Vol. 21, No. 1 Printed in U.S.A.

JOURNAL OF VIROLOGY, Jan. 1977, p. 338-346 Copyright © 1977 American Society for Microbiology

Purification and Molecular Characterization of Adenovirus Type 2 DNA-Binding Protein KENJI SUGAWARA,' ZVEE GILEAD, AND MAURICE GREEN* Institute for Molecular Virology, St. Louis University School of Medicine, St. Louis, Missouri 63110

Received for publication 16 July 1976

An adenovirus type 2 (Ad2) DNA-binding protein was purified by sequential DNA-cellulose, Sephadex G-200, and DEAE-Sephadex chromatography, with a yield of 120 ,ug of binding protein (95 to 99% homogeneity) starting with 2 x 109 infected cells. By omitting the Sephadex G-200 step, 400 to 600 ,ug of 95% pure binding protein was obtained. To obtain high yields of highly purified binding protein, it was necessary to include deoxycholate and Nonidet P-40 at selected stages during the preparation. The highly purified binding protein appeared to have retained its native state as indicated by: (i) binding to single-stranded but not native Ad2 DNA, (ii) almost complete precipitation by immunoglobulin G from hamsters immunized by extracts of tumors induced by Ad2-simian virus 40 hybrid viruses, and (iii) identical sedimentation coefficient with binding protein obtained from DNA-cellulose chromatography only. Zonal centrifugation in sucrose gradients and gel filtration revealed that purified binding protein has a sedimentation coefficient of 3.AS and a Stokes radius of 5.2 nm. Based on these two values, a molecular weight of 73,000 was calculated, in agreement with the estimate from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A frictional ratio of 1.88 was calculated, suggesting that the Ad2 DNA-binding protein does not have a typical globular protein structure. Proteins that bind to single-stranded but not Adl2 DBP (12). The DBP gene has been double-stranded DNA have been isolated from mapped in the Ad2 EcoRI-B fragment (map adenovirus type 2 (Ad2)-infected (14), Ad5-in- positions, 0.59 to 0.71) by cell-free translation fected (17), and Adl2-infected (13) cells early (7) and by using recombinants between H5ts125 after infection. The molecular weights of these and Ad2-simian virus 40 (SV40) nondefective proteins (by sodium dodecyl sulfate [SDS]-poly- hybrid ts mutants (T. Grodzicker, J. Sambrook, acrylamide gel electrophoresis) are 75,000 (75K) and C. W. Anderson, unpublished data cited in (Ad2), 72K (Ad5), and 58K (Adl2). Two or three reference 7). We have recently determined that the Ad2 smaller (41K to 48K) single-stranded binding proteins are also found in variable amounts in DBP undergoes post-translational modificaearly infected cells, which tryptic fingerprint tion. After a pulse with [35S]methionine (Met) analyses (12) and genetic studies (18) suggest followed by a chase period, this protein inare components (presumably proteolytic degra- creases in apparent molecular weight from 74K dation products) of the larger proteins. Studies to 77K, as determined by SDS-polyacrylamidewith the Ad5 and Adl2 temperature-sensitive gel electrophoresis; both forms are phosphoryl(ts) mutants H5tsl25 and H12tsA275 have indi- ated and bind to single-stranded DNA (Y-H. cated that the DNA-binding proteins (DBP) are Jeng, W. S. M. Wold, K. Sugawara, Z. Gilead, viral coded (13, 18) and are required for initia- and M. Green, submitted for publication). Pretion of viral DNA replication (15, 19). The Ad2 vious studies from our laboratory described two DBP has been shown to be viral-coded by cell- Ad2 DNA replication complexes, one from the free translation of hybridization-purified Ad2 inner nuclear membrane and the other solubiearly mRNA and by comparison of tryptic pep- lized from nuclei. Both contain the Ad2 DBP tides of the translation product with those of (14, 22; T. Yamashita, M. Q. Arens, and M. the protein isolated from infected cells (7). Pep- Green, submitted for publication). Since DBP tide analyses indicate that the Ad2 and Ad5 is required for initiation of viral DNA replicaDBPs are quite similar but distinct from the tion and since mutants (H5ts 125) defective in this protein are available, DBP together with Present address: Department of Microbiology, Faculty the DNA replication complexes provide powerof Medicine, Kyoto University, Konoe-cho, Yoshida, Sakyoful systems to evaluate the role of singleKu, Kyoto 606, Japan. I

