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Structure of the linkage between adenovirus DNA and the 55,000 molecular weight terminal protein
J. Mol.
Rio/.
(1981)
145, 319-337
Structure of the Linkage between Adenovirus DNA and the 55,000 Molecular Weight Terminal Protein STEPHEN
V. DESI~ERIO
ASU
THOMAS
J. KELLY
JH
Department of M ierobiolog y The Johns Hopkins University School of Medicine Baltimore, Md 21205, U.S.A. (Received
2 July
1980)
The 5’ terminus of each complementary strand of adenovirus DNA isolated from virions is covalently linked to a protein with an apparent molecular weight of 55,000. We have determined the structure of the protein-DNA linkage. The 55,000 M, protein, linked to a small [32P]oligonucleotide, was isolated after DNase digestion of uniformly 32P-labeled adenovirus 5 (Ad5) DNA-protein complex. The protein was digested with trypsin and the resulting [32P] peptides were analyzed with the following results. (1) Acid hydrolysis released a single phosphorylated amino acid which was identified as O-phosphoserine in four separate electrophoretic or chromatographic systems; (2) treatment with snake venom phosphodiesterase yielded exclusively dAMP, dCMP and dTMP as expected (there are no guanylate residues in the first 25 nucleotides at the 5’ ends of Ad5 DNA); (3) prior treatment of the [32P]peptide preparation with snake venom phosphodiesterase greatly reduced the yield of 0-phosphoserine upon subsequent acid hydrolysis. These results suggest that Ad5 DNA is bound to the terminal protein by a phosphodiester linkage to the B-OH of a serine residue. This conclusion is supported by the finding that the DNA-protein linkage is readily hydrolyzed in alkali. In 50 mM-NaOH at 70°C the half time for hydrolysis of the linkage is about ten minutes. After incubation of Ad.5 DNA under these conditions we were able to label the 5’ termini with 32P by sequential treatment with alkaline phosphatase and polynucleotide kinase. Digestion of the end-labeled DNA to 5’ mononucleotides yielded [32P]dCMP. Wt. conclude that the terminal protein is bound to Bd5 DNA by a phosphodiester linkage between the B-OH of a serine residue of the protein and the 5’-OH of the terminal deoxycytidine residue of the DNA.
1. Introduction The mature adenovirus type 5 genome isolated from virions is a linear duplex DN4 molecule containing about 35,000 base-pairs (Green et al., 1965). The 5’ end of each strand of the genome is covalently attached to a protein with a molecular weight of 55,000 (Rekosh et al., 1977; Carusi, 1977). Although the function of the terminal protein is not yet well understood, the available data suggest that it may participate in viral DNA replication. 319 P 1981 Acatlernir
Press IIN.
(London)
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One possible role for the terminal protein was suggested by studies of Ad5t DNA replication in vitro (Challberg & Kelly, 1979a,b; Kelly & Challberg, 1979). In this system Ad5 DNA with attached 55K protein is the only effective template for replicative synthesis. With this template the mechanism of adenovirus DNA replication in vitro closely resemblesthat observed in viwo. In particular, replication is initiated at the molecular termini and proceeds by a displacement mechanism. In contrast, when the terminal protein is removed by proteolysis, the resulting template supports only a limited repair-like reaction in vitro. On the basis of these observations, it has been suggested that the 55K terminal protein on parental DNA strands may serve to facilitate initiation of DNA replication at the molecular termini (Challberg & Kelly, 19796). A second role for the terminal protein in the initiation of adenovirus DNA replication was proposed several years ago on the basis of the known enzymatic properties of DNA polymerases (Rekosh et al., 1977). All such enzymes require a primer to initiate synthesis, and all catalyze chain elongation in the 5’ to 3’ direction. Since the adenovirus genome is linear, a special mechanism is required for the synthesis of the sequencesat the 5’ terminus of each strand. Rekosh et al. (1977) proposed that the terminal protein represents the primer for DNA synthesis at the extreme 5’ ends of adenovirus DNA strands. In support of this hypothesis it has been demonstrated that the 5’ terminus of each nascent daughter strand of Ad5 DNA replicated in vitro is covalently linked to a protein (Challberg et al., 1980). This protein has an apparent molecular weight of 80,000 and probably represents a precursor t’o the 55K terminal protein. Protein has also been detected at the ends of adenovirus replicative intermediates in IGO (Girard et al., 1977; Coombs et al., 1978: Kelly & Lechner, 1978: Stillman & Bellett, 1978). In view ofthe possible role of the terminal protein in adenovirus DNA replication, it was of interest to determine the precise chemical structure of the protein-DNA linkage. In this paper we present evidence that this linkage is a phosphodiester bond between the B-OH of a serine residue of the 55K protein and the 5’-OH of the terminal deoxyc-ytidine residue of the DNA.
2. Materials
and Methods
(a) Preparation of [3*Pl/Id,5 DSAprotein complex Ad5 was propagated in KB cell monolayers in 75 cm’ flasks as described by Lechner & Kelly (1977), except that the multiplicity of infection was 40 plaque-forming units/cell. At 12 h after infection the culture medium was changed to phosphate-free minimal essential Eagle’s medium (GIBCO) containing 2”/b (v/v) calf serum and 0.25 mCi [32P]orthophosphate/ml. At 40 h after infection the labeling medium was removed, and the cells in each flask were suspended in 2 ml of 150 mm-NaCl, 50 mM-HEPES (pH 7.5) by vigorous shaking. The cell suspensions were pooled and then frozen and thawed 3 times. Virions were purified from the resulting lysate as described previously (Lechner & Kelly, 1977). Ad5 DNA-protein complex was isolated free of other virion components by a modification of the method of Coombs et al. (1978). Purified virions, prepared as described above, were disrupted by incubation for 3 min at 100°C in 2% SDS, 0.1% X-mercaptoethanol and applied t Abbreviations dodecyl sulfate.
used: Ad5.
adenovirus
serotype
5; 55K prokin.
protein
of 55,000
M,;
SDS, sodium
ADENOVIRUS
DNA-PROTEIN
LINKAGE
321
to a Sepharose 2B column. Gel filtration was carried out at room temperature in 10 mMTrisCl (pH 8.0), 1 mM-EDTA, @l% SDS. The [32P]DNA-protein complex eluted in the void volume. It was collected by ethanol precipitation and redissolvc,d in 10 rnnn-Tris . Cl (pH 8.O), 1 mM-EDTA, 0.1 y. SDS (SDS facilitated resuspension of the pellet). The SDS was removed by extraction with aqueous isobutanol followed by extraction with ether. Deproteinized Ad5 DNA was prepared as previously described (Challberg & Kelly, 1979a). (b) Preparation
of [3”P]AdC5
terminal
protein
Ad5 [ 32P]DNA-protein complex (100 to 200 rg) was digested at 37°C for 2 h with DNasrb 1 (Worthington) in a 300 to 450 ~1 incubation mixture containing 20 m&l-MgCl,, 10 InM1 m&l-phenylmethylsulfonyl fluoride, and Tris .(‘I (pH 8.0), 10 mM-CaCl,, 1 mM-EDTA, 80 pg enzyme/ml. This mixture was then incubated for another hour at 37°C after the addition of 150 units exonuclease I/ml (a gift from Dr Paul Englund). The digestion was terminated by addition of SDS and NaCl to final concentrations of 20/b and 50 m&l. respectively, followed by incubation for 2 min at 100°C. The terminal [ 32P]protein was separated from [32P]oligonucleotides by gel filtration on Sephadex G50 in 50 mM-NaCl. 10 mM-Tris.Cl (pH 8.0), 1 mM-EDTA, 0.1% SDS. The radioactive material that emerged in t)he void volume was precipitated by addition of trichloroacetic acid to 15”/ (w/v). The precipitate was dissolved in 200 ~1 50 mM-Tris (pH 6.8), 2% SDS, 100/b glycerol, 0.001% phenol red, and electrophoresed on a 10% SDS/polyacrylamide gel as described by Laemmli (1970). A single radioactive band migrating with an apparent molecular weight of 55,000 was detected by autoradiography. The region of the gel containing this band was excised and crushed with a glass rod. The protein was eluted by incubation in 1 ml 50 mMNaH2P0,, 1 mM-phenylmethylsulfonyl flouride, 0.1% SDS for 12 h at 37°C. Acrylamide was removed by filtration through siliconized glass wool, and the retained acrylamide was washed with 0.3 ml 50 mmNaH,PG,, 1 mM-phenylmethylsulfonyl flouride, 0.1 y/o SDS. (irrat’clr than 90% of the protein was eluted from acrylamide by this method. amino acids Acid hydrolysis of proteins or peptides was carried out at 110°C for 2 h in 5.6 M-HCI. The HCl was removed in vacua, and samples were dissolved in 10 ~1 of a solution containing 1 mM each of O-phosphoserine, 0-phosphothreonine and 0-phosphotyrosine. (O-Phosphoserine and 0-phosphothreonine were obtained from Sigma; 0- phosphotyrosine was synthesized as described by Rothberg et al., 1978.) The samples were applied to 20 cm x 20 cm cellulose thinlayer plates (Kodak) and fractionated in 2 dimensions by 1 of 2 methods. Method (1): The hydrolysis products were separated in the 1st dimension by chromatography in isopropanol/concentrated HCl water (70:15:15, by vol: Sishimura, 1972). Aftrt allowing the plate to dry in air, fractionation in the 2nd dimension was carried out by electrophoresis in 75 mM-NH,COOH (pH 3.65) at 500 V for 90 min (Kelly & Smith, 1970). Method (2): Electrophoresis was performed in the 1st dimension in 2.5”/0 formic acid, 7.8($0 acetic acid (pH 2.0) at 1000 V for 90 min (Bitte & Kabat, 1974). Plates were dried in air and chromatography was performed in the 2nd dimension in isobutpric acid/O.5 M-ammonia (5: 3. v/v; Rothberg et al., 1978). Phosphorylated amino acid markers were detected by spraying the plates with a solution of 0.3% ninhydrin in I-butanol. Radioactivity was detected by autoradiography. (c) Acid
hydrolysis
and
identi$cation
of phosphorylatrd
(d) Preparation of 32P-labeled tryptic peptides of term&al proteirl The terminal [ 32P]protein, purified by SDS/polyacrylamide gel electrophoresis as drscribed above, was digested with trypsin in a 200 ~1 reaction mixture containing 50 mm NH,HCO, (pH 8.3), 50 rg bovine serum albumin, and 10 pg enzyme (Worthington) for 20 h at room temperature. The digestion products were lyophilized and fractionated I)> rlectrophoresis on thin-layer cellulose in 1-butanol/pyridine/acetic acid/water (2: 1: 1:36. by vol.) at 500 V for 2 h (Gibson, 1974). Radioactivity was detected by autoradiography. One major and 2 minor radioactive spots were found. Regions of the thin-layer plate)
3Z2
S. V. DESIDERIO
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T. tJ. KELLY
JR
containing [32P]peptides were cut out, and the cellulose was scraped from the Mylar backing. The peptides were eluted from the cellulose with water and lyophilized. A portion of each spot was digested with snake venom phosphodiesterase. In every case all detectable radioactivity was released as [32P]dCMP, [32P]dAMP, and [32P]dTMP (data not shown) as expected, since no guanylate residues are present in the first 25 nucleotides at the 5’ termini of Ad5 DNA (Steenbergh et al., 1977). This result suggested that each spot contained one or more tryptic peptides linked to a [32P]oligonucleotide. The heterogeneity of these 32P-labeled tryptic peptides may reflect heterogeneity in the length of the oligonucleotide linked to terminal protein, or may result from incomplete digestion of the terminal protein by trypsin. The peptides were pooled and used in subsequent enzymatic analysis. (e) D@estion
of (“P1peptide.s
by phosphodiesterase
and
phosphomonoesterase
