1-liter changes of Buffer B plus 0.2 M KCl for a total of 2.5 hours. The sample waa diluted 1 to 5 in successive 1-ml portions in Buffer B and applied to a phosphocellulose column (45 ml; 2.5-cm diameter) equilibrated in Buffer B, the column was washed with 45 ml of Buffer B and dnaC activity was eluted with a linear KC1 gradient (250 ml, 0.05 to 0.4 M in Buffer B). Pooled fractions (40 ml) precipitated with ammonium sulfate (0.39 g/ml) were dissolved in 4 ml of Buffer B containing 0.2 M KCI (Fraction 111) and stored in aliquots in liquid nitrogen where it was stable for at least 3 months. Fraction I1 may be stored at -20°, but is unstable at 0". I. Extract ................. 11. Ammonium sulfate .... 111. Phosphocelluloseb ....... Fraction x IO-' 284 214 156 Total units - Purifi- cation ___ 4.3 70 - 0 Calculation of yield and purification was based on the dnaC activity recovered in all of the ammonium sulfate fractions, assays in Fraction I were not reliable. * This step was carried out in a portion of Fraction I1 (5.5 ml) but is calculated for the entire preparation. I I 1 I WHAL'E HEMOGLOBIN MYOGLOBIN 1 0 I 50 FRACTION FIG. 3. Glycerol gradient sedimentation of dnaC protein. dnaC protein (75 pg, 300 units) was applied to a 3.8-ml10 to 25% glycerol gradient in 0.05 M imidazole-HC1 (pH 7.0)/0.2 M KCl/1 mM EDTA/1 mM dithiothreitol. Centrifugation was at 55,000 rpm for 24 hours at 0". Thirty-four fractions were collected, assayed, and marker positions were determined as described under "Methods." 5863 Protein mg/ml 25 1.9 0.23 0.14 0.043 TABLE V Purification of dnaB protein Escherichia coli H560 (1.5 liters, 300 g of frozen cells) was lysed to yield 1180 ml of Fraction I. Ammonium sulfate was added (0.226 g/ml) ; the precipitate was collected and resuspended in 118 ml of AS 0.20; the pellets were treated successively with 24-ml aliquots of AS 0.16, AS 0.15, and AS 0.14. The supernatant fluids from these last three washes were combined and the dnaB activity reprecipitated with ammonium sulfate (0.1 g/ml). The precipitate was collected and resuspended in Buffer D containing 0.25 M NaCl (Fraction 11, 10 ml). The sample was applied to a column of Bio-Gel A-1.5m (200 to 400 mesh, 475 ml, 3.8-cm diam- eter) equilibrated in and eluted with Buffer D containing 0.25 M NaCl. Fractions with dnaB activity (100 ml), eluted at a K,, of 0.36 to 0.64, were treated with ammonium sulfate (0.24 g/ml); the precipitate was dissolved in 3 ml of Buffer D containing 0.05 M NaCl (Fraction 111, 3.2 ml). The sample was applied to a column of Sephadex G-25 (40 ml, 2-cm diameter) equilibrated in Buffer D containing 0.05 M KCl and the void volume fractions were pooled (10 ml) and applied to a column of DEAE-cellulose (20 ml, 2-cm diameter) equilibrated in Buffer D (0.05 M NaC1). The column was then washed wit,h 10 ml of the same buffer fol- lowed by 30 ml of Buffer D containing 0.15 M NaCl; dnaB activity was eluted with a linear NaCl gradient (120 ml, 0.15 to 0.40 M in Buffer D) at about 0.35 M C1- and concentrated on DEAE-cellu- lose. Aliquots (2.5 ml) of the pooled activity (32 ml) were each diluted with 3 volumes of 20% glycerol/l mM EDTA and applied sequentially to a column of DEAE-cellulose (2 ml, 1-cm diameter) equilibrated in Buffer D. The activity was then step eluted with 10 ml of Buffer D + 0.40 M NaCl and 1-ml fractions were collected. The dnaB activity was pooled (Fraction IV, 2.8 ml) and dialyzed against three 500-ml changes of Buffer E for a total of 5 hours. The sample was applied to a column of phosphocellulose (10 ml, 1-cm diameter) equilibrated in Buffer E. The column was then washed with 20 ml of Buffer E and dnaB activity eluted with a linear KC1 gradient (30 ml, 0 to 0.40 M KCl in Buffer E) at about 0.3 M C1-. The pooled activity (Fraction V, 6.2 ml) was stored in aliquots in liquid nitrogen where it was stable for at least 3 months. Fraction I1 may be stored at -20". The activity is not stable at low ionic strength (<0.1 M C1-) at 0" for more than 1 day. Specific unildmg % x lo-' 0.013 7.3 60 12 21 27 17 370 12 activity ____- Fraction Purifi- cation .