338

VOL. 21, 1977

PURIFICATION OF DNA-BINDING PROTEIN

339

stranded DBP in viral DNA replication. The phy. All subsequent steps of purification were peravailability of pure protein is an obligatory re- formed at 0 to 4°C unless otherwise stated. Singlequirement for studies on function. In this report stranded DNA-cellulose was prepared using calf we describe the purification to near homogene- thymus DNA as described by Litman (8). DNAity and the partial characterization of the Ad2 cellulose (1.2 g) in buffer A was loaded on a column (10 by 1 cm) and washed with 20 ml of buffer A. DBP. Cytoplasmic extract was applied at a flow rate of 8 to MATERIAIS AND METHODS Materials. [3H]leucine (50 to 60 Ci/mmol) and [35S]Met (466 Ci/mmol) were obtained from New England Nuclear; calf thymus DNA (type I), DEAESephadex (A-50), Sephadex G-200, and bovine liver catalase were obtained from Sigma Chemical Co.; sodium deoxycholate was from EM Laboratories; cellulose powder (CF11) was from Whatman; acrylamide, bisacrylamide, and other materials for electrophoresis were purchased from Bio-Rad Laboratories; Escherichia coli ,B-galactosidase and yeast alcohol dehydrogenase were from Boehringer; and bovine serum albumin, human gamma globulin, ovalbumin, and horse heart cytochrome c were obtained from Schwarz/Mann. Cells, virus, and labeling of infected cells. Ad2 was purified as described using CsCl instead of RbCl gradients (5). A clonal line of KB cells was propagated in suspension in Eagle minimum essential medium (MEM) containing 5% horse serum. Cells were infected with 50 to 100 PFU of Ad2 (strain 38, plaque 4, free of adenovirus-associated virus) in serum-free MEM. After 1 h of absorption at 37°C, cells were centrifuged and resuspended at 3 x 105 cells/ml in leucine-free MEM supplemented with 5% horse serum and 2 ug of L-leucine per ml. [3H]leucine (1 1uCi/ml) and arabinosyl cytosine (AraC) (25 ug/ml) were added at 2 h postinfection (p.i.), and the cells were harvested at 24 h p.i. For [mS]Met labeling, infected cells were diluted after 1 h of absorption to 3 x 105 cells/ml in MEM supplemented with 5% horse serum. AraC (25 iLg/ml) was added at 2 h p.i. At 5 h p.i., cells were centrifuged, washed once in warm Met-free MEM, and resuspended in Met-free MEM supplemented with 5% horse serum, 25 ug of AraC per ml, and 5 ,LCi of [3S]Met per ml. Preparation of cytoplasmic extracts. In our general procedure, infected labeled cells (6 liters of culture, 1.8 x 109 cells), harvested 24 h p.i., were washed twice with phosphate-buffered saline (PBS) and resuspended in reticulocyte standard buffer (RSB) (10 mM Tris-hydrochloride, pH 7.4-10 mM NaCl-1.5 mM MgCl2) at 3 x 107 cells/ml. After 15 min at 4°C, cells were disrupted with 20 strokes of a tight-fitting Dounce homogenizer, and nuclei were removed by centrifuging at 800 x g for 15 min. The cytoplasm (supernatant) was digested with DNase I for 30 min at room temperature in RSB containing 5 mM MgCl2 and 1 mM 2-mercaptoethanol and dialyzed overnight in the cold against 100 volumes of buffer A (20 mM Tris-hydrochloride, pH 8.1-50 mM NaCl-5 mM EDTA-1 mM 2-mercaptoethanol-10% glycerol). Some precipitate appearing during dialysis was removed by centrifugation at 6,000 x g for 20 min. Isolation of DBP by DNA-cellulose chromatogra-