Peptides were digested with snake venom phosphodiesterase (Worthington) in a 15 ~1 reaction mixture containing 20 mM-TrisCl (pH 6%), 0.7 mM each of dCMP, dAMP, dGMP, dTMP and dUMP, 94 mM-dATP, and 93 unit enzyme. The mixture was incubated at 37°C for 12 h with readdition of 93 unit of enzyme after 3, 6 and 9 h. Peptides were treated with bacterial alkaline phosphatase (obtained from Worthington and repurified on DEAEcellulose as described by Weiss et al., 1968) in a 10 ~1 reaction mixture containing 10 mMTrisCI (pH 8.0), 95 mM-dTMP and 901 unit of enzyme. The reaction mixture was incubated at 37°C for 2 h, with readdition of 601 unit of enzyme after 1 h. A sample of each reaction was analyzed by cellulose thin-layer chromatography in saturated (NH,),SO,/l MNa acetate/isopropanol (40:9: 1, by vol.; Markham & Smith, 1952). Radioactivity was detected by autoradiography and deoxynucleoside 5’ monophosphate markers were detected under short wavelength U.V. light. (f) Alkaline hydrolysis of the DNA-protein linkage A sample of 32P-labeled Ad5 terminal protein was mixed with 7.5 ng of 3H-labeled +X174 replicative form DNA (spec. act. 1.7 x lo5 cts/min per pg) and the resulting solution was adjusted to 50 mM-NaOH in a final volume of 0.6 ml. A 0.1 ml portion was removed and neutralized by addition of 10 ~1 0.5 M-Tris . Cl (pH 6%) and 100 $50 mi+Tris .Cl (pH 8.0). The ratio of 32P radioactivity to 3H radioactivity in this sample (Rr) was determined. The remainder ofthe reaction mixture (0.5 ml) was incubated at 70°C. At various times during the incubation 0.1 ml samples were withdrawn and neutralized as above. After addition of 2 ~1 1% bovine serum albumin, each sample was extracted with 200 ~1 phenol. The ratio of “P radioactivit to 3H radioactivity in the aqueous phase (R,) was then determined. The amount of 9‘P radioactivity in each sample that partitioned into the aqueous phase was calculated from the equation : percent “P in aqueous phase = RJR, x 100. As a control, terminal [32P]protein was incubated under similar conditions in a reaction mixture containing 50 mM-NaCl instead of NaOH. (g) IdentiJication
of the nucleotide
linked
to the terminal
protein
The terminal SmuI restriction fragments, K and L, were isolated from Ad5 DNA as described below. Equimolar quantities of each fragment were incubated in 10 ~1 50 mMNaOH for zero time or for 1 h at 70°C. Samples were neutralized by the addition of 10 ~1 50 m&r-Hcl, 50 m&i-Tris.Cl (pH 8.0) and the DNA was dephosphorylated with 0.07 units bacterial alkaline phosphatase. After 1 h at 37”C, 907 unit additional enzyme was added and incubation was continued at 37°C for 2 h. An equivalent sample of each terminal fragment was incubated in NaOH at 70°C for 1 h, neutralized, and incubated at 37°C for 3 h in the absence of phos hatase. End labeling with P2P was carried out by a modification of the protocol of Maxam & Gilbert (1977) for labeling flush 5’ ends of duplex DNA. The DNA of each sample (0.13 pmol/sample) was precipiated with ethanol and resuspended in 10 ~1 20 mi%-Tris.Cl (pH 95), 06 miwspermidine, 0.1 mM-EDTA. DNA was denatured by heating samples to 100°C for 2 min followed by quick-freezing in a solid C02/ethanol bath. Samples were thawed by vortexing
ADENOVIRUH
DNA-PROTEIN
LINKAGE
323
and immediately transferred to tubes containing 4 pmol [Y-~‘P]ATP (spec. act. 12 Ci/mmol), 1.2 ~1 0.5 M-Tris.Cl (pH 9.5), 0.1 M-MgCl,, 50 mm-dithiothreitol, 50% (v/v) glycerol, and 4 units polynucleotide kinase (Boehringer-Mannheim). Samples were incubated at 37°C for 40 min. Reactions were stopped and DNA was precipitated as described by Maxam & Gilbert (1977). After precipitation with ethanol, end labeled DNA was dissolved in 10 ~1 1 miv-Tris.Cl (pH 8.0), 0.65% SDS. To 5.5 ~1 ofeach DNA sample were added 1 ~150 mM-NaGH and 3.5 ~1 50% glycerol, 25 mm-EDTA, 0.05% bromphenol blue. Strands were separated by electrophoresis through a 2.5% (w/v) agarose slab gel in a buffer containing 40 mru-Tris.Cl (pH 7.5). 5 mM-Na acetate, 1 mM-EDTA. The gel was autoradiographed. Regions of the gel containing 32P-labeled DNA strands were excised and DNA was eluted by the method of Maxam & Gilbert (1977). The 32P-labeled DNA strands were precipitated with ethanol and digested with DNase 1 and snake venom phosphodiesterase for 4 h at 37°C in a 25 ~1 reaction mixture containing 20 m&r-Tris.Cl (pH 6.8), 20 m&i-MgCl,, 0.1 mM each dAMP, dGMP, dTMP, dUMP, 0.1 mMdATP, 1 unit DNase I, and 0.01 unit of snake venom phosphodiesterase. Reaction products were separated by chromatography on thin-layer cellulose in saturated (NH&SO,/1 M-Na acetate/isopropanol (40:9: 1, by vol.). The cellulose thin-layer plate was dried in air and autoradiographed. (h) Restriction endmucleme dip.&n and gel electrophoresti Ad5 [ “P]DNA was digested with the Hind111 restriction enzyme (a gift from Dr H. 0. Smith) in a 0*3-ml reaction mixture containing 60 mna-NaCl, 10 mm-Tris.Cl (pH 8.5), 10 mMMgCl,, 2 mm2-mercaptoethanol, 40 pg DNA and 100 units of enzyme. After 2 h at 37°C the DNA was precipitated with ethanol. The precipitate was dissolved in 10% glycerol, 5 mMEDTA. O.OlO/o bromphenol blue and electrophoresed through a 1.476 agarose slab gel in a buffer containing 40 mm-Tris.Cl (pH 7.5) 5 mM-Na acetate, 1 mM-EDTA. The terminal Hind111 fragments, G and I (Sambrook et al., 1975) were located by autoradiography and eluted by the KI/hydroxylapatite method of Southern (1975). Digestion of Hind111 G and I fragments with the HhoI restriction enzyme (New England Biolabs) was performed in 100 ~1 reaction mixtures containing 50 mM-NaCl, 6 mmTris.Cl (pH 7.5), 6 mM-MgCl,, 6 mm-2mercaptoethanol, 100 mg bovine serum albumin/ml, and 8 units of enzyme. After 75 min at 37”C, 8 units additional enzyme were added and incubation at 37°C was continued for 75 min. The reaction products were electrophoresed through a polyacrylamide slab gel (8% acrylamide, 0.21 ye bisacrylamide) in a buffer containing 50 mm Tris-borate (pH 8.3) 1 mm-EDTA. The HhaI enzyme cleaves Ad5 DNA within the terminal repetition, producing an identical 73-base-pair terminal fragment at each end of the DNA (Steenbergh et al., 1977). These fragments and a 61-base-pair fragment, produced by Hhol cleavage of Hind111 I, were eluted by the method of Maxam & Gilbert (1977). Ad5 DNA was digested with the SmaI restriction enzyme (New England Biolabs) in a 30 ~1 reaction mixture containing 20 mru-KCl, 6 m&r-Tris.Cl (pH 8.0), 6 mM-MgCl,, 6 mM-2mercaptoethanol, 50 pg bovine serum albumin/ml, 14 units of enzyme and 30 rg DNA. After incubation at 37°C for 1 h, 14 additional units of enzyme were added and incubation at 37°C was continued for 1 h. The reaction products were resolved by electrophoresis through a 5% polyacrylamide slab gel in a buffer containing 50 mM-Tris-borate (pH 8.3), 1 mM-EDTA. The gel was stained with ethidium bromide and DNA bands were located under long-wave U.V. light. The terminal SwuzI fragments, K and L (Sambrook et al., 1975) were eluted by the method of Maxam & Gilbert (1977). The K fragment was separated from contaminating I fragment by electrophoresis through a 1.4% agarose slab gel in 40 mM-Tris’Cl (pH 7.5). 5 mM-Na acetate, 1 mM-EDTA and recovered from a minced agarose gel slice by soaking overnight at 37°C in a buffer containing 0.5 M-NH, acetate, 10 mM-Mg acetate, 1 mM-EDTA, and 0.1% SDS. The K fragment was collected by precipitation with ethanol and dissolved in 25 ~120 m&r-triethylammonium bicarbonate (TEABC) (pH 7.5). The resulting solution was applied to a column of DEAE-cellulose (Whatman DE52; bed volume 100 ~1) that had been II
324
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DESIDERIO
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equilibrated with 20 mm-TEABC (pH 7.5). The column was extensively washed with 5 ml 20 mM-TEABC and DNA was eluted with 1.0 M-TEABC. Fractions containing DNA (assayed by absorbance at 260 nm) were pooled and TEABC was removed in vacua.
3. Results (a) Purijication of Ad5 terminal
[32P] protein and releaseof O-[32P]phmphoserine by acid hydrolysis
Ad5 terminal [32P]protein was purified from disrupted virions by a simple threestep procedure. In the first step, the complex of Ad5 [32P]DNA and terminal protein was separated from other virion components by gel filtration on Sepharose 2B in the presence of SDS. In the second step, the isolated DNA-protein complex was digested with DNase, and the terminal protein, attached to a small nucleaseresistant oligonucleotide (Rekosh et al., 1977), was separated from low molecular weight digestion products by gel filtration on Sephadex G-50 in the presenceof SDS. In the final step, the terminal [32P]protein was further purified by preparative SDS/polyacrylamide gel electrophoresis. Figure 1 shows an autoradiogram of a typical preparative gel. As expected, most of the input radioactivity was recovered in a single specieswith an apparent molecular weight of 55,000 (Rekosh et al., 1977). As an approach to the identification of the amino acid residue involved in the protein-DNA linkage, terminal [32P]protein was subjected to partial acid hydrolysis in 5.6 M-HCl at 110°C for two hours (Rothberg et al., 1978). The hydrolysate was fractionated in two dimensions on a cellulose thin-layer plate as described in Materials and Methods (Fig. 2). The major radioactive product was [32P]orthophosphate, as expected, since under these hydrolysis conditions DNA is extensively degraded (Brown, 1974). A small fraction of the radioactivity, however, comigrated with the 0-phosphoserine marker. The identification of this minor radioactive species as 0-phosphoserine was confirmed by analysis of the hydrolysate in a second two-dimensional fractionation system (see Materials and Methods ; data not shown). Two possible sources of the 0-phosphoserine were considered, (1) that it was derived from the protein-DNA linkage, and (2) that it was derived from a serine phosphomonoester residue elsewhere in the polypeptide chain. Suggestive evidence in favor of the first possibility was obtained by analysis of protease-treated Ad5 DNA. A sample of [32P]DNA-protein complex was exhaustively digested with Pronase and extracted with phenol. This procedure removes most of the terminal protein from the DNA, but leaves one or more amino acid residues attached to each 5’ end (Carusi, 1977; Rekosh et al., 1977). The terminal HhaI restriction fragments, each 73 base-pairs in length (Steenbergh et al., 1977), were isolated from the Pronasetreated DNA as described in Materials and Methods and subjected to acid hydrolysis. Both terminal fragments yielded O-[32P]phosphoserine (Fig. 3). A 61 base-pair internal HhaI restriction fragment did not yield 0-phosphoserine when analyzed in an identical fashion. These results indicate that the O-[32P] phosphoserine is derived, at least in part, from the protein-DNA linkage or from the region of the terminal protein in proximity to the protein-DNA linkage.
ADEROVIRUS
DNA-PROTEIN
32.5
LINKAGE
-92
-5 K
-69K
-46K
-30
K
FIG. 1. Preparative SDS/polyacrylamide gel electrophoresis of terminal [32P]protein. I. Ad5 DNA-protein complex, uniformly labeled with 32P , was digested with DNase I and exonuclease and the products were chromatographed on Sephadex G50. The peak emerging in the void volume (0.03% of the input radioactivity) was pooled. Electrophoresis of this material was performed on a 10% SDS/polyacrylamide gel as described in Materials and Methods. An autoradiogram of the preparative gel is shown in the Figure; the positions of molecular weight markers are indicated. The ‘*Plabeled terminal protein has an apparent molecular weight of 55.000. The region of the gel containing terminal [32P]protein was excised and protein was eluted.