~ 590 980 2,180 29,600 Protein I1 I. Extract.. . .. . . . . . . . . 11. Ammonium sulfateo. . 111. DEAE-cellulose. . . . . . IV. Sephadex G-150. . . . . . V. Phosphocellulose. . . . . x lo-' 690 420 138 113 80 mg/ml 20 13.2 8.8 0.3 0.02 I. Extract ........ ...... 11. Ammonium sulfate@. . . 111. Bio-Gel A-1.5m.. . . . . . . IV. DEAE-cellulose , . . . . , . V. Phosphocellulose. . . . . . unitr/mg 0.08' x io-a 13 25 350 590 x 10-8 2000 1720 555 295 73 % 81 34 14 4 - Pur& cation 150 290 4020 6780 Calculation of yield and purification was based on the activity recovered in all of the ammonium sulfate fractions; assays in Fraction I were not reliable. have complicated the use of this approach to purify the deficient protein. Preliminary characterization of dnaB protein indicates a native molecular weight of about 250,000 in agreement with the pub- lished value (4). The function of dnaB protein has not yet been elucidated. Purification of Protein i Protein i was purified 30,000-fold by the procedure summarized in Table VI. Sodium dodecyl sulfate gel electrophoresis indicated a single major band which migrated slightly slower than DNA unwinding protein and thus had a monomer molecular weight of TABLE VI Purification of protein i Fraction I (2130 ml) prepared from a lysate of Escherichia coli H560 (3.0 liters, 600 g) was treated with ammonium sulfate as described for dnaB protein (Table V) except that 213 ml of AS 0.30 and 43 ml of the AS 0.16, AS 0.15, and AS 0.14 were used. The final precipitate (AS 0.14 pellet) which contained the bulk of protein i activity (as well as DNA polymerase 111 holoenzyme (12)) was resuspended in 25 ml of Buffer A (Fraction 11, 30 ml), clarified, and applied to a column of Sephadex G-25 (250 ml, 3- cm diameter) equilibrated in and eluted with Buffer C. The Sephadex G-25 void volume fractions of conductivity equivalent to Buffer C were pooled and applied to a column of DEAE-cellu- lose (50 ml, 2-cm diameter) equilibrated in Buffer C. Protein i activity was eluted with a linear NaCl gradient (500 ml, 0 to 0.3 M NaCl in Buffer C) at about 0.15 M C1- (Fraction 111, 50 ml), concentrated with ammonium sulfate (0.24 g/ml) and resuspended in 5 ml of Buffer D. This sample was applied to a column of Sephadex G-150 (250 ml, 2.5-cm diameter) equilibrated with Buffer D. Protein i activity was eluted at K,, = 0.25 (Fraction IV, 30 ml). This fraction was concentrated on a column of DEAE- cellulose (2.5 ml, 1.2-cm diameter). After equilibration in Buffer D, protein i activity was step eluted with 10 ml of Buffer D con- taining 0.2 M NaCl; protein i (5 ml) was dialyzed against two 500-ml changes of Buffer E for a total of 5 hours and immediately applied to a column of phosphocellulose (5 ml, 1-em diameter) equilibrated in Buffer E. The column was washed with 4 ml of Buffer E and protein i activity eluted with a linear KCI gradient (50 ml, 0 to 0.4 M KCl in Buffer E) at about 0.2 M C1-. The pooled activity (Fraction V, 5 ml) was stored in aliquots in liquid nitro- gen where it was stable for at least 6 months. Fraction I1 may be stored at -20°, but once dissolved should be chromatographed on DEAE-cellulose immediately. Fraction Total units Calculation of yield and purification was based on the ac- tivity recovered in all of the ammonium sulfate fractions; assays in Fraction I were not reliable. about 20,000.' Glycerol gradient sedimentation of protein i (Fig. 4) gave an S value of 3.0 corresponding to a molecular weight of about 35,000 to 40,000 indicating protein i is either an asymmetric monomer or a dimer. From the data of Table VI we estimate about 100 to 150 molecules of protein i per cell. No function for protein i has yet been determined. Based on similarities in the purification procedure and physical properties it appears that