10 ml/h, washed with 20 ml of buffer A, and eluted with 30 ml of buffer A containing 0.4 M NaCl, followed by 30 ml of buffer A containing 0.6 M NaCl. Radioactivity of column fractions was monitored by counting small portions, and peak fractions eluted with 0.6 M NaCl were pooled (0.6 M eluate). Purification of DBP by Sephadex G-200 chromatography. The 0.6 M NaCl eluate from DNA-cellulose was concentrated by vacuum dialysis against buffer B (20 mM Tris-hydrochloride, pH 8.1-0.6 M NaCl-1 mM 2-mercaptoethanol-10% glycerol-0.02% Nonidet P-40 [NP40]). Dialyzed material was then chromatographed on a Sephadex G-200 column (95 by 2.5 cm) pre-equilibrated with buffer B, and the column was eluted with buffer B at a flow rate of 10 ml/h. Fractions (2 ml) were collected, and the radioactivity of 50-,lI portions was determined. Purification of DBP by DEAE-Sephadex chromatography. Pooled fractions containing DBP from Sephadex G-200 (G-200 pool) after concentration by vacuum dialyses were treated with sodium deoxycholate (DOC) (1% final concentration) for 1 h at 4°C and dialyzed against 50 volumes of buffer C (10 mM Tris-hydrochloride, pH 7.5-50 mM NaCl-1 mM 2mercaptoethanol-0.02% NP40-0.4 M urea-10% glycerol) for 24 h with one buffer change. The dialyzed material was chromatographed on a DEAE-Sephadex column (6 by 0.9 cm) equilibrated in buffer C. After washing with 15 ml of buffer C, the column was eluted with a 120-ml linear gradient generated with equal amounts of buffer C and buffer C containing 0.3 M NaCl. Fractions (2 ml) were collected, and the radioactivity of 50-,lI portions was determined. In an abbreviated procedure to provide higher yields of DBP of slightly lower purity, the Sephadex G-200 step was omitted, and the concentrated 0.6 M eluate from DNA-cellulose was treated with DOC, applied to a larger DEAE-Sephadex column (10 by 0.9 cm), and eluted with a 160-ml linear gradient. SDS-gel electrophoresis. Polyacrylamide gel electrophoresis in 0.1% SDS was performed as described by Maizel (10). Samples for electrophoresis were concentrated by vacuum dialysis or by precipitation with 7% trichloroacetic acid and heated at 100°C for 2 min in 0.01 M phosphate buffer containing 1% SDS, 1% 2-mercaptoethanol, and 0.007% bromophenol blue. Sucrose was then added to 10%, and samples were loaded onto 6% polyacrylamide gels. Electrophoresis was performed at 8 mA/gel, and gels were fractionated into 2-mm slices with a Gilson gel fractionator. For direct visualization of proteins, gels were stained with 0.05% Coomassie blue in methanol-acetic acid-water (5:1:5) at 37°C for 2 h and then destained with 10% methanol-7% acetic acid for 2 to 3 days. E. coli 8-galactosidase (molecular weight, 135K), bovine serum albumin (67K), human