(b) Enzymatic
analysis
of the protein-DNA
linkage
Direct evidence that the 0-phosphoserine is involved in a phosphodiester linkage was obtained by analysis of the products produced by digestion of terminal [32P]protein with snake venom phosphodiesterase. This enzyme is an exonuclease that-cleaves ribo- or deoxyribonucleoside 5’ monophosphates from the 3’ ends of oligonucleotides. In addition, compounds in which a 5’ ribo- or deoxyribonucleotide
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FIG. 2. Release of O-[32P]phosphoserine by acid hydrolysis of terminal [32P]protein. in Materials and Methods. The 32P-labeled terminal protein was hydrolyzed in HCI as described hydrolysate was combined with 0-phosphotyrosine, 0-phosphothreonine, and 0-phosphoserine markers and the mixture was applied to a cellulose thin-layer plate. Separation was performed in the 1st dimension by chromatography in isopropanol/concentrated HCl/water (70: 15: 15, by vol.) (Nishimura, 1972) and in the 2nd dimension by electrophoresis in 75 mM-ammonium formate at pH 3.65 (Kelly & Smith, 1970). The anode is to the right. An autoradiogram of the cellulose plate is shown in the Figure. The positions of the markers, which were detected with ninhydrin, are indicated by dotted outlines. The order of the migration of the markers is, from right to left: 0-phosphoserine, 0-phosphothreonine and Ophosphotyrosine. The major radioactive species migrates as inorganic phosphate. The minor species comigrates with authentic 0-phosphoserine,
is linked by a phosphodiester bond to a variety of other moieties are also hydrolyzed, yielding the nucleoside 5’ monophosphate (Laskowski, 1966). The enzyme does not hydrolyze phosphomonoesters. Our initial attempts to release [32P]deoxyribonucleoside 5’ monophosphates from terminal [32P]protein by nuclease digestion were not successful, presumably because of steric hindrance. This problem was overcome by cleavage of the terminal protein with trypsin. The resulting [32P]peptides were found to be completely susceptable to digestion with snake venom phosphodiesterase. The digestion products were analyzed in two ways (Figs 4 and 5). First, a sample was fractionated
(b)
--
FIG. 3. Acid hydrolysis of 32P-labeled restriction fragments of The 73.base-pair terminal HhaI restriction fragments and a 61.base-pair internal HhaI fragment described in Materials and Methods. The fragments were hydrolyzed in acid and hydrolysates were cellulose as described in the legend to Fig. 2 and in Materials and Methods. (a) and (b) Hydrolysates of 73-base-pair HhaI restriction fragments from the right and left ends (c) Hydrolysate of a 61 -base-pair internal restriction fragment. The minor radioactive species indicated phosphoserine.
(a)
Cc)
of the genome, by the arrow
respectively. in (a) and (b) comigrated
with
authentic
O-
adenovirus DNA. were isolated from Pronase-treated Ad5 [3ZP]DNA as analyzed by 2-dimensional fractionation on thin-layer
- I
-C-T-
-A-
-orI-
+
(4
+
W
Fro. 4. Release of [32P]mononucleotides from labeled tryptic peptides of terminal protein by snake venom phosphodiesterase. The 3fP-labeled tryptic peptides of terminal protein, isolated by electrophoresis on thin-layer cellulose (see Materials and Methods), were combined and concentrated by lyophilization. Identical quantities of this material were incubated in snake venom phosphodiesterase cocktail in the presence or absence of phosphodiesterase; similarly, identical samples were incubated in bacterial alkaline phosphatase cocktail with or without addition of phosphatase (for conditions, see Materials and Methods). After incubation, 0.1 of each of the 4 samples was applied to a cellulose thin-layer plate and reaction products were separated by chromatography, as described in Materials and Methods. The thin-layer plate was dried in air and autoradiographed. (a) Chromatography of [32P]peptides treated (+) with snake venom phosphodiesterase and an untreated (-) control. (b) Chromatography of a similar preparation treated ( + ) with alkaline phosphatase and an untreated ( - ) control. The positions of deoxyribonucleoside 5’ monophosphate (C, T, 6, A) and inorganic phosphate (P,) markers are indicated. The remainder of each of the 4 samples was hydrolyzed in HCl and analyzed by 2dimensional fractionation (see Materials and Methods and legend to Fig. 5).
ADENOVIRUS
(a)
DNA-PROTEIN
LINKACE
(b)
Fn:. 5. Susceptibility of the DNA-protein linkage to digestion with venom phosphodiesterase or alkaline phosphatase. Equivalent samples of the 32P-labe1ed tryptic peptide preparation were incubated in snake venom phosphodiesterase cocktail with (b) or without (a) addition of phosphodiesterase; similar samples were incubated in bacteria1 alkaline phosphatase cocktail with (d) or without (c) phosphatase. A portion of each reaction mixture was analyzed by thin-layer chromatography (Fig. 4). The remainder of each reaction mixture was then hydrolyzed in HC1 (for details see Materials and Methods). The 4 hydrolysates were analyzed by 2-dimensional fractionation and autoradiography a8 described in the legend to Fig. 2. The minor radioactive species indicated by the arrow in (a), (c) and (d) comigrated in each case with authentic 0-phosphoserine.
by thin-layer chromatography to identify the [32P]nucleotides released by phosphodie&erase. Essentially all of the radioactivity was recovered in three spots corresponding to dAMP, dCMP and dTMP (Fig. 4). The absence of dGMP was expected since there are no guanylate residues in the first 25 nucleotides at the 5’ ends of Ad5 DNA (Steenbergh et al., 1977). A second sample of the phosphodiesterase digestion products was subjected to acid hydrolysis and then analyzed for O-[32P]phosphoserine. As shown in Figure 5, digestion with snake venom phosphodiesterase resulted in a greatly reduced yield of O-[32P]phosphoserine as compared to a sample incubated in the absence of the enzyme. The simplest
330
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interpretation of these results is that each 5’ end of Ad5 DNA is linked to the terminal protein by a phosphodiester bond to serine. As a control, [32P]tryptic peptides of terminal protein were digested with bacterial alkaline phosphatase, a phosphomonoesterase. This treatment did not result in the release of detectable [32P]orthophosphate (Fig. 4) nor did it affect the yield of 0-phosphoserine obtained upon subsequent acid hydrolysis (Fig. 5). (c) Alkaline
hydrolysis
of the DNA-protein
linkage
The conclusion that the DNA-protein linkage involves a phosphodiester bond to serine was supported by a study of the stability of the linkage under alkaline conditions. It is known that phosphodiester bonds to serine (or threonine) are alkali labile, whereas a number of other candidate linkages (e.g. phosphodiester bonds to tyrosine or phosphoamide bonds to lysine) are relatively alkali stable (Majerus et al., 1965; Juodka et al., 1968; Shabarova, 1970; Gumport & Lehman, 1971; Rothberg et aE., 1978). In the experiment whose results are displayed in Figure 6 a sample of
2 ‘a s
I? i .E a. 2 s 5 %
loo/
l -------.
+ NaOH
l
0
75
50
25
z
l -a’
-NaOH
30
60 Time
90
120
(mini
FIG. 6. Alkaline hydrolysis of the DNA-protein linkage. Terminal [32P]protein was incubated with +X174 RF 13H]DNA in 50 mM-NaOH at 7OOC. After 0, 15, 30, 60 and 120 min of incubation portions were extracted with phenol and the percentage of 32P radioactivity partitioning into the aqueous phase was calculated a8 described in Materials and Methods. This value is plotted as a function of time ( +NaOH). A control sample of terminal [32P]protein was incubated in 50 mM-NaCl at 70°C and assayed in the same manner at 0 and 60 min ( -NaOH).
purified terminal [32P]protein was incubated in 50 mM-NaGH at 70°C and monitored for the release of [32P]oligonucleotide by a phenol extraction assay. This assay was based upon the finding that [32P]oligonucleotide bound to terminal protein partitioned into the phenol phase during extraction, whereas unbound oligonucleotide partitioned into the aqueous phase. Under the incubation conditions that we employed, the [32P]oligonucleotide was quantitatively released from the terminal protein with a half-time of about ten minutes. Thus the behavior of the DNA-protein linkage in alkali is similar to that of other phosphodiester bonds to
ADENOVIRUS
DNA-PROTEIN
331
LINKAGE
serine (Majerus et al., 1965; Juodka et al., 1968). Incubation of terminal [ 32P]protein for 60 minutes at 70°C in 50 mM-NaCl, instead of NaOH, did not result in the release of any detectable radioactivity into the aqueous phase. (d) Ident@cation
of
the nucleotide
linked
to terminal
protein
The lability of the DNA-protein linkage in alkali made it possible to identify the nucleotide bound to terminal protein by 5’ end labeling methods. Ad5 DNA from Pronase-treated virions was digested with the Smal restriction enzyme and the terminal restriction fragments (K and L) were isolated by gel electrophoresis. Samples of each fragment were made 50 mM in NaOH and were either neutralized immediately or incubated at 70°C for one hour prior to neutralization. DNA from these samples was dephosphorylated with bacterial alkaline phosphatase and the 5’ ends were labeled with 32P by incubation with polynucleotide kinase and [y“P]ATP (Weiss et al., 1968). The DNA was then denatured and complementary strands were separated by agarose gel electrophoresis. In the case of DNAs not treated at 70°C with alkali, radioactivity was detected in only one strand of each terminal restriction fragment (Fig. 7(c) and (f)). This was expected, since the 5’ end of one strand is covalently attached to protein and therefore blocked to end labeling. When, however, terminal fragments were incubated in alkali prior to end labeling. 32P was incorp orated equally into both strands (Fig. 7(a) and (d)), indicating that the blocking peptide had been removed. This finding is consistent with the results of Carusi (1977), who reported that alkaline treatment of Ad DNA increased the efficiency of 5’ end labeling. When the phosphatase step was omitted, three- t,o fivefold less32P was incorporated into each strand (Fig. 7 (b) and (e)). Thus, at least in the majority of molecules, alkaline hydrolysis of the DNA-protein linkage leaves a 5’ phosphoryl group at the ends of the DNA. The low level of 32P incorporated in t’he absence of dephosphorylation may be the result of a phosphate exchange reaction catalyzed by polynucleotide kinase (Van de Sande et al., 1973). Alternatively, it is possible that some 5’ phosphoryl groups are removed during incubation in alkali. DNA strands labeled with 32P were eluted from the agarose gel and digested to 5’ mononucleotides with DNase I and snake venom phosphodiesterase. Digestion products were analyzed by thin-layer chromatography and autoradiography. Almost all of the radioactivity in each strand was recovered as a single [32P]deoxy ribonucleoside 5’ monophosphate representing the 5’.terminal nucleotide of each strand. For both the K and L fragments, the strand susceptible to end labeling without prior alkaline treatment (the slowly migrating strand) yielded almost exclusively [32P]dGMP (Fig. 8(a),(c),(d) and (f)), as predicted by the known sequence of the SmaI cleavage site (Roberts, 1980). The complementary strand, which was labeled only after prior incubation in alkali, released almost all of its radioactivity as [32P]dCMP (Fig. 8(b) and (e)). In addition, sequencing of the endlabeled DNA by the procedure of Maxam & Gilbert (1977) gave the same5’.terminal sequence as reported by Steenbergh et al. (1977) (data not shown). These results indicate that the nucleotide covalently bound to the Ad5 terminal protein is a deoxycytidylyl residue. From the results taken as a whole we conclude that the terminal protein is bound to Ad5 DNA by a phosphodiester linkage between the
332
S.
V. (4
DESIDERIO (b)
AND (cl
T. (4
J.
KELLY (e)
JR (f)
-r
FIG. 7. Separated strands of 5’ end-labeled terminal restriction fragments. The terminal SmaI restriction fragments K (lanes (a) to (c)) and L (lanes (d) to (f)) were isolated from Pronase-treated Ad5 DNA. Samples of each fragment were adjusted to 50 mM-NaOH and were either neutralized immediately or incubated at 70°C for 1 h prior to neutralization. The samples were then dephosphorylated by treatment with alkaline phosphatase and labeled at their 5’ termini by polynucleotide kinase and [y-32P]ATP. In some cases (indicated below) the phosphatase step was omitted. All kinase reaction mixtures contained the same quantity of DNA fragment (@13pmol). Following 5’ end labeling a portion of each sample was denatured and the complementary strands were separated by electrophoresis on a 2.5% agarose gel. The positions of the r and 2 strands of uniformly labeled terminal.
ADENOVIRUS
DNA-PROTEIN
LINKAGE
/l-OH of a serine residue of the protein and the 5’-OH of the terminal residue of the DNA.