protein i corresponds to the Factor X recently reported by Wickner and Hurwitz (9). Purijication of Protein n Protein n was purified about 2,000-fold by the procedure in Table VII. Sodium dodecyl sulfate gel electrophoresis indicated a single major band at 82,000 daltons and minor bands at 25,000 and 50,000 daltons. The activity appeared to te associated with the major band. Fraction VI was estimated to be about 80% pure by Coomassie blue staining. From the data of Table VI1 about * R. McMacken and A. Kornberg, unpublished work. 5864 25 25 z 0 0 Y v) 15 a a . f 10 3 5 I I I HEMOGLOBIN MYOGLOBIN 1 + n I " 10 20 30 40 50 FRACTION FIG 4. Glycerol gradient sedimentation of protein i. Protein i (2.4 pg, 900 units) was applied to a 3.8-ml, 10 to 30% glycerol gradi- ent in: 0.05 M Tris-HCI (pH 7.5)/0.20 M NaCl/l mM EDTA. Cen- trifugation was at 55,000 rpm for 16 hours at 0". Forty-three frac- tions were collected, assayed, and marker positions were deter- mined as described under "Methods." 200 molecules of prot,ein II are present per cell. Protei11 11 aggre- gates at protein concentrations greater than 0.1 mg/ml and salt concentrations less than 0.25 M making estimation of the native molecular weight inaccurate. The protein binds specifically to single-stranded DNA dramatically altering the contour length.' DISCUSSION What seemed to be a simple operation of copying the small, single-stranded DNA circle of 4x174 to form the duplex requires a multienzyme system. The many proteins which make up this system hold great interest because they appear to be the compo- nents which the uninfected Escherichia coli uses to initiate and sustain the replication of its own chromosome. Thus, the resolu- tion and reconstitution of this system made possible by the sim- plicity of the phage DNA template has implications for under- standing replication of duplex DNA of E. coli. In an analogous way the filamentous phage 1113 has been a useful probe for another replicative system in E. coli, one which appears to be reserved for replication of extrachromosomal elements (13). It simplifies discussion to consider three major stages in the synthesis of a DNA chain : initiation, elongation, and termina- tion. Both the 4x174 and h113 replication systems are similar in their requirement for an RNA-primer fragment synthesized in the initiation stage, for the DNA polymerase 111 holoenzyme and DNA unwinding protein to sustain the elongation stage, and for DNA polymerase I and ligase to carry out the termination stage (q. RF I1 conversion to RF 1) (8). The two systems differ in the mechanism used for synthesis of the RNA primer. Initia- tion OIL M13 DNA depends on a form of the multisubunit RNA polymerase and so is sensitive to inhibition by rifampicin. The 4x174 system, on the other hand, is resistant to rifampicin and depends on a novel RNA synthetic enzyme.2 Eight proteins have been resolved from extracts of E. coli that are needed for reconstituting the 4x174 single strand con- version to RF 11. Available information about their physical and functional properties, summarized in Table VIII, is incom- 4 J. H. Weiner and A. Kornberg, unpublished work. TABLE VI1 Purification of protein n Escherichia coli H560 (700 ml, 140 g) was lysed to yield Fraction I (575 ml). Ammonium sulfate was added (0.226 g/ml) and the precipitate collected and resuspended in 58 ml of AS 0.24. The precipitate was collected and the procedure repeated with 11-ml aliquots of AS 0.20 and AS 0.18. The final precipitate was col- lected, dissolved in 8.5 ml of Buffer D containing 1 mM dithio- threitol, clarified and applied to a Sephadex G-25 column (150 ml, 3-cm diameter) equilibrated in and eluted with Buffer D (1 mM dithiothreitol). To the pooled void volume fraction NaCl was added to a con- centration of 0.2 M. Fraction I1 (34 ml) was applied to a DEAE- cellulose column (10 ml, 2-cm diameter) equilibrated in and eluted with the same buffer. The protein fractions