340

SUGAWARA, GILEAD, AND GREEN

gamma globulin (50K and 25K), ovalbumin (45K), and cytochrome c (12K) were used as molecular weight standards. Radioimmune precipitation. The indirect radioimmune precipitation assay with antisera from immunized or tumor-bearing hamsters and the source of antisera have been published (4). The following antisera were used: (i) pooled sera from hamsters immunized with extracts of tumors induced by Ad2-SV40 hybrid virus (Ad2 T serum); (ii) pooled sera from hamsters bearing tumors induced by Ad12 (Adl2 T serum); (iii) pooled sera from hamster bearing tumors induced by SV40 virus (SV40 T serum). The immunoglobulin (IgG) fractions were isolated from the sera by DEAE-cellulose column chromatography as previously described (4). 35S-labeled purified 75K DBP protein was mixed with varying amounts of immune IgG, the mixtures were incubated at 370C for 1 h, and goat serum anti-hamster IgG was added. The precipitates formed after 2 h of incubation at 37°C were centrifuged, washed twice, dissolved in 0.3 M acetic acid, and counted. DNA binding activity of DBP. Binding of purified DBP to Ad2 DNA was examined by zonal centrifugation in sucrose gradients. ['4C]thymidine-labeled Ad2 DNA (1,286 cpm/,ug) was prepared as previously described (6). Ad2 DNA was denatured at 1000C for 10 min and rapidly cooled in ice water. 3Hlabeled DBP and Ad2 14C-labeled DNA were mixed and incubated at 00C for 1 h in binding buffer (10 mM Tris-hydrochloride, pH 7.5-50 mM NaCl-1 mM EDTA). The mixture was layered on a 5 to 30% linear sucrose gradient prepared in binding buffer containing 0.02% NP40 and centrifuged at 4°C for 3 h at 40,000 rpm in a Spinco SW50.1 rotor. Fractions were collected, and radioactivity was determined. Determination of sedimentation coefficient of DBP protein. The procedure of Martin and Ames (11) was used for determination of sedimentation coefflcient of DBP protein. DBP preparations were layered on 5 to 20% linear sucrose gradients containing 20 mM Tris-hydrochloride (pH 7.5), 0.6 M NaCl, 1 mM 2-mercaptoethanol, and 0.02% NP40 and sedimented at 40,000 rpm for 24 h at 40C in a Spinco 50.1 rotor. Marker proteins included bovine serum albumin (4.4S), ovalbumin (3.6S), and whale sperm myoglobin (2.0S). Bovine albumin and ovalbumin were detected by absorbance at 280 nm, and myoglobin was determined by absorbance at 410 nm. Determination of the Stokes radius of DBP. DBP (0.4 ml) was applied to a column (97 by 1.0 cm) of Sephadex G-200. The column was pre-equilibrated and eluted with 20 mM Tris-hydrochloride (pH 8.1)0.6 M NaCl-1 mM 2-mercaptoethanol-1 mM EDTA0.02% NP40 at a flow rate of 2.6 ml/h. Fractions (0.88 ml) were collected, and the elution position of DBP was determined by assaying samples for radioactivity. Excluded and included column volumes were determined by measuring absorbance of blue dextran (absorbance at 254 nm) and phenol red (absorbance at 560 nm). The column was calibrated with proteins of known Stokes radii: bovine liver catalase (5.2 nm), yeast alcohol dehydrogenase (4.6 nm), and bovine serum albumin (3.5 nm) (16). Catalase activity was assayed by the method of Beers and

J. VIROL. Sizer (3). Alcohol dehydrogenase activity was assayed by increased absorbance at 340 nm upon incubation of column fraction in 0.1 M glycine-NaOH (pH 9.0), 0.35 M ethanol, and 1 mM nicotinamide adenine dinucleotide. Bovine albumin was detected by absorbance at 280 nm. Other methods. Protein concentration was estimated by the method of Lowry et al. (9). DNA was quantitated by absorbance at 260 nm. Radioactivity was counted in Aquasol (New England Nuclear) using a Beckman liquid scintillation counter.