X33
deoxycytidine
4. Discussion In this paper we have described the results of experiments that define the precise chemical structure of the linkage between Ad5 DNA and the 55K terminal protein. Terminal protein, bound to a small [32P]oligonucleotide, was isolated from Ad5 [“P]DNA-protein complex and digested with trypsin. The resulting [32P]peptides were analyzed with the following results : (1) partial acid hydrolysis released a single phosphorylated amino acid which was identified as 0-phosphoserine; (2) treatment with snake venom phosphodiesterase yielded dAMP, dCMP and dTMP as the only radiolabeled products; and (3) prior phosphodiesterase treatment greatly reduced the yield of 0-phosphoserine upon subsequent acid hydrolysis. These results indicated that Ad5 DNA is linked to the terminal protein by a phosphodiester bond to the P-OH of serine. This conclusion was supported by the observation that the DNA-protein linkage is alkali-labile. The lability of the linkage in alkali is also of considerable practical importance because it permits complete removal of the protein under conditions in which DNA is not significantly degraded. In 50 mMNaOH at 70°C the rate constant for hydrolysis of the linkage was approximately 7 x low2 min-‘. After incubation of Ad5 DNA under these conditions we were able to label the 5’ termini with 32P by polynu cl eotide kinase. Digestion of the endlabeled DNA to completion with DNase I and snake venom phosphodiesterase yielded [32P]dCMP. We conclude that the DNA-protein linkage is deoxycytidylyl(5’+0)-serine. In interpreting the results of our experiments we have considered the possibility that the 0-phosphoserine found in acid hydrolysates of terminal [32P]protein could have arisen from an N to 0 migration of phosphorus during hydrolysis. This type of rearrangement has been observed during acid hydrolysis of model compounds containing a nucleotide bound by a phosphoamide (P-N) linkage to the amino group of serine. The products of hydrolysis of such compounds include the nucleoside and 0-phosphoserine (Shabarova, 1970). Two lines of evidence, however, argue strongly against the possibility that adenovirus DNA is linked to terminal protein by a phosphoamide bond. First, the DNA-protein linkage is susceptible to cleavage by
SmaI fragments are indicated. The K and L fragments are derived from the left and right ends. respectively, of the Ad5 genome (Sambrook et al., 1975). (a) K fragment incubated in alkali. (b) K fragment incubated in alkali; phosphatase step omitted. (c) K fragment not incubated in alkali. (d) L fragment incubated in alkali. (e) L fragment incubated in alkali; phosphatase step omitted. (f) L fragment not incubated in alkali. Regions of the gel corresponding to labeled strands were cut out and assayed for radioactivity with the following results. (a) r strand, 232 ctsjmin; 1 strand, 214 cts/min; (b) r strand, 50 cts/min; 1 strand, 66cts/min: (c) r strand, 170 cts/min: 2 strand, 24 cts/min; (d) r strand, 117 cts/min; 2 strand, 145 cts/min: (e) r strand. 65 cts/min: 1 strand, 54 cts/min: (f) r strand, 19 cts/min: 1 strand, 150 cts/min.
334
S.
V.
DESIDERIO
AND
T.
J.
KELLY
JR
C-
T-
G-
A-
ori -
(a)
(b)
(c)
(d)
(4
(f)
FIG. 8. Identification of the nucleotide linked to terminal protein. After alkaline treatment, both complementary strands of each SnmI terminal restriction fragment could be end-labeled with 32P (Fig. 7(a) and (d)). Without prior alkaline treatment, only one strand of each fragment was labeled (Fig. 7(c) and (f)). Labeled strands were eluted from an agarose gel identical to that shown in Fig. 7 and digested to 5’ mononucleotides with DNase I and snake venom phosphodie&erase (see Materials and Methods). Digestion products were resolved by thin-layer chromatography in saturated (NH&SO.& M-Na acetate/isopropanol (49:9:1, by vol.) and detected by autoradiography. The positions of the-4 deoxyribonucleoside 5’ monophosphate markers (C, T, A, G) are indicated. (a) K fragment, incubated in alkali, r strand. (b) K fragment, incubated in alkali, 1 strand.
ADENOVIRUH
DNA-PROTEIN
335
LINKAGE
snake venom phosphodiesterase. This enzyme is specific for phosphodiester bonds and is reported to be inactive against phosphoamide linkages (Bogdanov et al., 1962). Second, under alkaline conditions in which the DNA-protein linkage is retidily hydrolyzed, phosphoamide bonds are generally stable (Little et al., 1967; Juodka et al., 1969). Several examples of covalent complexes between DNA or RNA and protein are known. Aside from adenovirus, such linkages are known to exist in the genomes of poliovirus (Lee et al., 1977; Nomoto et al., 1977) and the bacteriophage 429 (Ortin et al., lQ71), in replicative intermediates ofthe parvovirus H-l (Revie et al., 1979) and the bacteriophage 4X174 (Eisenberg et al., 1979), and in relaxation complexes of bacterial plasmids such as ColEl (Blair & Helinski, 1975). In addition, reactions of a number of topoisomerases with DNA have been shown to involve covalent protein-DNA intermediates (Depew et al., 1978; Champoux, 1977; Morrison & (‘ozzarelli, 1979). In the case of poliovirus, the RNA genome is attached to a virion protein by a phosphodiester bond between a uridylate residue and the phenolic hydroxyl group of tyrosine (Ambros & Baltimore, 1978; Rothberg et al, 1978). It is of interest to note that in the caseof bacteriophage $29, whose DNA replicates by a mechanism similar to that of adenovirus, a 27,000 M, protein is bound to the genome by a phosphodiester linkage between the /?-OH group of serine and the 5’.OH of the terminal deoxyadenosine residue (Hermoso $ Salas, 1980). The function of the adenovirus terminal protein is not yet clear. Studies performed in this laboratory (Challberg & Kelly, 1979b)have provided evidence that the 55K protein bound to the ends of parental DNA strands may play an essential role in DNA replication; adenovirus DNA from which the protein has been removed by proteolytic treatment is ineffective as a template for replication in vitro. The 55K protein on parental strands may facilitate initiation of DNA replication at the molecular termini, perhaps via interactions with other replication factors. We have recent,ly demonstrated (Challberg et al., 1980) that the 5’ terminus of each nascent’ Ad,5 daughter strand synthesized in vitro is also covalently linked to a protein. This protein has an apparent molecular weight of 80,000 and represents a precursor to t,he 55K protein. The 80K protein is also attached to DNA by a phosphodiester bond t,o serine. These observations are consistent with a second possible role for the t)rrminal protein in adenovirus DNA replication, namely, that it serves as the primer for daughter strand synthesis (Rekosh et aZ., 1977). Thus, the formation of an ester linkage between the r-phosphoryl group of dCTP and the P-OH of a serine residue in the 80K protein may be the primary initiation event in adenovirus DNA replication (Challberg et al.. 1980).
(c) K fragment, (d) L fragment, (e) L fragment, (f) L fragment, The ,SmaI K and (Sambrook ~4 al.,
not incubated in alkali, r strand. incubated in alkali, 2 strand. incubated in alkali, r strand. not incubated in alkali, I strand. L fragments are derived from the left 197.5).
and
right
ends
of the Ad5
genome,
respectively
336
8. V. DESIDERIO
AND
T. J. KELLY
JR
We thank Michael Stern for technical assistance and Drs Mark Challberg, Gary Ketner and Hamilton Smith for helpful discussions. This work was supported by United States Public Health Service grant CA16519 from the National Cancer Institute. One of the authors (S. V. D.) is a scholar of the Insurance Medical Scientist Scholarship Fund. REFERENCES Ambros, V. & Baltimore, D. (1978). J. Btil. Chem. 253, 5263-5266. Bitte, L. & Kabat, D. (1974). Methods Enzymol. 30, 563-590. Blair, D. G. & Helinski, D. R. (1975). J. Bzbl. Chem. 250, 8785-8789. Bogdanov, A. A., Antonovich, Y. G., Terganova, G. V. & Prokofiev, M. A. (1962). Biokhimiya, 27,1054-1060. Brown, D. M. (1974). Basic PrincipZes in NucEeic Acid Chemistry (Tso, P. 0. P., ed.), vol. 2, pp. l-90, Academic Press, New York. Carusi, E. A. (1977). ViroZogy, 76, 386394. Challberg, M. D. t Kelly, T. J. Jr (1979a). Proc. Nat. Acud. Sci., U.S.A. 76, 655-659. Challberg, M. D. & Kelly, T. J. Jr (1979b). J. Mol. Biol. 135, 999-1012. Challberg, M. D., Desiderio, S. V. & Kelly, T. J. Jr (1980). Proc. Nut. ilead. Sci., U.S.A. 77, 51055109. Champoux, J. J. (1977). Proc. Nat. Acud. Sci., U.S.A. 74, 3800-3804. Coombs, D. H., Robinson, A. J., Bodnar, J. W., Jones, C. J. & Pearson, G. D. (1978). Cold Spring Harbor Symp. Quunt. Biol. 43, 741-753. Depew, R. E., Liu, L. F. & Wang, J. C. (1978). J. Biol. Chem. 253, 511-518. Eisenberg, S., Scott, J. F. & Kornberg, A. (1979). Cold Spring Harbor Symp. Quant. Bill. 43, 295302. Gibson, W. (1974). Virology, 62, 319-336. Girard, M., Bouche, J. P., Marty, L., Revet, B. & Berthelot, N. (1977). Virology, 83, 34-55. Green, M., Pina, M., Kimes, R. C., Wensink, P. C., MacHattie, L. A. & Thomas, C. A. Jr (1965). Proc. Nat. Acad. Sci., U.S.A. 57, 1302-1309. Gumport, R. I. & Lehman, I. R. (1971). Proc. Nut. Acad. Sci., U.S.A. 68, 2559-2563. Hermoso, J. M. & Salas, M. (1980). Proc. Nat. Acud. Sci., [T.S.A. In the press. Juodka, B. A., Saveliev, E. P., Shabarova, Z. A. & Prokofiev, M. A. (1968). Biokhimiyu, 33, 907-915. Juodka, B. A., Obruchnikov, I. V., Nedbye, V. K., Shabarova, Z. ii. & Prokofiev, M. A. (1969). Biokhimiya, 34, 647-654. Kelly, T. J. Jr & Challberg, M. D. (1979). ICN/UCLA Symposiumon Extrachromosomal DNA (Stevens, J. G., Todaro, G. J. & Fox, C. F., eds), pp. 165176, Academic Press, New York. Kelly, T. J. Jr & Lechner, R. L. (1978). Cold Spring Harbor Symp. Quant. Biol. 43, 721-727. Kelly, T. J. Jr & Smith, H. 0. (1970). J. Mol. Biol. 51, 393-409. Laemmli, U. K. (1970). Nature (London), 227, 68&685. Laskowski, M. S. Sr (1966). Procedures in Nucleic ilcids Research (Cantoni, G. L. & Davies, D. R., eds), vol. 1, pp. 154187, Harper and Row, New York. Lechner, R. L. & Kelly, T. J. Jr (1977). Cell, 12, 1007-1020. Lee, Y, Ii‘., Nomoto, A., Detjen, B. M. & Wimmer, E. (1977). Proc. Vat. Acad. Sci., U.S.A. 74, 59-63. Little, J. W., Zimmerman, S. B., Oshinski, C. & Gellert, M. (1967). Proc. Nat. Acad. Sci., U.S.A. 58, 2004-2011. Majerus, P. W., Alberts, A. W. & Vagelos, P. R. (1965). Proc. Nat. Acad. Sci., U.S.A. 53, 41 O-417. Markham, R. & Smith, J. D. (1952). B&hem. J. 52, 55&565. Maxam, A. M. & Gilbert, W. (1977). Proc. Nut. Acad. Sci., U.S.A. 74, 566564. Morrison, A. & Cozzarelli, N. R. (1979). Cell, 17, 175184. Nishimura, S. (1972). Prog. Nucl. Acids Res. Mol. Biol. 12, 49-85. Nomoto, A., Detjen, B., Pozzatti, R. & Wimmer, E. (1977). Nature (London), 268,208213. Ortin, J., Vinuela, E., Sales, M. $ Vasquez, C. (1971). Nature New Btil. 234, 276277.