in the flow- through were pooled, precipitated with ammonium sulfate (0.313 g/ml), and the precipitate was collected and resuspended in 3.3 ml of Buffer B. This fraction was then applied to a Sephadex G-25 column (45-ml, 2-cm diameter) equilibrated in and eluted with Buffer B. The pooled Sephadex G-25 void volume protein (Fraction 111, 7.5 ml) was applied to a DNA-cellulose column (12 ml, 4-cm diameter) equilibrated in Buffer B. The column was washed with 100 ml of Buffer B and the protein n activity was eluted with 40 ml of Buffer B containing 2 M NaCl. The eluate was precipitated with ammonium sulfate (0.226 g/ml) and dis- solved in Buffer D (1 mM dithiothreitol) (Fraction IV, 4 ml). This sample was dialyzed against 1 liter of Buffer D (1 mM dithio- threitol) for 12 hours and the precipitate which formed was col- lected by centrifugation at 41,000 X g for 60 min and suspended by homogenizing it in 4 ml of Buffer B (Fraction V). The pre- cipitate was again collected and the wash was repeated. The final protein pellet was resuspended in 2 ml of Buffer B contain- ing 0.25 M NaCl, clarified, and the supernatant (Fraction VI) withdrawn. This fraction was stored in aliquots in liquid nitrogen where it was stable for at least 6 months. At 0" Fraction VI is stable for at least 2 months. Ammonium sulfate precipitates of each of the fractions were stable on storage at 0" for at least 2 weeks. Fraction I. Extract.. . .. . . . . . . . . . 11. Ammonium sulfate". . I I I. DEAE-cellulose . . . . . . IV. DNA-cellulose.. . . . . . . V. Low ionic strength precipitation . . . . . . . . . VI. Wash .... . . . . . . . . . . . . . - Total units x 10-1 190 128 48 35 29 12 Protein mghl 16.7 2.9 6.4 2.3 0.36 0.17 Spe+fic activity unifslmg x lo-' 0.016 1.3 1 .o 3.8 20 34 __ Yield 5% 87 34 24 20 8 Purifi- cation 90 70 250 1,320 2,160 Calculation of yield and purification was based on the ac- tivity recovered in all of the ammonium sulfate fractions; assays in Fraction I were not reliable. plete in most instances. It seems clear that the polymerase III* and copolymerase III* (6) subunits of the DNA polymerase I11 holoenzyme are needed for DNA synthesis and that binding of the DNA template by the DNA unwinding protein is required both for initiation and elongation. A strong clue to the function of the dnaG protein has been furnished recently by its action in the conversion of the viral single-stranded DNA of G4, a phage related to &~X174.~ In this case, the RNA primer is synthesized by the dnaG protein in the presence of DNA unwinding protein;z the other proteins needed 6K. Zechel, J.-P. Bouch6, and A. Kornberg (1975) J. Biol. Chem. 150, in press. 5865 dnaC. ............ dnaB ............. Protein i.. ........ Protein n.. ....... DNA polymerase I11 holoenzymed dnaG.. ........... DNA-unwinding protein ......... TABLE VI11 Summary of protein properties -__ + + f - + + + + + - +- -- Protein dnaC dnaB ? ? -~ 20,000 250,W 40,000 80,000 Gene Molecular locus 1 weight dnaE 330,0006 dnaG 1 65,0001 ? I 76,m Function ?b ? ? DNA binding DNA synthesis RNA synthesis DNA binding a Denatured calf thymus DNA-cellulose and 50 mM imidazole- HCl (pH 7.0). ?, unknown. Ref. 4. DNA polymerase III* (dnaE locus) + copolymerase III* (77,000-dalton subunit, gene locus unknown). o Ref. 12. I Footnote 2. Ref. 11. for 4x174 are dispensable (8). It seems reasonable that dnuG protein would perform the RNA synthetase function of 4x174 DNA as it does for G4 DNA and that the four additional proteins may serve by orienting regions of the 4x174 DNA into a form suitable as an origin ("promoter" for RNA synthesis). Current studies of a complex of protein n with 4x174 DNA show a dramatic change in conformation of the DNA. It is re- duced 6- to 7-fold in contour length and appears as a string of beads, represented by about 25 molecules of protein n. As for the functions of protein i, dnuB protein, and dnuC protein nothing is known as yet. Protein i, like