RESULTS

Purification of DBP. Previous preparations of Ad2 DBP (4, 14) were obtained by a single DNA-cellulose chromatography step (0.6 M NaCl eluate; see above). Such preparations contain DBP as a major radioactive component (50 to 80% of radioactivity) as shown by polyacrylamide gel electrophoresis (e.g., Fig. 3A). But analysis of gels stained with Coomassie blue revealed numerous protein bands that are either unlabeled or lightly labeled (e.g., Fig. 4B). The 0.6 M eluate from DNA-cellulose was therefore subjected to further purification. Numerous trials, including purification of DBP in the presence of various concentrations of ionic and nonionic detergents, gave rise to the following protocol that allowed purification of DBP in good yields with high purity. Typically, the 0.6 M eluate prepared as described in Materials and Methods was concentrated by vacuum dialysis, dialyzed against buffer B, and then chromatographed on a Sephadex G-200 column. DBP eluted as a prominent peak (Fig. 1). The peak fractions were pooled and concentrated by vacuum dialysis (G-200 pool). Analysis of the G200 pool by SDS-gel electrophoresis (see Fig. 3B) indicated that DBP now accounted for 85 to 90% of the radioactivity in the preparation. Further purification was achieved by DEAESephadex chromatography. The concentrated G-200 pool was first treated with DOC. DOC treatment was necessary, apparently to prevent aggregation of contaminating proteins with DBP. The presence of NP40 in the column buffer was essential for good recovery of DBP from DEAE-Sephadex. Urea was also found to increase yields significantly. The DOC-treated G-200 pool was dialyzed against buffer C, which contains both NP40 and urea (see Materials and Methods) and then chromatographed on a DEAE-Sephadex column using a linear gradient from 50 mM to 300 mM NaCl (Fig. 2). The main radioactive peak eluting at 200 mM NaCl was pooled, concentrated by vacuum dialysis (DEAE-Sephadex pool), and dialyzed for 24 h against 200 volumes of 10 mM Tris-hydrochloride (pH 7.5)-200 mM NaCl-1 mM 2-mercapto-

PURIFICATION OF DNA-BINDING PROTEIN

VOL. 21, 1977

341

x

2

0C-)

=i_

30

40

50

60

70

FRACTION NUMBER FIG. 1. Sephadex G-200 chromatography of DBP present in 0.6 M eluate from DNA-cellulose. The 0.6 M eluate (18 ml) was concentrated to 3.2 ml by vacuum dialysis, dialyzed against buffer B, and chromatographed on a Sephadex G-200 column (95 by 2.5 cm). Fractions (5 ml) were collected, and the radioactivity present in 50-pJ portions was determined. Fractions 36 to 42 were pooled for further purification. The arrow indicates the position of the excluded column volume.

^°

12

0? C.)

.5-

C DEAE-sephadex

0.3

C-)~~~~~~~~~~~~~~~~. CT 6 ~~~~~~~~~~~0.1

10

30 20 40 FRACTION NUMBER

50

FIG. 2. DEAE-Sephadex chromatography of the G-200 pool. Pool fractions 32 to 42 of Fig. 1 were treated with 1 % sodium deoxycholate (see Materials and Methods) and dialyzed against buffer C. The dialyzed fraction was loaded on a DEAE-Sephadex column (6 by 0.9 cm) and eluted with a linear 120-ml gradient (50 to 300 mM NaCl in buffer C). Fractions (2 ml) were collected, and the radioactivity in 50-pl portions was counted. Fractions 34 to 42 were pooled

(DEAE-Sephadex pool).

ethanol-10% glycerol, with one buffer change. This preparation contained a single homogeneous component that accounted for 95 to 99% of the radioactivity when analyzed by SDS-gel electrophoresis (Fig. 30). Roughly 1 to 2% of the radioactivity was present in several minor

1

10

20

30

40

SLICE NUMBER FIG. 3. Polyacrylamide gel electrophoresis of labeled DBP at various stages ofpurification. Samples of various preparations (2.9 x 104 cpm of DNAcellulose pool, 3.6 x 104 cpm of G-200 pool, and 104 cpm of DEAE-Sephadex pool) were subjected to electrophoresis on 6% SDS-polyacrylamide gels at 8 mAl gel for 6 h as described in Materials and Methods. The radioactivity present in 2-mm slices was determined. The arrow indicates the position of the tracking dye.

342

J. VIROL.

SUGAWARA, GILEAD, AND GREEN

peaks, and sometimes as much as 4% in a 45Kdalton polypeptide, presumably the known breakdown product of DBP (12). This species could have been generated during the DEAESephadex purification procedure. Several studies (unpublished data) showed that the 45K polypeptide chromatographed at the leading edge of the DBP peak on DEAE-Sephadex. Recovery of radioactivity and protein during a typical purification is given in Table 1. The final purified material (DEAE-Sephadex pool) contained almost pure DBP (Fig. 3C). A 10 to 20% yield of DBP was obtained starting with the DNA-cellulose step. For physicochemical studies where DBP is monitored by radioactivity and where high purity is not necessary, the Sephadex G-200 step may be omitted and the 0.6 M eluate applied directly to a larger DEAESephadex (10 by 0.9 cm) column. This yielded a labeled DBP contaminated only 5% by other labeled species and with considerably higher yields of labeled DBP (400 to 600 ,ug from 2 x 109 infected cells, compared to 120 ,ug by the longer procedure). The DBP obtained this way yielded a single band in SDS-gel electrophoresis (Fig. 4A). For comparison, a stained gel of the 0.6 M eluate containing multiple proteins is shown in Fig. 4B. All the physicochemical studies to be described below were performed with DBP purified by the abbreviated procedure (without the Sephadex G-200 step). Antigenic specificity. Previous studies have indicated that Ad2 DBP reacts immunologically with sera from hamsters immunized with extracts of tumors induced in hamsters by the Adl-SV40 and Ad2-SV40 viruses (4). Over 70% of purified 35S-labeled DBP was precipitated by IgG isolated from Ad2 T serum but not that from SV40 T sera (Fig. 5). The small degree of precipitability with Ad12 T serum could possibly indicate some cross-reactivity between Ad2 (group C) and Adl2 (group A) DBPs. The results indicate that purified DBP has retained TABLE 1. Purification of DBP from cytoplasmic extracts of Ad2-infected cells Fraction

Total Vol- Total cpm protein reume recovered (x 105) covered (ml)

(mg) Cytoplasmic extract (1.8 x 109 cells) DNA-cellulose (0.6 M eluate) Concentration and dialysis Sephadex G-200 Concentration and dialysis DEAE-Sephadex Concentration and dialysis

53 18 3.2 36 8.0 21 3.6

15,370 51 41 20 15 7.3 5.1

600

2.3

0.12

A

-d

B

I6 S;

FIG. 4. SDS-polyacrylamide gel electropherogram of (A) purified DBP (DEAE-Sephadex pool) and (B) 0.6 M eluate from DNA-cellulose. Purified DBP (10 Mg) and 0.6 M eluate from DNA-cellulose (30 pg) were subjected to electrophoresis at 8 mA/gel for 6 h. The gels were stained with Coomassie blue.

its immunological reactivity and is the component in the 0.6 M eluate shown previously to react with the Adl-SV40 or Ad2-SV40 serum. DNA binding activity. Retention of biological activity by the purified DBP was also studied in binding experiments that examined the interaction of the protein with native and denatured Ad2 DNA. Purified DBP was mixed with native or denatured Ad2 '4C-labeled DNA and the binding was analyzed by zonal centrifugation in sucrose gradients as described above. Native Ad2 DNA sedimented at 31S, whereas all of the DBP sedimented at a much lower rate (Fig. 6B). However, when DBP was mixed with denatured Ad2 DNA (Fig. 6A), all of the pro-

PURIFICATION OF DNA-BINDING PROTEIN

VOL. 21, 1977

343

dard proteins with known sedimentation coefficients. The symmetrical profile and S value of the peak was not altered by centrifugation in sucrose gradients containing 0.15 to 0.6 M A

15

3-

F

40 cLI

x

C')