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Rekosh, D. M. K., Russell, W. C., Bellett, A. J. D. & Robinson, A. J. (1977). Cell, 11,283-295. Revie, D., Tseng, B. Y., Grafstrom, R. H. t Goulian, M. (1979). Proc. Nut. Acad. Sk., t’.&Y.Q. 76. 5539-5543. Roberts, R. J. (1980). NucE. Acids Res. 8, r63-r80. Rothberg, P. G., Harris, T. J. R., Nomoto, A. & Wimmer, E. (1978). Proc. Nut. Acad. Sci., C7.S.A. 75, 4868-4872. Sambrook. J.. Williams, J., Sharp, P. A. & Grodzicker, T. (1975). J. Mol. Rd. 97, 369-390. Shabarova, Z. A. (1970). Prog. LVucl. Acids Res. Mol. Biol. 10, 145182. Southern, E. M. (1975). J. Mol. Biol. 94, 51-69. Steenbergh, P. H.. Maat, J., Van Ormondt, H. & Sussenbach, ,J. 8. (1977). Nucl. Acids Res. 4. 4371-4389. Stillman, A. & Bellett, A. ,J. D. (1978). Cold Spring Harbor Symp. &ant. Riol. 43, 729-739. Van de Sande, J. H., Kleppe, K. & Khorana, H. G. (1973). Biochemistry, 12, 5050-5055. Weiss. B.. Live. T. R. & Richardson, C. C. (1968). J. Bid. Chem. 243, 4530-4542.
Rio/.
(1981)
145, 319-337
Structure of the Linkage between Adenovirus DNA and the 55,000 Molecular Weight Terminal Protein STEPHEN
V. DESI~ERIO
ASU
THOMAS
J. KELLY
JH
Department of M ierobiolog y The Johns Hopkins University School of Medicine Baltimore, Md 21205, U.S.A. (Received
2 July
1980)
The 5’ terminus of each complementary strand of adenovirus DNA isolated from virions is covalently linked to a protein with an apparent molecular weight of 55,000. We have determined the structure of the protein-DNA linkage. The 55,000 M, protein, linked to a small [32P]oligonucleotide, was isolated after DNase digestion of uniformly 32P-labeled adenovirus 5 (Ad5) DNA-protein complex. The protein was digested with trypsin and the resulting [32P] peptides were analyzed with the following results. (1) Acid hydrolysis released a single phosphorylated amino acid which was identified as O-phosphoserine in four separate electrophoretic or chromatographic systems; (2) treatment with snake venom phosphodiesterase yielded exclusively dAMP, dCMP and dTMP as expected (there are no guanylate residues in the first 25 nucleotides at the 5’ ends of Ad5 DNA); (3) prior treatment of the [32P]peptide preparation with snake venom phosphodiesterase greatly reduced the yield of 0-phosphoserine upon subsequent acid hydrolysis. These results suggest that Ad5 DNA is bound to the terminal protein by a phosphodiester linkage to the B-OH of a serine residue. This conclusion is supported by the finding that the DNA-protein linkage is readily hydrolyzed in alkali. In 50 mM-NaOH at 70°C the half time for hydrolysis of the linkage is about ten minutes. After incubation of Ad.5 DNA under these conditions we were able to label the 5’ termini with 32P by sequential treatment with alkaline phosphatase and polynucleotide kinase. Digestion of the end-labeled DNA to 5’ mononucleotides yielded [32P]dCMP. Wt. conclude that the terminal protein is bound to Bd5 DNA by a phosphodiester linkage between the B-OH of a serine residue of the protein and the 5’-OH of the terminal deoxycytidine residue of the DNA.
1. Introduction The mature adenovirus type 5 genome isolated from virions is a linear duplex DN4 molecule containing about 35,000 base-pairs (Green et al., 1965). The 5’ end of each strand of the genome is covalently attached to a protein with a molecular weight of 55,000 (Rekosh et al., 1977; Carusi, 1977). Although the function of the terminal protein is not yet well understood, the available data suggest that it may participate in viral DNA replication. 319 P 1981 Acatlernir
Press IIN.
(London)
Lttl
320
S. V. DESIDERIO
AND
T. J. KELLY
JR
One possible role for the terminal protein was suggested by studies of Ad5t DNA replication in vitro (Challberg & Kelly, 1979a,b; Kelly & Challberg, 1979). In this system Ad5 DNA with attached 55K protein is the only effective template for replicative synthesis. With this template the mechanism of adenovirus DNA replication in vitro closely resemblesthat observed in viwo. In particular, replication is initiated at the molecular termini and proceeds by a displacement mechanism. In contrast, when the terminal protein is removed by proteolysis, the resulting template supports only a limited repair-like reaction in vitro. On the basis of these observations, it has been suggested that the 55K terminal protein on parental DNA strands may serve to facilitate initiation of DNA replication at the molecular termini (Challberg & Kelly, 19796). A second role for the terminal protein in the initiation of adenovirus DNA replication was proposed several years ago on the basis of the known enzymatic properties of DNA polymerases (Rekosh et al., 1977). All such enzymes require a primer to initiate synthesis, and all catalyze chain elongation in the 5’ to 3’ direction. Since the adenovirus genome is linear, a special mechanism is required for the synthesis of the sequencesat the 5’ terminus of each strand. Rekosh et al. (1977) proposed that the terminal protein represents the primer for DNA synthesis at the extreme 5’ ends of adenovirus DNA strands. In support of this hypothesis it has been demonstrated that the 5’ terminus of each nascent daughter strand of Ad5 DNA replicated in vitro is covalently linked to a protein (Challberg et al., 1980). This protein has an apparent molecular weight of 80,000 and probably represents a precursor t’o the 55K terminal protein. Protein has also been detected at the ends of adenovirus replicative intermediates in IGO (Girard et al., 1977; Coombs et al., 1978: Kelly & Lechner, 1978: Stillman & Bellett, 1978). In view ofthe possible role of the terminal protein in adenovirus DNA replication, it was of interest to determine the precise chemical structure of the protein-DNA linkage. In this paper we present evidence that this linkage is a phosphodiester bond between the B-OH of a serine residue of the 55K protein and the 5’-OH of the terminal deoxyc-ytidine residue of the DNA.
2. Materials
and Methods
(a) Preparation of [3*Pl/Id,5 DSAprotein complex Ad5 was propagated in KB cell monolayers in 75 cm’ flasks as described by Lechner & Kelly (1977), except that the multiplicity of infection was 40 plaque-forming units/cell. At 12 h after infection the culture medium was changed to phosphate-free minimal essential Eagle’s medium (GIBCO) containing 2”/b (v/v) calf serum and 0.25 mCi [32P]orthophosphate/ml. At 40 h after infection the labeling medium was removed, and the cells in each flask were suspended in 2 ml of 150 mm-NaCl, 50 mM-HEPES (pH 7.5) by vigorous shaking. The cell suspensions were pooled and then frozen and thawed 3 times. Virions were purified from the resulting lysate as described previously (Lechner & Kelly, 1977). Ad5 DNA-protein complex was isolated free of other virion components by a modification of the method of Coombs et al. (1978). Purified virions, prepared as described above, were disrupted by incubation for 3 min at 100°C in 2% SDS, 0.1% X-mercaptoethanol and applied t Abbreviations dodecyl sulfate.
used: Ad5.
adenovirus
serotype
5; 55K prokin.
protein
of 55,000
M,;
SDS, sodium
ADENOVIRUS
DNA-PROTEIN
LINKAGE
321
to a Sepharose 2B column. Gel filtration was carried out at room temperature in 10 mMTrisCl (pH 8.0), 1 mM-EDTA, @l% SDS. The [32P]DNA-protein complex eluted in the void volume. It was collected by ethanol precipitation and redissolvc,d in 10 rnnn-Tris . Cl (pH 8.O), 1 mM-EDTA, 0.1 y. SDS (SDS facilitated resuspension of the pellet). The SDS was removed by extraction with aqueous isobutanol followed by extraction with ether. Deproteinized Ad5 DNA was prepared as previously described (Challberg & Kelly, 1979a). (b) Preparation
of [3”P]AdC5
terminal
protein
Ad5 [ 32P]DNA-protein complex (100 to 200 rg) was digested at 37°C for 2 h with DNasrb 1 (Worthington) in a 300 to 450 ~1 incubation mixture containing 20 m&l-MgCl,, 10 InM1 m&l-phenylmethylsulfonyl fluoride, and Tris .(‘I (pH 8.0), 10 mM-CaCl,, 1 mM-EDTA, 80 pg enzyme/ml. This mixture was then incubated for another hour at 37°C after the addition of 150 units exonuclease I/ml (a gift from Dr Paul Englund). The digestion was terminated by addition of SDS and NaCl to final concentrations of 20/b and 50 m&l. respectively, followed by incubation for 2 min at 100°C. The terminal [ 32P]protein was separated from [32P]oligonucleotides by gel filtration on Sephadex G50 in 50 mM-NaCl. 10 mM-Tris.Cl (pH 8.0), 1 mM-EDTA, 0.1% SDS. The radioactive material that emerged in t)he void volume was precipitated by addition of trichloroacetic acid to 15”/ (w/v). The precipitate was dissolved in 200 ~1 50 mM-Tris (pH 6.8), 2% SDS, 100/b glycerol, 0.001% phenol red, and electrophoresed on a 10% SDS/polyacrylamide gel as described by Laemmli (1970). A single radioactive band migrating with an apparent molecular weight of 55,000 was detected by autoradiography. The region of the gel containing this band was excised and crushed with a glass rod. The protein was eluted by incubation in 1 ml 50 mMNaH2P0,, 1 mM-phenylmethylsulfonyl flouride, 0.1% SDS for 12 h at 37°C. Acrylamide was removed by filtration through siliconized glass wool, and the retained acrylamide was washed with 0.3 ml 50 mmNaH,PG,, 1 mM-phenylmethylsulfonyl flouride, 0.1 y/o SDS. (irrat’clr than 90% of the protein was eluted from acrylamide by this method. amino acids Acid hydrolysis of proteins or peptides was carried out at 110°C for 2 h in 5.6 M-HCI. The HCl was removed in vacua, and samples were dissolved in 10 ~1 of a solution containing 1 mM each of O-phosphoserine, 0-phosphothreonine and 0-phosphotyrosine. (O-Phosphoserine and 0-phosphothreonine were obtained from Sigma; 0- phosphotyrosine was synthesized as described by Rothberg et al., 1978.) The samples were applied to 20 cm x 20 cm cellulose thinlayer plates (Kodak) and fractionated in 2 dimensions by 1 of 2 methods. Method (1): The hydrolysis products were separated in the 1st dimension by chromatography in isopropanol/concentrated HCl water (70:15:15, by vol: Sishimura, 1972). Aftrt allowing the plate to dry in air, fractionation in the 2nd dimension was carried out by electrophoresis in 75 mM-NH,COOH (pH 3.65) at 500 V for 90 min (Kelly & Smith, 1970). Method (2): Electrophoresis was performed in the 1st dimension in 2.5”/0 formic acid, 7.8($0 acetic acid (pH 2.0) at 1000 V for 90 min (Bitte & Kabat, 1974). Plates were dried in air and chromatography was performed in the 2nd dimension in isobutpric acid/O.5 M-ammonia (5: 3. v/v; Rothberg et al., 1978). Phosphorylated amino acid markers were detected by spraying the plates with a solution of 0.3% ninhydrin in I-butanol. Radioactivity was detected by autoradiography. (c) Acid
hydrolysis
and
identi$cation
of phosphorylatrd
(d) Preparation of 32P-labeled tryptic peptides of term&al proteirl The terminal [ 32P]protein, purified by SDS/polyacrylamide gel electrophoresis as drscribed above, was digested with trypsin in a 200 ~1 reaction mixture containing 50 mm NH,HCO, (pH 8.3), 50 rg bovine serum albumin, and 10 pg enzyme (Worthington) for 20 h at room temperature. The digestion products were lyophilized and fractionated I)> rlectrophoresis on thin-layer cellulose in 1-butanol/pyridine/acetic acid/water (2: 1: 1:36. by vol.) at 500 V for 2 h (Gibson, 1974). Radioactivity was detected by autoradiography. One major and 2 minor radioactive spots were found. Regions of the thin-layer plate)
3Z2
S. V. DESIDERIO
AND
T. tJ. KELLY
JR