protein n and the DNA unwinding protein, is required in the reaction at a level of approximately 25 molecules/DNA circle; the dnuB protein may be needed at a lower ratio and is known to have an ATPase activity stimulated by DNA (14). Probably some or all of these proteins interact physically to form the assembly unit required for the initiation events. Among the eight proteins discussed, four are still unassigned to genetic loci. Mutants defective in proteins i or n, copolymerase III*, or the DNA unwinding protein have yet to be discovered or may be identified with one of the recently reported dnaH (15), dnaI (16), or dnaZ (17) thermosensitive replication mutants. The product of the dnuA gene of the established loci for DNA replica- tion, is not among those needed for reconstituting the 4x174 conversion. Nevertheless a dependence on a dnuA gene product was clearly implied in earlier complementation studies with un- fractionated cell extracts (2). Its function may prove to be indirect, such as the neutralization of an inhibitory effect of the defective dnuA product contributed by the mutant extract. The requirement for spermidine, as well as for the DNA un- winding protein (8) is puzzling in view of the antagonistic func- tions these molecules generally perform. Binding of spermidine to DNA, which favors helix formation may be essential for main- taining a certain conformation at a particular stage in the reac- tion, whereas the unwinding protein may serve by binding the single-stranded part of the molecule for other operations in the reaction. Whether the participation of either the polyamine or the unwinding protein in this enzyme system is an accurate guide to their physiological roles must remain undecided. The fully single-stranded DNA template provided in the in vitro system is not found in the cell. Instead decapsidation of the phage particle and penetration of the DNA into the cell is tightly coupled to its replication. Current studies designed to reconstitute the mo!ecu- lar details of these early stages of infection may clarify some of these questions. Note Added in Proof-Procedures for stabilization and purifi- cation of the dnaC protein now yield a preparation about 20% pure with a specific activity of 80,000 units/mg (see Table IV for comparison). REFERENCES 1. WICKNER, W., BRUTLAQ, D., SCHEKMAN, R., AND KORNBERG, A. (1972) Proc. Natl. Acad. Sci. U. S. A. 69,965-969 2 SCHEKMAN, R., WICKNER, W., WESTERGAARD, O., BRUTLAG, D., GEIDER, K., BERTSCH, L., AND KORNBERG, A. (1972) Proc. Natl. Acad. Sei. U. S. A. 69, 2691-2695 3. WICKNER, R. B., WRIGHT, M., WICKNER, S., AND HURWITZ, J. (1972) Proc. Natl. Acad. Sei. U. S. A. 69, 3233-3237 4. WRIGHT, M., WICKNER, S., AND HURWITZ, J. (1973) Proc. Natl. Acad. Sci. U. S. A. 70, 312Cb3124 5. WICKNER, S., BERKOWER, I., WRIGHT, M., AND HURWITZ, J. (1973) Proc. Natl. Acad. Sei. U. S. A. 70, 2369-2373 6. WICKNER, W., SCHEKMAN, R., GEIDER, K., AND KORNBERG, A. (1973) Proc. Nat2. Acad. Sci. U. S. A. 70, 1764-1767 7. WICKNER, S., WRIGHT, M., AND HURWITZ, J. (1973) Proc. Nafl. Aead. Sei. U. S. A. 70, 1613-1618 8. SCHEKMAN, R., WEINER, A., AND KORNBERG, A. (1974) Science 186, 987-993 U. S. A. 71, 4120-4124 198-217 Biol. Chem. 260, 1972-1980 6244-6249 3999-4005 Acad. Sci. U. S. A. 71, 783-787 ing of Japan, Kyoritsu Shuppan Co., Tokyo Bacteriol. 118, 783-787 AND WALKER, J. R. (1974) J. Bucteriol. 119. 443-449 9. WICKNER, S., AND HURWITZ, J. (1974) Proc. Natl. Acad. Sci. 10. ALBERTS, B., AND HERRICK, G. (1971) Methods Enzymol. 21, 11. WEINER, J. H., BERTSCH, L. L., AND KORNBERG, A. (1975) J. 12. WICKNER, W., AND KORNBERQ, A. (1974) J. Biol. Chem. 249, 13. GEIDER, K., AND KORNBERG, A. (1974) J. Biol. Chem. 249, 14. WICKNER, S., WRIGHT, M., AND HURWITZ, J. (1974) Proc. Nutl. 15. KOMANO, T., AND SAKAI, H. (1972) in Molecular Biology Meet- 16. BOYERSMANN, D., MESSER, w., AND SCHLICHT, M. (1974) J. 17. FILIP, C. C., ALLEN, J. S., GUSTAFSON, R. A., ALLEN, R. G.,