20

2

-

\

C') \x5

Cm) wt

5 I

10

30 50 jig IMMUNOGLOBULIN INPUT

FIG. 5. Radioimmune precipitation of purified DBP by Ad2-SV40 T serum. Portions (5,400 cpm) of 35S-labeled DBP (DEAE-Sephadex pool) were mixed with varying amounts of immune IgG. Normal IgG was added to provide a final concentration of 80 pg of protein in each tube. After 1 h of incubation at 37°C, goat anti-hamster IgG was added, and the incubation was continued for 2 h at 37°C. The immunoprecipitates were sedimented and washed with PBS containing 1% Triton X-100 and 0.5% DOC. The precipitates were dissolved in acetic acid (0.3 M), and the radioactivity was determined. Symbols: *, Ad2SV40 T serum; 0, Adl2 T serum; A, SV40 T serum.

tein cosedimented with viral DNA at approximately 43S. Molecular weight and hydrodynamic properties of DBP. Molecular weight of purified DBP was estimated by co-electrophoresis in SDS-polyacrylamide gels with marker proteins of known molecular weights. A value of 74K to 76K was found (Fig. 7), in agreement with previous estimates with partially purified preparations. When examined by sucrose gradient centrifugation, purified DBP sedimented as a single symmetrical peak (Fig. 8A). For comparison, the sedimentation profile of 0.6 M eluate is shown (Fig. 8B). DBP present in the 0.6 M eluate sedimented at the same position as purified DBP. These results suggest that purified DBP is not degraded or denatured by the detergent and urea treatment used for purification and retains the conformation possessed by the DBP in 0.6 M eluate. A sedimentation coefficient (S20,w) of 3.4 was calculated based on stan-

B 15

6 ~~~~~~~~~0

*

10 N0

4

radioactivity-)as determined.

5 -2

a SpnoS5.

oo.Fatin 10 20eecletd n FRACTION NUMBER FIG. 6. Binding of purified DBP to singlestranded Ad2 DNA. Purified 3H-labeled DBP was mixed with (A) denatured Ad2 14C-labeled DNA (7.2 Mg ofprotein and 5.6 Mg of DNA) and (B) native Ad2 "4C-labeled DNA (3.0 pg of protein and 9.4 pg of DNA) in binding buffer. After 1 h at O0C, the reaction mixtures were layered on 5 to 30% linear sucrose gradients made in binding buffer containing 0.02% NP40 and centrifuged at 40,000 rpm for 3 h at 40C in a Spinco SW50.1 rotor. Fractions were colkected, and radioactivity was determined.

344

J. VIROL.

SUGAWARA, GILEAD, AND GREEN

LU

p - galactosidase 10 xt Bovine serum albumin 1 6

DBP

CZi

4

-

Y-globulin (heavy)

Ovalbumin

-J C)

>< -globulin (light) 2-

Cytochrome 1

1

0.2

0.4

0.8 0.6 RELATIVE MOBILITY

1.0

FIG. 7. Estimation ofmolecular weight ofpurified DBP by SDS-polyacrylamide gel electrophoresis. Purified DBP (DEAE-Sephadex pool) was subjected to electrophoresis on 6% polyacrylamide gels, and gels were stained with Coomassie blue. Marker proteins were cytochrome c (molecular weight, 12,400), human gamma globulin (25,000 and 50,000), ovalbumin (45,000), bovine albumin (67,000), and /3 galactosidase (135,000).

NaCl. The sedimentation profile was not affected by the omission of EDTA, NP40, or 2mercaptoethanol. Excellent recovery from the gradient was obtained using 0.6 M NaCl; lower concentrations of NaCl gave low and variable recovery. A sedimentation coefficient of 3.4S would correspond to a molecular weight of 30K to 40K for a globular protein. However, a molecular weight for the DBP subunit determined by SDS-gel electrophoresis was about 75K. To further investigate this discrepancy, we attempted to determine the Stokes radius of purified DBP by analytical gel filtration. Unfortunately, purified DBP gave variable elution profiles. The concentrated DEAE-Sephadex pool contains a high level of NP40 that could not be removed by dialysis. The NP40 seemed to result in aggregation of DBP upon exposure to the eluting column buffer. These aggregates eventually dispersed but gave false and variable elution profiles. Therefore, DBP in the 0.6 M eluate (50 to 80% pure by radioactivity) was used for analysis by analytical gel filtration. This substitution is justified since DBP in the 0.6 M eluate and DEAE-Sephadex pool are identical as shown by their sedimentation in sucrose gradients (Fig. 8). As shown in Fig. 9, DBP was

0

20 30 10 FRACTION NUMBER

40

FIG. 8. Determination of the sedimentation coefficients of (A) purified DBP (DEAE-Sephadex pool) and (B) DBP in 0.6 M eluate from DNA-cellulose. Samples were diluted in 20 mM Tris-hydrochloride (pH 7.5)-0.6 M NaCl-1 mM 2-mercaptoethanol0.02% NP40 and layered on 5 to 20% linear sucrose gradients prepared in the same buffer. Tubes were centrifuged at 40,000 rpm for 24 h at 4°C in a Spinco 50.1 rotor. The fractions were collected, and radioactivity was determined. Marker proteins included (I) bovine serum albumin (4.4S); (II) ovalbumin (3.68); and (III) myoglobin (2.0S). The positions of these markers are indicated by arrows.

VOL. 21, 1977

345

PURIFICATION OF DNA-BINDING PROTEIN

eluted at the same position as catalase that has a Stokes radius of 5.2 nm (16). From the data obtained by sucrose gradient centrifugation and by gel filtration, the molecular weight and frictional ratio of DBP were calculated from the equations (16): M = [a(67rrzqN)s]/[1-4p] and f/fo = a/[3vM/47rN]113, where M = molecular weight, a = Stokes radius, s = sedimentation coefficient, v = partial specific volume, f/fo = fractional ratio, q = viscosity of medium, p = density of medium, and N = Avogadro's number). For this calculation we assume that the partial specific volume is 0.725 cm3/g (16). The results of the calculation indicated that the molecular weight of DBP is 73K and the frictional ratio is 1.88. DISCUSSION The availability of native, highly purified DBP is a prerequisite for studies on properties and function. We have devised two purification procedures for the DBP, starting with the partially purified material isolated by DNA-cellulose chromatography. These were a one-step procedure (DEAE-Sephadex chromatography) and a two-step procedure (Sephadex G-200 and DEAE-Sephadex chromatography). A high yield of DBP, about 95% purity, was obtained from the one-step procedure. A somewhat purer preparation but with a lower yield resulted from the two-step procedure; this may be useful for studies that require greatest purity. The purified DBP seemed to have retained its native state in spite ofthe use of detergents and urea, as evidenced by radioimmune precipitation and binding to single-stranded DNA. This was further suggested by identical sedimentation rate and chromatographic behavior of the purified DBP and crude DBP (0.6 M eluate). A trace of Ad2-specific 45K DBP is still apparent in the purest DBP preparations. It was difficult to completely eliminate this polypeptide because its chromatographic behavior on DEAESephadex was similar to that of DBP. The sedimentation coefficient of DBP is 3AS, as determined by sucrose gradient centrifugation, and its Stokes radius is 5.2 nm, as determined by gel filtration on Sephadex G-200. From these two parameters, we can calculate the molecular weight and frictional ratio of DBP if we assume the partial specific volume to be 0.725 cm3/g (16). The calculated molecular weight, 73K, correlates well with that obtained from SDS-gel electrophoresis. The frictional ratio is 1.88, indicating that DBP is not a typical globular protein. Bacteriophage T4 DBP (gene 32 protein) has a relatively high frictional coefficient. Therefore this protein may also have a nonspherical shape (1). Detergent was found to

44 -

Ve

+

Catalase BSA ADH Obalbumin

Vi

+ 1, +

4

0 I

w

0.).4