containing [32P]peptides were cut out, and the cellulose was scraped from the Mylar backing. The peptides were eluted from the cellulose with water and lyophilized. A portion of each spot was digested with snake venom phosphodiesterase. In every case all detectable radioactivity was released as [32P]dCMP, [32P]dAMP, and [32P]dTMP (data not shown) as expected, since no guanylate residues are present in the first 25 nucleotides at the 5’ termini of Ad5 DNA (Steenbergh et al., 1977). This result suggested that each spot contained one or more tryptic peptides linked to a [32P]oligonucleotide. The heterogeneity of these 32P-labeled tryptic peptides may reflect heterogeneity in the length of the oligonucleotide linked to terminal protein, or may result from incomplete digestion of the terminal protein by trypsin. The peptides were pooled and used in subsequent enzymatic analysis. (e) D@estion
of (“P1peptide.s
by phosphodiesterase
and
phosphomonoesterase
Peptides were digested with snake venom phosphodiesterase (Worthington) in a 15 ~1 reaction mixture containing 20 mM-TrisCl (pH 6%), 0.7 mM each of dCMP, dAMP, dGMP, dTMP and dUMP, 94 mM-dATP, and 93 unit enzyme. The mixture was incubated at 37°C for 12 h with readdition of 93 unit of enzyme after 3, 6 and 9 h. Peptides were treated with bacterial alkaline phosphatase (obtained from Worthington and repurified on DEAEcellulose as described by Weiss et al., 1968) in a 10 ~1 reaction mixture containing 10 mMTrisCI (pH 8.0), 95 mM-dTMP and 901 unit of enzyme. The reaction mixture was incubated at 37°C for 2 h, with readdition of 601 unit of enzyme after 1 h. A sample of each reaction was analyzed by cellulose thin-layer chromatography in saturated (NH,),SO,/l MNa acetate/isopropanol (40:9: 1, by vol.; Markham & Smith, 1952). Radioactivity was detected by autoradiography and deoxynucleoside 5’ monophosphate markers were detected under short wavelength U.V. light. (f) Alkaline hydrolysis of the DNA-protein linkage A sample of 32P-labeled Ad5 terminal protein was mixed with 7.5 ng of 3H-labeled +X174 replicative form DNA (spec. act. 1.7 x lo5 cts/min per pg) and the resulting solution was adjusted to 50 mM-NaOH in a final volume of 0.6 ml. A 0.1 ml portion was removed and neutralized by addition of 10 ~1 0.5 M-Tris . Cl (pH 6%) and 100 $50 mi+Tris .Cl (pH 8.0). The ratio of 32P radioactivity to 3H radioactivity in this sample (Rr) was determined. The remainder ofthe reaction mixture (0.5 ml) was incubated at 70°C. At various times during the incubation 0.1 ml samples were withdrawn and neutralized as above. After addition of 2 ~1 1% bovine serum albumin, each sample was extracted with 200 ~1 phenol. The ratio of “P radioactivit to 3H radioactivity in the aqueous phase (R,) was then determined. The amount of 9‘P radioactivity in each sample that partitioned into the aqueous phase was calculated from the equation : percent “P in aqueous phase = RJR, x 100. As a control, terminal [32P]protein was incubated under similar conditions in a reaction mixture containing 50 mM-NaCl instead of NaOH. (g) IdentiJication
of the nucleotide
linked
to the terminal
protein
The terminal SmuI restriction fragments, K and L, were isolated from Ad5 DNA as described below. Equimolar quantities of each fragment were incubated in 10 ~1 50 mMNaOH for zero time or for 1 h at 70°C. Samples were neutralized by the addition of 10 ~1 50 m&r-Hcl, 50 m&i-Tris.Cl (pH 8.0) and the DNA was dephosphorylated with 0.07 units bacterial alkaline phosphatase. After 1 h at 37”C, 907 unit additional enzyme was added and incubation was continued at 37°C for 2 h. An equivalent sample of each terminal fragment was incubated in NaOH at 70°C for 1 h, neutralized, and incubated at 37°C for 3 h in the absence of phos hatase. End labeling with P2P was carried out by a modification of the protocol of Maxam & Gilbert (1977) for labeling flush 5’ ends of duplex DNA. The DNA of each sample (0.13 pmol/sample) was precipiated with ethanol and resuspended in 10 ~1 20 mi%-Tris.Cl (pH 95), 06 miwspermidine, 0.1 mM-EDTA. DNA was denatured by heating samples to 100°C for 2 min followed by quick-freezing in a solid C02/ethanol bath. Samples were thawed by vortexing
ADENOVIRUH
DNA-PROTEIN
LINKAGE
323
and immediately transferred to tubes containing 4 pmol [Y-~‘P]ATP (spec. act. 12 Ci/mmol), 1.2 ~1 0.5 M-Tris.Cl (pH 9.5), 0.1 M-MgCl,, 50 mm-dithiothreitol, 50% (v/v) glycerol, and 4 units polynucleotide kinase (Boehringer-Mannheim). Samples were incubated at 37°C for 40 min. Reactions were stopped and DNA was precipitated as described by Maxam & Gilbert (1977). After precipitation with ethanol, end labeled DNA was dissolved in 10 ~1 1 miv-Tris.Cl (pH 8.0), 0.65% SDS. To 5.5 ~1 ofeach DNA sample were added 1 ~150 mM-NaGH and 3.5 ~1 50% glycerol, 25 mm-EDTA, 0.05% bromphenol blue. Strands were separated by electrophoresis through a 2.5% (w/v) agarose slab gel in a buffer containing 40 mru-Tris.Cl (pH 7.5). 5 mM-Na acetate, 1 mM-EDTA. The gel was autoradiographed. Regions of the gel containing 32P-labeled DNA strands were excised and DNA was eluted by the method of Maxam & Gilbert (1977). The 32P-labeled DNA strands were precipitated with ethanol and digested with DNase 1 and snake venom phosphodiesterase for 4 h at 37°C in a 25 ~1 reaction mixture containing 20 m&r-Tris.Cl (pH 6.8), 20 m&i-MgCl,, 0.1 mM each dAMP, dGMP, dTMP, dUMP, 0.1 mMdATP, 1 unit DNase I, and 0.01 unit of snake venom phosphodiesterase. Reaction products were separated by chromatography on thin-layer cellulose in saturated (NH&SO,/1 M-Na acetate/isopropanol (40:9: 1, by vol.). The cellulose thin-layer plate was dried in air and autoradiographed. (h) Restriction endmucleme dip.&n and gel electrophoresti Ad5 [ “P]DNA was digested with the Hind111 restriction enzyme (a gift from Dr H. 0. Smith) in a 0*3-ml reaction mixture containing 60 mna-NaCl, 10 mm-Tris.Cl (pH 8.5), 10 mMMgCl,, 2 mm2-mercaptoethanol, 40 pg DNA and 100 units of enzyme. After 2 h at 37°C the DNA was precipitated with ethanol. The precipitate was dissolved in 10% glycerol, 5 mMEDTA. O.OlO/o bromphenol blue and electrophoresed through a 1.476 agarose slab gel in a buffer containing 40 mm-Tris.Cl (pH 7.5) 5 mM-Na acetate, 1 mM-EDTA. The terminal Hind111 fragments, G and I (Sambrook et al., 1975) were located by autoradiography and eluted by the KI/hydroxylapatite method of Southern (1975). Digestion of Hind111 G and I fragments with the HhoI restriction enzyme (New England Biolabs) was performed in 100 ~1 reaction mixtures containing 50 mM-NaCl, 6 mmTris.Cl (pH 7.5), 6 mM-MgCl,, 6 mm-2mercaptoethanol, 100 mg bovine serum albumin/ml, and 8 units of enzyme. After 75 min at 37”C, 8 units additional enzyme were added and incubation at 37°C was continued for 75 min. The reaction products were electrophoresed through a polyacrylamide slab gel (8% acrylamide, 0.21 ye bisacrylamide) in a buffer containing 50 mm Tris-borate (pH 8.3) 1 mm-EDTA. The HhaI enzyme cleaves Ad5 DNA within the terminal repetition, producing an identical 73-base-pair terminal fragment at each end of the DNA (Steenbergh et al., 1977). These fragments and a 61-base-pair fragment, produced by Hhol cleavage of Hind111 I, were eluted by the method of Maxam & Gilbert (1977). Ad5 DNA was digested with the SmaI restriction enzyme (New England Biolabs) in a 30 ~1 reaction mixture containing 20 mru-KCl, 6 m&r-Tris.Cl (pH 8.0), 6 mM-MgCl,, 6 mM-2mercaptoethanol, 50 pg bovine serum albumin/ml, 14 units of enzyme and 30 rg DNA. After incubation at 37°C for 1 h, 14 additional units of enzyme were added and incubation at 37°C was continued for 1 h. The reaction products were resolved by electrophoresis through a 5% polyacrylamide slab gel in a buffer containing 50 mM-Tris-borate (pH 8.3), 1 mM-EDTA. The gel was stained with ethidium bromide and DNA bands were located under long-wave U.V. light. The terminal SwuzI fragments, K and L (Sambrook et al., 1975) were eluted by the method of Maxam & Gilbert (1977). The K fragment was separated from contaminating I fragment by electrophoresis through a 1.4% agarose slab gel in 40 mM-Tris’Cl (pH 7.5). 5 mM-Na acetate, 1 mM-EDTA and recovered from a minced agarose gel slice by soaking overnight at 37°C in a buffer containing 0.5 M-NH, acetate, 10 mM-Mg acetate, 1 mM-EDTA, and 0.1% SDS. The K fragment was collected by precipitation with ethanol and dissolved in 25 ~120 m&r-triethylammonium bicarbonate (TEABC) (pH 7.5). The resulting solution was applied to a column of DEAE-cellulose (Whatman DE52; bed volume 100 ~1) that had been II
324
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DESIDERIO
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equilibrated with 20 mm-TEABC (pH 7.5). The column was extensively washed with 5 ml 20 mM-TEABC and DNA was eluted with 1.0 M-TEABC. Fractions containing DNA (assayed by absorbance at 260 nm) were pooled and TEABC was removed in vacua.
3. Results (a) Purijication of Ad5 terminal
[32P] protein and releaseof O-[32P]phmphoserine by acid hydrolysis
Ad5 terminal [32P]protein was purified from disrupted virions by a simple threestep procedure. In the first step, the complex of Ad5 [32P]DNA and terminal protein was separated from other virion components by gel filtration on Sepharose 2B in the presence of SDS. In the second step, the isolated DNA-protein complex was digested with DNase, and the terminal protein, attached to a small nucleaseresistant oligonucleotide (Rekosh et al., 1977), was separated from low molecular weight digestion products by gel filtration on Sephadex G-50 in the presenceof SDS. In the final step, the terminal [32P]protein was further purified by preparative SDS/polyacrylamide gel electrophoresis. Figure 1 shows an autoradiogram of a typical preparative gel. As expected, most of the input radioactivity was recovered in a single specieswith an apparent molecular weight of 55,000 (Rekosh et al., 1977). As an approach to the identification of the amino acid residue involved in the protein-DNA linkage, terminal [32P]protein was subjected to partial acid hydrolysis in 5.6 M-HCl at 110°C for two hours (Rothberg et al., 1978). The hydrolysate was fractionated in two dimensions on a cellulose thin-layer plate as described in Materials and Methods (Fig. 2). The major radioactive product was [32P]orthophosphate, as expected, since under these hydrolysis conditions DNA is extensively degraded (Brown, 1974). A small fraction of the radioactivity, however, comigrated with the 0-phosphoserine marker. The identification of this minor radioactive species as 0-phosphoserine was confirmed by analysis of the hydrolysate in a second two-dimensional fractionation system (see Materials and Methods ; data not shown). Two possible sources of the 0-phosphoserine were considered, (1) that it was derived from the protein-DNA linkage, and (2) that it was derived from a serine phosphomonoester residue elsewhere in the polypeptide chain. Suggestive evidence in favor of the first possibility was obtained by analysis of protease-treated Ad5 DNA. A sample of [32P]DNA-protein complex was exhaustively digested with Pronase and extracted with phenol. This procedure removes most of the terminal protein from the DNA, but leaves one or more amino acid residues attached to each 5’ end (Carusi, 1977; Rekosh et al., 1977). The terminal HhaI restriction fragments, each 73 base-pairs in length (Steenbergh et al., 1977), were isolated from the Pronasetreated DNA as described in Materials and Methods and subjected to acid hydrolysis. Both terminal fragments yielded O-[32P]phosphoserine (Fig. 3). A 61 base-pair internal HhaI restriction fragment did not yield 0-phosphoserine when analyzed in an identical fashion. These results indicate that the O-[32P] phosphoserine is derived, at least in part, from the protein-DNA linkage or from the region of the terminal protein in proximity to the protein-DNA linkage.
ADEROVIRUS
DNA-PROTEIN
32.5
LINKAGE
-92
-5 K
-69K
-46K
-30
K
FIG. 1. Preparative SDS/polyacrylamide gel electrophoresis of terminal [32P]protein. I. Ad5 DNA-protein complex, uniformly labeled with 32P , was digested with DNase I and exonuclease and the products were chromatographed on Sephadex G50. The peak emerging in the void volume (0.03% of the input radioactivity) was pooled. Electrophoresis of this material was performed on a 10% SDS/polyacrylamide gel as described in Materials and Methods. An autoradiogram of the preparative gel is shown in the Figure; the positions of molecular weight markers are indicated. The ‘*Plabeled terminal protein has an apparent molecular weight of 55.000. The region of the gel containing terminal [32P]protein was excised and protein was eluted.
(b) Enzymatic
analysis
of the protein-DNA
linkage
Direct evidence that the 0-phosphoserine is involved in a phosphodiester linkage was obtained by analysis of the products produced by digestion of terminal [32P]protein with snake venom phosphodiesterase. This enzyme is an exonuclease that-cleaves ribo- or deoxyribonucleoside 5’ monophosphates from the 3’ ends of oligonucleotides. In addition, compounds in which a 5’ ribo- or deoxyribonucleotide
326
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DESIDERIO
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FIG. 2. Release of O-[32P]phosphoserine by acid hydrolysis of terminal [32P]protein. in Materials and Methods. The 32P-labeled terminal protein was hydrolyzed in HCI as described hydrolysate was combined with 0-phosphotyrosine, 0-phosphothreonine, and 0-phosphoserine markers and the mixture was applied to a cellulose thin-layer plate. Separation was performed in the 1st dimension by chromatography in isopropanol/concentrated HCl/water (70: 15: 15, by vol.) (Nishimura, 1972) and in the 2nd dimension by electrophoresis in 75 mM-ammonium formate at pH 3.65 (Kelly & Smith, 1970). The anode is to the right. An autoradiogram of the cellulose plate is shown in the Figure. The positions of the markers, which were detected with ninhydrin, are indicated by dotted outlines. The order of the migration of the markers is, from right to left: 0-phosphoserine, 0-phosphothreonine and Ophosphotyrosine. The major radioactive species migrates as inorganic phosphate. The minor species comigrates with authentic 0-phosphoserine,
is linked by a phosphodiester bond to a variety of other moieties are also hydrolyzed, yielding the nucleoside 5’ monophosphate (Laskowski, 1966). The enzyme does not hydrolyze phosphomonoesters. Our initial attempts to release [32P]deoxyribonucleoside 5’ monophosphates from terminal [32P]protein by nuclease digestion were not successful, presumably because of steric hindrance. This problem was overcome by cleavage of the terminal protein with trypsin. The resulting [32P]peptides were found to be completely susceptable to digestion with snake venom phosphodiesterase. The digestion products were analyzed in two ways (Figs 4 and 5). First, a sample was fractionated
(b)
--
FIG. 3. Acid hydrolysis of 32P-labeled restriction fragments of The 73.base-pair terminal HhaI restriction fragments and a 61.base-pair internal HhaI fragment described in Materials and Methods. The fragments were hydrolyzed in acid and hydrolysates were cellulose as described in the legend to Fig. 2 and in Materials and Methods. (a) and (b) Hydrolysates of 73-base-pair HhaI restriction fragments from the right and left ends (c) Hydrolysate of a 61 -base-pair internal restriction fragment. The minor radioactive species indicated phosphoserine.
(a)
Cc)
of the genome, by the arrow
respectively. in (a) and (b) comigrated
with
authentic
O-
adenovirus DNA. were isolated from Pronase-treated Ad5 [3ZP]DNA as analyzed by 2-dimensional fractionation on thin-layer
- I
-C-T-
-A-
-orI-
+
(4
+
W
Fro. 4. Release of [32P]mononucleotides from labeled tryptic peptides of terminal protein by snake venom phosphodiesterase. The 3fP-labeled tryptic peptides of terminal protein, isolated by electrophoresis on thin-layer cellulose (see Materials and Methods), were combined and concentrated by lyophilization. Identical quantities of this material were incubated in snake venom phosphodiesterase cocktail in the presence or absence of phosphodiesterase; similarly, identical samples were incubated in bacterial alkaline phosphatase cocktail with or without addition of phosphatase (for conditions, see Materials and Methods). After incubation, 0.1 of each of the 4 samples was applied to a cellulose thin-layer plate and reaction products were separated by chromatography, as described in Materials and Methods. The thin-layer plate was dried in air and autoradiographed. (a) Chromatography of [32P]peptides treated (+) with snake venom phosphodiesterase and an untreated (-) control. (b) Chromatography of a similar preparation treated ( + ) with alkaline phosphatase and an untreated ( - ) control. The positions of deoxyribonucleoside 5’ monophosphate (C, T, 6, A) and inorganic phosphate (P,) markers are indicated. The remainder of each of the 4 samples was hydrolyzed in HCl and analyzed by 2dimensional fractionation (see Materials and Methods and legend to Fig. 5).
ADENOVIRUS
(a)
DNA-PROTEIN
LINKACE
(b)
Fn:. 5. Susceptibility of the DNA-protein linkage to digestion with venom phosphodiesterase or alkaline phosphatase. Equivalent samples of the 32P-labe1ed tryptic peptide preparation were incubated in snake venom phosphodiesterase cocktail with (b) or without (a) addition of phosphodiesterase; similar samples were incubated in bacteria1 alkaline phosphatase cocktail with (d) or without (c) phosphatase. A portion of each reaction mixture was analyzed by thin-layer chromatography (Fig. 4). The remainder of each reaction mixture was then hydrolyzed in HC1 (for details see Materials and Methods). The 4 hydrolysates were analyzed by 2-dimensional fractionation and autoradiography a8 described in the legend to Fig. 2. The minor radioactive species indicated by the arrow in (a), (c) and (d) comigrated in each case with authentic 0-phosphoserine.
by thin-layer chromatography to identify the [32P]nucleotides released by phosphodie&erase. Essentially all of the radioactivity was recovered in three spots corresponding to dAMP, dCMP and dTMP (Fig. 4). The absence of dGMP was expected since there are no guanylate residues in the first 25 nucleotides at the 5’ ends of Ad5 DNA (Steenbergh et al., 1977). A second sample of the phosphodiesterase digestion products was subjected to acid hydrolysis and then analyzed for O-[32P]phosphoserine. As shown in Figure 5, digestion with snake venom phosphodiesterase resulted in a greatly reduced yield of O-[32P]phosphoserine as compared to a sample incubated in the absence of the enzyme. The simplest
330
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DESIDERIO
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interpretation of these results is that each 5’ end of Ad5 DNA is linked to the terminal protein by a phosphodiester bond to serine. As a control, [32P]tryptic peptides of terminal protein were digested with bacterial alkaline phosphatase, a phosphomonoesterase. This treatment did not result in the release of detectable [32P]orthophosphate (Fig. 4) nor did it affect the yield of 0-phosphoserine obtained upon subsequent acid hydrolysis (Fig. 5). (c) Alkaline
hydrolysis
of the DNA-protein
linkage
The conclusion that the DNA-protein linkage involves a phosphodiester bond to serine was supported by a study of the stability of the linkage under alkaline conditions. It is known that phosphodiester bonds to serine (or threonine) are alkali labile, whereas a number of other candidate linkages (e.g. phosphodiester bonds to tyrosine or phosphoamide bonds to lysine) are relatively alkali stable (Majerus et al., 1965; Juodka et al., 1968; Shabarova, 1970; Gumport & Lehman, 1971; Rothberg et aE., 1978). In the experiment whose results are displayed in Figure 6 a sample of
2 ‘a s
I? i .E a. 2 s 5 %
loo/
l -------.
+ NaOH
l
0
75
50
25
z
l -a’
-NaOH
30
60 Time
90
120
(mini
FIG. 6. Alkaline hydrolysis of the DNA-protein linkage. Terminal [32P]protein was incubated with +X174 RF 13H]DNA in 50 mM-NaOH at 7OOC. After 0, 15, 30, 60 and 120 min of incubation portions were extracted with phenol and the percentage of 32P radioactivity partitioning into the aqueous phase was calculated a8 described in Materials and Methods. This value is plotted as a function of time ( +NaOH). A control sample of terminal [32P]protein was incubated in 50 mM-NaCl at 70°C and assayed in the same manner at 0 and 60 min ( -NaOH).
purified terminal [32P]protein was incubated in 50 mM-NaGH at 70°C and monitored for the release of [32P]oligonucleotide by a phenol extraction assay. This assay was based upon the finding that [32P]oligonucleotide bound to terminal protein partitioned into the phenol phase during extraction, whereas unbound oligonucleotide partitioned into the aqueous phase. Under the incubation conditions that we employed, the [32P]oligonucleotide was quantitatively released from the terminal protein with a half-time of about ten minutes. Thus the behavior of the DNA-protein linkage in alkali is similar to that of other phosphodiester bonds to
ADENOVIRUS
DNA-PROTEIN
331
LINKAGE
serine (Majerus et al., 1965; Juodka et al., 1968). Incubation of terminal [ 32P]protein for 60 minutes at 70°C in 50 mM-NaCl, instead of NaOH, did not result in the release of any detectable radioactivity into the aqueous phase. (d) Ident@cation
of
the nucleotide
linked
to terminal
protein
The lability of the DNA-protein linkage in alkali made it possible to identify the nucleotide bound to terminal protein by 5’ end labeling methods. Ad5 DNA from Pronase-treated virions was digested with the Smal restriction enzyme and the terminal restriction fragments (K and L) were isolated by gel electrophoresis. Samples of each fragment were made 50 mM in NaOH and were either neutralized immediately or incubated at 70°C for one hour prior to neutralization. DNA from these samples was dephosphorylated with bacterial alkaline phosphatase and the 5’ ends were labeled with 32P by incubation with polynucleotide kinase and [y“P]ATP (Weiss et al., 1968). The DNA was then denatured and complementary strands were separated by agarose gel electrophoresis. In the case of DNAs not treated at 70°C with alkali, radioactivity was detected in only one strand of each terminal restriction fragment (Fig. 7(c) and (f)). This was expected, since the 5’ end of one strand is covalently attached to protein and therefore blocked to end labeling. When, however, terminal fragments were incubated in alkali prior to end labeling. 32P was incorp orated equally into both strands (Fig. 7(a) and (d)), indicating that the blocking peptide had been removed. This finding is consistent with the results of Carusi (1977), who reported that alkaline treatment of Ad DNA increased the efficiency of 5’ end labeling. When the phosphatase step was omitted, three- t,o fivefold less32P was incorporated into each strand (Fig. 7 (b) and (e)). Thus, at least in the majority of molecules, alkaline hydrolysis of the DNA-protein linkage leaves a 5’ phosphoryl group at the ends of the DNA. The low level of 32P incorporated in t’he absence of dephosphorylation may be the result of a phosphate exchange reaction catalyzed by polynucleotide kinase (Van de Sande et al., 1973). Alternatively, it is possible that some 5’ phosphoryl groups are removed during incubation in alkali. DNA strands labeled with 32P were eluted from the agarose gel and digested to 5’ mononucleotides with DNase I and snake venom phosphodiesterase. Digestion products were analyzed by thin-layer chromatography and autoradiography. Almost all of the radioactivity in each strand was recovered as a single [32P]deoxy ribonucleoside 5’ monophosphate representing the 5’.terminal nucleotide of each strand. For both the K and L fragments, the strand susceptible to end labeling without prior alkaline treatment (the slowly migrating strand) yielded almost exclusively [32P]dGMP (Fig. 8(a),(c),(d) and (f)), as predicted by the known sequence of the SmaI cleavage site (Roberts, 1980). The complementary strand, which was labeled only after prior incubation in alkali, released almost all of its radioactivity as [32P]dCMP (Fig. 8(b) and (e)). In addition, sequencing of the endlabeled DNA by the procedure of Maxam & Gilbert (1977) gave the same5’.terminal sequence as reported by Steenbergh et al. (1977) (data not shown). These results indicate that the nucleotide covalently bound to the Ad5 terminal protein is a deoxycytidylyl residue. From the results taken as a whole we conclude that the terminal protein is bound to Ad5 DNA by a phosphodiester linkage between the
332
S.
V. (4
DESIDERIO (b)
AND (cl
T. (4
J.
KELLY (e)
JR (f)
-r
FIG. 7. Separated strands of 5’ end-labeled terminal restriction fragments. The terminal SmaI restriction fragments K (lanes (a) to (c)) and L (lanes (d) to (f)) were isolated from Pronase-treated Ad5 DNA. Samples of each fragment were adjusted to 50 mM-NaOH and were either neutralized immediately or incubated at 70°C for 1 h prior to neutralization. The samples were then dephosphorylated by treatment with alkaline phosphatase and labeled at their 5’ termini by polynucleotide kinase and [y-32P]ATP. In some cases (indicated below) the phosphatase step was omitted. All kinase reaction mixtures contained the same quantity of DNA fragment (@13pmol). Following 5’ end labeling a portion of each sample was denatured and the complementary strands were separated by electrophoresis on a 2.5% agarose gel. The positions of the r and 2 strands of uniformly labeled terminal.
ADENOVIRUS
DNA-PROTEIN
LINKAGE
/l-OH of a serine residue of the protein and the 5’-OH of the terminal residue of the DNA.
X33
deoxycytidine
4. Discussion In this paper we have described the results of experiments that define the precise chemical structure of the linkage between Ad5 DNA and the 55K terminal protein. Terminal protein, bound to a small [32P]oligonucleotide, was isolated from Ad5 [“P]DNA-protein complex and digested with trypsin. The resulting [32P]peptides were analyzed with the following results : (1) partial acid hydrolysis released a single phosphorylated amino acid which was identified as 0-phosphoserine; (2) treatment with snake venom phosphodiesterase yielded dAMP, dCMP and dTMP as the only radiolabeled products; and (3) prior phosphodiesterase treatment greatly reduced the yield of 0-phosphoserine upon subsequent acid hydrolysis. These results indicated that Ad5 DNA is linked to the terminal protein by a phosphodiester bond to the P-OH of serine. This conclusion was supported by the observation that the DNA-protein linkage is alkali-labile. The lability of the linkage in alkali is also of considerable practical importance because it permits complete removal of the protein under conditions in which DNA is not significantly degraded. In 50 mMNaOH at 70°C the rate constant for hydrolysis of the linkage was approximately 7 x low2 min-‘. After incubation of Ad5 DNA under these conditions we were able to label the 5’ termini with 32P by polynu cl eotide kinase. Digestion of the endlabeled DNA to completion with DNase I and snake venom phosphodiesterase yielded [32P]dCMP. We conclude that the DNA-protein linkage is deoxycytidylyl(5’+0)-serine. In interpreting the results of our experiments we have considered the possibility that the 0-phosphoserine found in acid hydrolysates of terminal [32P]protein could have arisen from an N to 0 migration of phosphorus during hydrolysis. This type of rearrangement has been observed during acid hydrolysis of model compounds containing a nucleotide bound by a phosphoamide (P-N) linkage to the amino group of serine. The products of hydrolysis of such compounds include the nucleoside and 0-phosphoserine (Shabarova, 1970). Two lines of evidence, however, argue strongly against the possibility that adenovirus DNA is linked to terminal protein by a phosphoamide bond. First, the DNA-protein linkage is susceptible to cleavage by
SmaI fragments are indicated. The K and L fragments are derived from the left and right ends. respectively, of the Ad5 genome (Sambrook et al., 1975). (a) K fragment incubated in alkali. (b) K fragment incubated in alkali; phosphatase step omitted. (c) K fragment not incubated in alkali. (d) L fragment incubated in alkali. (e) L fragment incubated in alkali; phosphatase step omitted. (f) L fragment not incubated in alkali. Regions of the gel corresponding to labeled strands were cut out and assayed for radioactivity with the following results. (a) r strand, 232 ctsjmin; 1 strand, 214 cts/min; (b) r strand, 50 cts/min; 1 strand, 66cts/min: (c) r strand, 170 cts/min: 2 strand, 24 cts/min; (d) r strand, 117 cts/min; 2 strand, 145 cts/min: (e) r strand. 65 cts/min: 1 strand, 54 cts/min: (f) r strand, 19 cts/min: 1 strand, 150 cts/min.
334
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DESIDERIO
AND
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KELLY
JR
C-
T-
G-
A-
ori -
(a)
(b)
(c)
(d)
(4
(f)
FIG. 8. Identification of the nucleotide linked to terminal protein. After alkaline treatment, both complementary strands of each SnmI terminal restriction fragment could be end-labeled with 32P (Fig. 7(a) and (d)). Without prior alkaline treatment, only one strand of each fragment was labeled (Fig. 7(c) and (f)). Labeled strands were eluted from an agarose gel identical to that shown in Fig. 7 and digested to 5’ mononucleotides with DNase I and snake venom phosphodie&erase (see Materials and Methods). Digestion products were resolved by thin-layer chromatography in saturated (NH&SO.& M-Na acetate/isopropanol (49:9:1, by vol.) and detected by autoradiography. The positions of the-4 deoxyribonucleoside 5’ monophosphate markers (C, T, A, G) are indicated. (a) K fragment, incubated in alkali, r strand. (b) K fragment, incubated in alkali, 1 strand.
ADENOVIRUH
DNA-PROTEIN
335
LINKAGE
snake venom phosphodiesterase. This enzyme is specific for phosphodiester bonds and is reported to be inactive against phosphoamide linkages (Bogdanov et al., 1962). Second, under alkaline conditions in which the DNA-protein linkage is retidily hydrolyzed, phosphoamide bonds are generally stable (Little et al., 1967; Juodka et al., 1969). Several examples of covalent complexes between DNA or RNA and protein are known. Aside from adenovirus, such linkages are known to exist in the genomes of poliovirus (Lee et al., 1977; Nomoto et al., 1977) and the bacteriophage 429 (Ortin et al., lQ71), in replicative intermediates ofthe parvovirus H-l (Revie et al., 1979) and the bacteriophage 4X174 (Eisenberg et al., 1979), and in relaxation complexes of bacterial plasmids such as ColEl (Blair & Helinski, 1975). In addition, reactions of a number of topoisomerases with DNA have been shown to involve covalent protein-DNA intermediates (Depew et al., 1978; Champoux, 1977; Morrison & (‘ozzarelli, 1979). In the case of poliovirus, the RNA genome is attached to a virion protein by a phosphodiester bond between a uridylate residue and the phenolic hydroxyl group of tyrosine (Ambros & Baltimore, 1978; Rothberg et al, 1978). It is of interest to note that in the caseof bacteriophage $29, whose DNA replicates by a mechanism similar to that of adenovirus, a 27,000 M, protein is bound to the genome by a phosphodiester linkage between the /?-OH group of serine and the 5’.OH of the terminal deoxyadenosine residue (Hermoso $ Salas, 1980). The function of the adenovirus terminal protein is not yet clear. Studies performed in this laboratory (Challberg & Kelly, 1979b)have provided evidence that the 55K protein bound to the ends of parental DNA strands may play an essential role in DNA replication; adenovirus DNA from which the protein has been removed by proteolytic treatment is ineffective as a template for replication in vitro. The 55K protein on parental strands may facilitate initiation of DNA replication at the molecular termini, perhaps via interactions with other replication factors. We have recent,ly demonstrated (Challberg et al., 1980) that the 5’ terminus of each nascent’ Ad,5 daughter strand synthesized in vitro is also covalently linked to a protein. This protein has an apparent molecular weight of 80,000 and represents a precursor to t,he 55K protein. The 80K protein is also attached to DNA by a phosphodiester bond t,o serine. These observations are consistent with a second possible role for the t)rrminal protein in adenovirus DNA replication, namely, that it serves as the primer for daughter strand synthesis (Rekosh et aZ., 1977). Thus, the formation of an ester linkage between the r-phosphoryl group of dCTP and the P-OH of a serine residue in the 80K protein may be the primary initiation event in adenovirus DNA replication (Challberg et al.. 1980).
(c) K fragment, (d) L fragment, (e) L fragment, (f) L fragment, The ,SmaI K and (Sambrook ~4 al.,
not incubated in alkali, r strand. incubated in alkali, 2 strand. incubated in alkali, r strand. not incubated in alkali, I strand. L fragments are derived from the left 197.5).
and
right
ends
of the Ad5
genome,
respectively
336
8. V. DESIDERIO
AND
T. J. KELLY
JR
We thank Michael Stern for technical assistance and Drs Mark Challberg, Gary Ketner and Hamilton Smith for helpful discussions. This work was supported by United States Public Health Service grant CA16519 from the National Cancer Institute. One of the authors (S. V. D.) is a scholar of the Insurance Medical Scientist Scholarship Fund. REFERENCES Ambros, V. & Baltimore, D. (1978). J. Btil. Chem. 253, 5263-5266. Bitte, L. & Kabat, D. (1974). Methods Enzymol. 30, 563-590. Blair, D. G. & Helinski, D. R. (1975). J. Bzbl. Chem. 250, 8785-8789. Bogdanov, A. A., Antonovich, Y. G., Terganova, G. V. & Prokofiev, M. A. (1962). Biokhimiya, 27,1054-1060. Brown, D. M. (1974). Basic PrincipZes in NucEeic Acid Chemistry (Tso, P. 0. P., ed.), vol. 2, pp. l-90, Academic Press, New York. Carusi, E. A. (1977). ViroZogy, 76, 386394. Challberg, M. D. t Kelly, T. J. Jr (1979a). Proc. Nat. Acud. Sci., U.S.A. 76, 655-659. Challberg, M. D. & Kelly, T. J. Jr (1979b). J. Mol. Biol. 135, 999-1012. Challberg, M. D., Desiderio, S. V. & Kelly, T. J. Jr (1980). Proc. Nut. ilead. Sci., U.S.A. 77, 51055109. Champoux, J. J. (1977). Proc. Nat. Acud. Sci., U.S.A. 74, 3800-3804. Coombs, D. H., Robinson, A. J., Bodnar, J. W., Jones, C. J. & Pearson, G. D. (1978). Cold Spring Harbor Symp. Quunt. Biol. 43, 741-753. Depew, R. E., Liu, L. F. & Wang, J. C. (1978). J. Biol. Chem. 253, 511-518. Eisenberg, S., Scott, J. F. & Kornberg, A. (1979). Cold Spring Harbor Symp. Quant. Bill. 43, 295302. Gibson, W. (1974). Virology, 62, 319-336. Girard, M., Bouche, J. P., Marty, L., Revet, B. & Berthelot, N. (1977). Virology, 83, 34-55. Green, M., Pina, M., Kimes, R. C., Wensink, P. C., MacHattie, L. A. & Thomas, C. A. Jr (1965). Proc. Nat. Acad. Sci., U.S.A. 57, 1302-1309. Gumport, R. I. & Lehman, I. R. (1971). Proc. Nut. Acad. Sci., U.S.A. 68, 2559-2563. Hermoso, J. M. & Salas, M. (1980). Proc. Nat. Acud. Sci., [T.S.A. In the press. Juodka, B. A., Saveliev, E. P., Shabarova, Z. A. & Prokofiev, M. A. (1968). Biokhimiyu, 33, 907-915. Juodka, B. A., Obruchnikov, I. V., Nedbye, V. K., Shabarova, Z. ii. & Prokofiev, M. A. (1969). Biokhimiya, 34, 647-654. Kelly, T. J. Jr & Challberg, M. D. (1979). ICN/UCLA Symposiumon Extrachromosomal DNA (Stevens, J. G., Todaro, G. J. & Fox, C. F., eds), pp. 165176, Academic Press, New York. Kelly, T. J. Jr & Lechner, R. L. (1978). Cold Spring Harbor Symp. Quant. Biol. 43, 721-727. Kelly, T. J. Jr & Smith, H. 0. (1970). J. Mol. Biol. 51, 393-409. Laemmli, U. K. (1970). Nature (London), 227, 68&685. Laskowski, M. S. Sr (1966). Procedures in Nucleic ilcids Research (Cantoni, G. L. & Davies, D. R., eds), vol. 1, pp. 154187, Harper and Row, New York. Lechner, R. L. & Kelly, T. J. Jr (1977). Cell, 12, 1007-1020. Lee, Y, Ii‘., Nomoto, A., Detjen, B. M. & Wimmer, E. (1977). Proc. Vat. Acad. Sci., U.S.A. 74, 59-63. Little, J. W., Zimmerman, S. B., Oshinski, C. & Gellert, M. (1967). Proc. Nat. Acad. Sci., U.S.A. 58, 2004-2011. