THE JOURNAL OF BIOLOCICAI. CHEMISTRY Vol. 254, No. 12, Issue of June 25, pp. 5328-5332, 1979 Printed in U.S.A.

Purification and Characterization of @X174 Gene A Protein A MULTIFUNCTIONAL ENZYME OF DUPLEX DNA REPLICATION*

(Received for publication, December 22, 1978)

Shlomo Eisenbergt and Arthur Kornberg

From the Department of Biochemistry, Stanford University School of Medicine, Stanford, California 94305

Synthesis of +X174 viral (+) strand circles in vitro requires gene A protein, rep protein, DNA binding pro- tein, and DNA polymerase III holoenzyme (Eisenberg, S., Scott, J. F., and Kornberg, A., (1976) Proc. Natl. Acad Sci. U. S. A. 73, 3151-3155). We have used this reaction as an assay to isolate gene A protein in greater than 90% purity. Its molecular weight under denaturing conditions is 59,000. The protein tends to aggregate and lose activity at low ionic strength. Tritium-labeled gene A protein cleaves the +X174 duplex replicative form and is bound to it in a 1:l ratio as part of an active replication complex. The attachment, at the 5’ phos- phoryl end of the cleavage point, is apparently cova- lent. The complex was not dissociated by: (i) banding in CsCl, (ii) treatment with 0.2 M NaOH, or (iii) boiling in 1% sodium dodecyl sulfate and electrophoresis on a sodium dodecyl sulfate-acrylamide gel; only micrococ- cal nuclease digestion of the DNA released the protein.

Gene A (cistron A) of +X174 bacteriophage codes for two polypeptides read in the same frame: gene A protein (59 to 62 kilodaltons) and gene A* protein (35 kilodaltons) (1). The larger polypeptide was implicated in the initiation of duplex RF’ replication (2) and viral (+) strand DNA synthesis (3). The function of the smaller polypeptide, gene A* protein, is uncertain.

In uiuo experiments (4), as well as studies with partially purified gene A protein (5), have suggested that it is an endonuclease that cleaves specifically the viral (+) strand in the RF I superhelical molecule. Soluble extracts which sup- port replication of superhelical RF I DNA template have been used to obtain partially purified gene A protein as well as rep protein (6-8), the enzyme that catalytically separates strands of the duplex. Replication of +XRF, reconstituted with DNA polymerase III holoenzyme, DNA binding protein, rep protein, and gene A protein, results in synthesis of viral (+), single- stranded circles in quantities far exceeding the amount of input superhelical template (9, 10). This reaction was used as an assay for the purification of rep protein (11) and gene A

* This work was supported in part by grants from the National Institutes of Health and the National Science Foundation. This is Paper 7 in the series “An Enzyme System for the Replication of Duplex Circular DNA: The Replicative Form of Phage $X174.” Paper 6 is Ref. 13. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 USC. Section 1734 solely to indicate this fact.

$ Present address, Department of Biochemistry, Weizmann Insti- tute, Rehovot, Israel.

’ The abbreviations used are: RF, duplex circular replicative form of 4X DNA; albumin, bovine serum albumin; $X, +X174; gene A. RF II complex, gene A protein. RF II complex; Hepes, 4-(2.hydroxy- ethyl)-1-piperazineethanesulfonic acid; SDS, sodium dodecyl sulfate.

protein and for investigating their functions (10, 12, 13). Four activities of gene A protein in RF replication were

identified (12): (i) nicking of the viral (+) strand at the origin of replication to initiate a round of replication, (ii) complexing with RF II to support fork movement in strand separation and replication, (iii) cleaving the regenerated origin to produce unit-length DNA, and (iv) ligating the newly generated 3’ hydroxyl end to the complexed 5’ phosphoryl end to form a circular viral molecule.

In this paper, we report the purification and further char- acterization of gene A protein.

MATERIALS AND METHODS

The organisms used were Escherichia coli 4720 and 4X174 am3, as previously described (6). The cells were grown to AW = 1 and infected with phage at a multiplicity of 5 to 10 (6). Thirty minutes after infection, the culture was chilled to 15°C. The cells were har- vested by centrifugation in a Sharples continuous-flow centrifuge, resuspended in 250 ml of Buffer A (see below) per 190 to 200 g (wet weight) of cells, divided into 15.ml portions in plastic vials, and frozen in liquid nitrogen. Such cells have been stored at -70°C for many months without loss of gene A protein activity.

Chemicals and Buffers-Sources of chemicals were as previously noted (14). Buffer A contained 50 mM Tris.Cl (pH 7.5) and 10% sucrose. Buffer B contained 50 mM imidazole.Cl (pH 6.8), 1 mM EDTA, and 5 rniw dithiothreitol. Buffer C contained 100 mM Hepes (pH 8.0), 0.1 mM EDTA, 5 mM dithiothreitol, and 20% glycerol. Enzyme dilution buffer for assays contained 50 mu Tris. Cl (pH 7.5), 50% glycerol, 5 ITIM dithiothreitol, 1 mM EDTA, and 0.2 mg/ml of albumin.

Assay for Gene A Protein-A 25.~1 reaction contained 25 IIIM Tris. Cl (pH 7.5); 5% sucrose; 5 mM dithiothreitol; 0.1 mg/ml of albumin; 10 mM MgC12; 40 pM (each) dATP, dCTP, dGTP, and [“H]dTTP (120 to 200 cpm/pmol of total nucleotide); 0.5 pg of RF I DNA; 1.6 fig of DNA binding protein; 500 units of DNA polymerase III holoenzyme; 3600 units of rep protein; and gene A protein to be assayed. Incubation was for 10 min at 30°C. The reaction was stopped by chilling the mixture on ice and adding 1 ml of 10% trichloroacetic acid containing 20 mM sodium pyrophosphate. The DNA was collected on Whatman GF/C fdters, washed three times (5 ml each) with 1 M HCl containing 100 mM sodium pyrophosphate and once with 5 ml ethanol, and dried. Radioactivity was measured in a toluene-based scintillation fluid. One unit of gene A protein activity is defined as 1 pmol of total nucleotide incorporated per min.

Preparation of 32P-labeled +X RF I DNA-From E. coli C infected in a Tris/pyruvate/glucose medium (15), RF I DNA was extracted as before (16).

Preparation of ‘H-labeled Gene A Protein-The protein (6 X 10’ units of Fraction V; Table I) was transferred to a Hepes-based buffer by adsorption on a hydroxyapatite column (0.5 ml; equilibrated with Buffer C + 0.6 M NaCl) and elution with 0.3 M potassium phosphate (pH 8.0) in the same buffer. The gene A protein (2 x 10’ units in 0.7 ml) was diluted by addition of an equal volume of Buffer C, mixed with formaldehyde and “H-labeled sodium borohydride (7 to 12 Ci/ mmol) to final concentrations of 15 and 10 mM, respectively, and kept for 2 min on ice. Then, 10.6 ml of Buffer B containing 50% glycerol and 0.75 M NaCl was added. The diluted ‘H-labeled gene A protein solution was applied to a hydroxyapatite column (0.5 ml), washed with 20 ml of Buffer B containing 20% glycerol and 0.75 M NaCl, and

5328

by guest on January 18, 2020http://w

ww

.jbc.org/D

ownloaded from

@Xl 74 Gene A Protein in Duplex DNA Replication 5329

eluted with Buffer B containing 50% glycerol, 0.75 M NaCl, and 0.3 M potassium phosphate (pH 6.8). Recovery of gene A protein activity was 60%; the specific activity (4 x lo7 units/mg) was reduced by one- third; the specific radioactivity was 1.9 X lo5 cpm/pg.

RESULTS

Purification of Gene A Protein-The procedure, as before (6), was based on a reconstitution assay for viral (+) strand synthesis. Three modifications were introduced (Table 1): (i) cells were not treated with chloramphenicol after infection, thereby increasing yields of gene A protein activity in Fraction I by nearly lo-fold; (ii) Fraction II (ammonium sulfate) was centrifuged in the Ti 45 rotor to remove phage particles, phage DNA, and other nucleic acids, thus making the next step in the purification (Bio-Rex 70) more reproducible; (iii) precau-

tions were taken to avoid aggregation and activity loss by

TABLE I

Purification of gene A protein All operations were carried out at 4°C unless indicated otherwise.

I. Lysate: Frozen cells (400 g wet paste) were thawed at 24”C, diluted to 630 ml with Buffer A, divided into four 250-ml centrifuge bottles, and chilled on ice to 0°C. Solutions of 0.1 M dithiothreitol, 1.25 M

NaCl, 0.125 M EDTA, and 0.25 M spermidine were added to each bottle to final concentrations of 5, 50, 5, and 10 InM, respectively. Lysozyme (120 pg/ml, final concentration) was added, and the sus- pension was kept on ice for 45 min. The bottles were immersed in a water bath at 37°C and inverted every minute over a 4-min period, then chilled on ice. Cold, 5 M NaCl was added slowly, to a final concentration of 1 M, and the lysate was centrifuged for 60 min at 14,000 rpm in the J-21B Beckman centrifuge. The supernatant solu- tion is Fraction I. ZZ. Ammonium sulfate: Solid ammonium sulfate (0.220 g/ml of Fraction I) was added over a period of 30 min at O”C, stirred on a magnetic stirrer for an additional 30 min, and centrifuged at 13,000 rpm for 30 min in the same centrifuge. The pellet was dissolved in 85 ml of Buffer B + 20% glycerol and 1 M NaCl, and the solution was centrifuged in the Ti 45 rotor at 35,000 rpm for 12 h. The supernatant solution is Fraction II. ZZZ. Bio-Rex 70: Fraction II was diluted with Buffer B + 50% glycerol to a conductivity equivalent to that of buffer + 0.2 M NaCl and was applied to a Bio-Rex 70 column (4 X 6.5 cm) equilibrated with Buffer B + 20% glycerol and 0.2 M

NaCl. The column was washed with 1 column volume of Buffer B + 20% glycerol and 0.2 M NaCl, followed by a wash with 10 column volumes of Buffer B + 20% glycerol and 0.4 M NaCl. Gene A protein activity was eluted with Buffer B + 50% glycerol and 0.75 M NaCl. IV. DNA-cellulose: Fraction III was diluted with Buffer B + 50% glycerol to a conductivity equivalent to that of Buffer B + 0.2 M NaCl and was applied to a DNA-cellulose column (2.5 x 5.0 cm) equilibrated with Buffer B + 20% glycerol and 0.2 M NaCl. The column was washed with 10 column volumes of Buffer B + 20% glycerol and 0.4 M NaCl; the activity was eluted with Buffer B + 50% glycerol and 0.75 M NaCl. V. Hydroxyupatite: Fraction IV was applied directly on a column (1.2 x 5 cm) equilibrated with Buffer B + 20% glycerol and 0.75 M NaCl. The column was washed with 10 column volumes of Buffer B + 20% glycerol, 0.75 M NaCl, and 10 mM potassium phosphate (pH 6.8). The activity was eluted with a 50-ml linear gradient of 10 to 300 mM potassium phosphate (pH 6.8) in Buffer B + 20% glycerol and 0.75 M NaCl. Active fractions were diluted with 1.25 volumes of Buffer B + 70% glycerol and stored at -20°C. The gene A protein was stored at -20°C for 6 months without loss in activity.

Fraction Total protein Total units Specific ac- tivity Recovery

w x10-6 lo6 lJ/mg % I. Lysate 20,300 (728)" (0.036)"

II. Ammonium 2,110 728 0.35 uw sulfate

III. Bio-Rex 70 112 696 6.2 96 IV. DNA- 15 530 35 73

cellulose V. Hydroxy- 10.2 610 60 84

apatite

a Since assays in Fraction I were not reproducible, the activity is taken as that determined for Fraction II.

Gene A-

-92.51< -67

-55

-47

-35

FIG. 1. SDS-acrylamide gel electrophoresis of gene A protein. A slab gel was prepared by mixing, at room temperature, equal volumes of 60% acrylamide, 1% bisacrylamide, and a solution containing, per 100 ml, 24 ml of 1 M HCl, 18.5 g of Tris, 0.46 ml of N,N,N’,N’- tetramethylethylenediamine, and 200 mg of ammonium persulfate. Samples were precipitated prior to electrophoresis with an equal volume of 10% trichloroacetic acid and sedimented for 15 mm at 16,000 rpm at OQC in the J-21B centrifuge. Pellets were dissolved with 50 ~1 of a solution containing 0.4 M Tris . Cl (pH 8.0), 30% glycerol, 40 mM dithiothreitol, 1% SDS, and bromophenol blue. Samples were kept at room temperature for 10 min, then heated for 2 min at 100°C. Electrophoresis was in a Tris. glycine system (17) containing 0.1% SDS in the upper chamber at a constant current of 25 mA at room temperature for 2.5 h. The bands were stained in a solution of 40% methanol, 10% acetic acid, and 0.25% Coomassie brillant blue for 2 h at 37”C, and destained overnight in 10% methanol + 7% acetic acid. The right lane contained 5 pg of each of the following standards phosphorylase a (92.5 kilodaltons), albumin (67 kilodaltons), E. coli elongation factor (EF Tu) (47 kilodaltons), and lactate dehydrogenase (35 kilodaltons). The left lane contained 5 pg of tritiated gene A protein.

maintaining the salt concentration at or above 0.6 M NaCl throughout the purification procedure unless 50% glycerol was present. In Buffer B containing 20% glycerol and less than 0.4 M NaCl, the loss of gene A protein activity was rapid.

The present procedure (Table I) has produced gene A protein in higher yields (84%) and higher purity. Homogeneity of Fraction V has varied from 50 to 70% in different prepara- tions. A major contaminant observed by SDS-polyacrylamide gel analysis was a band of 35 kilodaltons, most probably gene A* protein; most of this component could be removed by repeating the last hydroxyapatite step twice.

Physical Properties of Gene A Protein-Over 90% of the protein migrated under denaturing conditions on SDS-acryl- amide gels as a single band of 59 kilodaltons (Fig. 1). Sedi- mentation properties of gene A protein under nondenaturing conditions were determined in glycerol gradients (Fig. 2). In a gradient containing 0.2 M NaCl (Fig. 2A), a large fraction of 3H-labeled gene A protein migrated faster than, or coincident with, the peak of DNA polymerase I activity, a standard of

by guest on January 18, 2020http://w

ww

.jbc.org/D

ownloaded from

5330 @Xl 74 Gene A Protein in Duplex DNA Replication

t _ A. N&I, 0.2M

4

6

4

2

I

7 0 2

; 8

I m -

4

2

0

-.-Gene A Activity

B. NaCI, 1M

t

10 20 30 40

I 1

3

2

1

%I 7 z ; 8 7 “- 2

1

FRACTION NUMBER

FIG. 2. Sedimentation properties of gene A protein on glycerol gradients. Tritiated gene A protein (2.5 pg) was mixed at 0°C with 5 pg of DNA polymerase I in a 50+1 mixture containing 50 mM imidazole (pH 6.8), 10% glycerol, 1 M NaCl, 1 miw EDTA, and 10 InM 2- mercaptoethanol. The mixture was placed on a linear (20 to 40%) glycerol gradient containing 50 ITIM imidazole (pH 6.8), 1 mM EDTA, 10 mM 2-mercaptoethanol, and either 0.2 M NaCl (Panel A) or 1.0 M NaCl, (Panel B). Centrifugation in the SW56 rotor was at 50,000 rpm at 0°C for 24.5 h with sedimentation in the direction of the arrow; 0.1.ml fractions were collected. Gene A protein activity was deter- mined by the assay described under “Materials and Methods.” DNA polymerase I activity was assayed with ‘H-deoxynucleotides using poly(dAT) as the primer-template (18). Radioactivity in the tritiated gene A protein was determined in 25-~1 aliquots of each fraction.

109 kilodaltons. In 1 M NaCl (Fig. 2B), the rapidly sediment- ing, 3H-labeled gene A protein shifted toward the position expected for the monomeric form. At low or high salt (Fig. 2A, 2B), most of the gene A protein, measured by its replica- tive activity, sedimented as the monomeric form.

Stoichiometry of Gene A Protein in an Active Gene A Protein. RF II Complex-Specific cleavage of superhelical RF I DNA by gene A protein produced a protein. DNA complex active in replication (8, 12). The complex formed from [““PI- DNA and tritiated gene A protein was sedimented through a neutral sucrose gradient (Figs. 3 and 4). More than 50% of the RF I DNA was converted to RF II. A peak of tritiated gene A protein was associated with the RF II DNA. These fractions were active in replication without further addition of gene A

I “i 0 ; 4 a K

-I 16

(B) RFI RFII ’ I 24

1 1

! - 11 ;’ II

FRACTION NUMBER

FIG. 3. Formation of gene A protein.RF II DNA complex. 32P- labeled $X RF I DNA (1 to 2 pg) was incubated with 3H-gene A protein (0.5 pg) for 10 min at 30°C in a 25+1 reaction mixture containing 10 mu MgC12,0.2 mg/ml of albumin, 30 mu NaCl, 12 mu potassium phosphate, and 50 mu Tris.Cl (pH 8.0). EDTA was added to 50 mM final concentration, and the mixture was layered on a linear (5 to 20%) sucrose gradient containing 5 mu EDTA, 0.2 mg/ml of albumin, 1 M NaCl, and 50 mu Tris.Cl (pH 8.0). Centrifugation was in a SW56 rotor, at 50,000 rpm at 10°C for 3 h. Fractions of 0.1 ml were assayed for radioactivity. Panels A and B represent control and experimental incubations, without and with gene A protein, respec- tively.

I I I I I

FRACTION NUMBER

FIG. 4. Stoichiometry of gene A protein to RF II DNA in gene A + RF II complex. (3H, 32P)-labeled gene A.RFII complex was prepared as in Fig. 3 in a 4-fold larger reaction mixture and purified through a sucrose gradient. It was then dialyzed against 50 InM Tris. Cl buffer (pH 8.0) containing 1 mM EDTA, 0.2 M NaCl, and 5 mM dithiothreitol for 60 min at 4”C, and sedimented at 0°C through a 5 to 20% neutral sucrose gradient as in Fig. 3, except that the gradient contained 0.2 M NaCl rather than 1 M NaCl. Fractions of 0.1 ml were collected and 70-,al aliquots were assayed for radioactivity. 3H-labeled gene A pro- tein (59 kilodaltons) contained 1.9 x lo” cpm/pg; RF1 [““PIDNA (3600 kilodaltons) contained 8 x lo3 cpm/pg. Synthesis of DNA was assayed in lo-p1 aliquots as described under “Materials and Methods,” except that gene A protein was not added.

by guest on January 18, 2020http://w

ww

.jbc.org/D

ownloaded from

+X1 74 Gene A Protein in Duplex DNA Replication

1 I I 16s14s 1 I -

tt

II ‘8

A. CsCl B. Alk. Sucrose

0 10 20 0 10 20 FRACTION NUMBER

6

I “; 4 0

:-

; B 2 g

FIG. 5. Stability of gene A-RF II complex in CsCl and alkali. Panel A: An aliquot of (3H, 32P)-labeled complex (pre- pared as in Fig. 3) was mixed with CsCl (1.25 g of CsCl/g of solution) and centri- fuged at 20°C for 48 h in a Ti 50 rotor at 40,000 rpm. Fractions of 0.1 ml were as- sayed for radioactivity. Panel B: An ali- quot of doubly labeled complex was in- cubated with 40 mM EDTA, 1% SDS, salmon sperm DNA (0.24 pg/ml), and 0.15 M NaOH at 37°C for 30 min. The mixture was layered on a 5-20% alkaline sucrose gradient containing 0.8 M NaCl, 5 mM EDTA, 0.2 mg/ml of albumin and 0.2 M NaOH, and then centrifuged at 20°C for 4 h in a SW56 rotor at 50,000 rpm. Fractions were collected and as- sayed for radioactivity.

Gene A-RI1 + Gene A-RF11 NUCLEASE Gene A

a b c d

FIG. 6. Gene A protein released from RF II complex by nuclease. Samples a, b, and c containing (3H, 32P)-labeled gene A. RF II complex (prepared as in Fig. 3) were incubated at 37°C for 2 h in mixtures containing 25 mu Tris.HCl buffer (pH 8.0), 5 mM CaC12, and 0.1 mg/ ml of albumin. Sample c alsO contained micrococcal nucleate at 270 units/ml. The reaction was stopped by addition of an equal volume of 10% trichloroacetic acid, 20 nm sodium pyrophosphate. Precipi- tates were collected by centrifugation, washed with 0.2 ml of 10% trichloroacetic acid, centrifuged, and dissolved in 50 ~1 of buffer containing 0.5M Tris. HCl (pH 8.1), 50 mu dithiothreitol, 40% glyc- erol, and 1% SDS. Prior to electrophoresis, the dissolved precipitates were treated as follows: (a) (32P, 4700 cpm; 3H, 5200 cpm) incubated 2 min at 100°C; (b) (32P, 5100 cpm; ‘H, 6200 cpm) incubated 30 min at 30°C; (c) (no detectable 32P; 3H, 5400 cpm) incubated 2 min at 100°C. Sample d was 3H-labeled gene A protein (7500 cpm) in 0.1 mg/ml of albumin, diluted with an equal volume of trichloroacetic acid/pyrophosphate (above) incubated 2 min at 100°C. Electropho- resis of all samples was on 15% bisacrylamide gel (see Fig. 1) at 25 mA for 15 h. The slab gel was impregnated with scintillant 2,5-diphenyl- oxazole and exposed to Kodak XR5 flhn for 4 days at -70°C.

protein (Fig. 4). Replication resulted in net DNA synthesis: 8 to 10 single-stranded circles were produced per RF II. The stoichiometry of gene A protein to RF II in this complex was approximately 1.0 (Fig. 4).

Gene A Protein Is Linked to DNA by a Covalent Bond- Isolation of an active gene A protein. RF II complex by sedimentation through a neutral sucrose gradient containing 1 M NaCl (Fig. 3) indicates a tight association of gene A protein with the DNA. The gene A protein also remained associated with the RF II DNA after: (i) banding in CsCl (48 h at 2O’C) (Fig. 5A); (ii) incubation in 0.15 M NaOH at 37°C for 30 min; (iii) sedimentation through an alkaline sucrose gradient (Fig. 5B) with a value of 14S, indicative of protein association with linear strands of +X unit length, and presum- ably attached to a chain end, and (iv) treatment with 1% SDS and 40 IIIM dithiothreitol (30 min at 25”C, or 2 min at 100°C) and electrophoresis on SDS-acrylamide gel (as in Fig. 6). The protein was released from the gene A. RF II complex only upon digestion of the DNA by micrococcal nuclease (Fig. 6).

These data provide evidence that gene A protein is cova- lently bound to a chain end of RF II DNA. In view of the capacity of the complex to support replication, it may be inferred that gene A protein does not block the 3’ end (12). Furthermore, the catalytic activity of gene A protein in form- ing closed viral circles clearly implies a linkage to the 5’ end that preserves the free energy of the phosphodiester bond.

DISCUSSION

The gene A product was identified by SDS-gel electropho- resis as a polypeptide of about 60 kilodaltons absent from cells infected with gene A mutants (19). Its function was suggested, fist, by the predominance of nicked replicative forms (RF II) in cells infected with wild type phage, as compared to the largely intact duplexes (RF I) found in infections with the mutant, and second, by the capacity of a partially purified gene A protein to cleave specifically the viral (+) strand of +X174 RF I (l-5).

Assay of replication of superhelical RF I by extracts of cells infected with wild type, but not gene A mutant, phage also served to identify gene A protein (6-10). This gene A protein preparation contained a 60-kilodalton polypeptide as the prin- cipal component which nicked the viral (+) strand of 4X RF I (8, 9). Furthermore, the nicked complex contained gene A protein in a stable and functionally active form. In conjunction with rep protein to separate the strands of the duplex, and DNA polymerase III holoenzyme to synthesize DNA from the free 3’ hydroxyl end, single-stranded viral circles were pro- duced rapidly and catalytically.

Thus the gene A protein proved to be a multifunctional catalytic protein with a capacity to (i) initiate replication by cleavage of the viral strand at a particular phosphodiester bond (12, 20) called the origin, (ii) form a mobile replication complex oriented on the template strand, (iii) cleave again at the regenerated origin, and (iv) ligate the unit-length viral

by guest on January 18, 2020http://w

ww

.jbc.org/D

ownloaded from

5332 @Xl 74 Gene A Protein in Duplex DNA Replication

strand to form and release a single-stranded circle. The mul- molecules of gene A protein within 30 min after infection. tiple properties of this nearly homogeneous gene A protein do Possibly, high concentrations are needed for rapid formation not differ from those described for the less purified prepara- of the gene A. RF II complex and still other functions of gene tion (12). A protein in the viral life cycle that remain to be discovered.

The previous purification procedure (6) had not freed the preparation of gene A* protein, a 35-kilodalton polypeptide coded by the distal half of the A gene and read from the same message in the same frame (1). The yields were insufficient to label the protein and determine its stoichiometry in the RF II complex. The chief difficulties in purification of gene A protein are the need to maintain high ionic strength (equivalent to at least 0.3 M NaCl) to prevent aggregation and inactivation, and the similarity in properties of gene A* protein. Purification steps described in this paper produce an enzyme at least 90% pure, in very good yield (Table I), and essentially free of gene A* protein. Tritium-labeling of the enzyme by reductive meth- ylation was achieved with nearly full retention of activity at a specific radioactivity of about 2 x lo5 cpm/pg.

REFERENCES

1. 2. 3. 4. 5.

Linney, E., and Hayashi, M. (1973) Nature New Biol. 245, 6-8 Tessman, E. S. (1966) J. Mol. Biol. 17, 218-236 Fujisawa, H., and Hayashi, M. (1976) J. Viral. 19, 416-424 Francke, B., and Ray, D. S. (1971) J. Mol. Biol. 61,565-586 Henry, T. J., and Knippers, R. (1974) Proc. Natl. Acad. Sci. U. 5’.

A. 71,1549-1553 Eisenberg, S., Scott, J. F., and Kornberg, A. (1976) Proc. Natl.

Acad. Sci. U. S. A. 73, 1594-1597 Sumida-Yasumoto, C., Yudelevich, A., and Hurwitz, J. (1976)

Proc. Natl. Acad. Sci. U. S. A. 73, 1887-1891 Ikeda, J., Yudelevich, A., and Hurwitz, J. (1976) Proc. Natl. Acad.

Sci. U. S. A. 73,2669-2673

The isolated, functionally active, gene A.RF II complex contains one gene A protein molecule per RF II circle. Thus, the catalytic properties of gene A protein in the complex, entailing two successive nicking events (12), must be ascribed to two active sites in the polypeptide rather than to two subunits. Resistance of the protein/DNA linkage to heat, alkali, and detergents clearly indicates a covalent attachment. The linkage preserves the energy of the phosphodiester bond, but its precise nature is still in doubt. The covalent linkage is presumed to be similar for two distinct sites on the gene A protein molecule, if the proposed mechanism, which employs two active sites alternately in successive rounds of replication, is correct.

6.

7.

8.

9.

10.

11.

12.

13.

14. 15.

16.

17.

18.

19. 20.

Eisenberg, S., Scott, J. F., and Kornberg, A. (1976) Proc. Natl. Acad. Sci. U. S. A. 73, 3151-3155

Scott, J. F., Eisenberg, S., Bertsch, L. L., and Kornberg, A. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 193-197

Scott, J. F., and Kornberg, A. (1978) J. Biol. Chem. 253, 3292- 3297

Eisenberg, S., Griffith, J., and Kornberg, A. (1977) Proc. Natl. Acad. Sci. U. S. A. 74,3198-3202

Kornberg, A., Scott, J. F., and Bertsch, L. L. (1978) J. Biol. Chem. 253, 3298-3304

Rowen, L., and Kornberg, A. (1978) J. Biol. Chem. 253, 758-764 Denhardt. D. T.. Dressler. D. H.. and Hathawav. A. (1967) Proc.

Natl. Acad. Sk. U. S. A. 57,8i3-820 ” Eisenberg, S., Harbers, B., Hams, C., and Denhardt, D. T. (1975)

J. Mol. Biol. 99, 107-123 Marco, R.; Jazwinski, S. M., and Kornberg, A. (1974) Virology

62,209-223 The view that one or a few gene A. RF II complexes can

account for the synthesis of several hundred viral circles is consistent with the observation that in uivo a parental master template serves for all progeny RF molecules (19). However, it is difficult to explain the need to accumulate 2000 to 3000

Richardson, C. C., Schildkraut, C. L., Aposhian, H. V., and Korn- berg, A. (1964) J. Biol. Chem. 239,227-232

Denhardt, D. T. (1975) CRC Crit. Rev. Microbial. 4, 161-223 Langeveld, S. A., van Mansfeld, A. D. M., Baas, P. D., Jansz, H.

S., van Arkel, G. A., and Weisbeek, P. J. (1978) Nature 271, 417-420

by guest on January 18, 2020http://w

ww

.jbc.org/D

ownloaded from

S Eisenberg and A Kornbergenzyme of duplex DNA replication.

Purification and characterization of phiX174 gene A protein. A multifunctional

1979, 254:5328-5332.J. Biol. Chem. 

  http://www.jbc.org/content/254/12/5328Access the most updated version of this article at

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/254/12/5328.full.html#ref-list-1

This article cites 0 references, 0 of which can be accessed free at

by guest on January 18, 2020http://w

ww

.jbc.org/D

ownloaded from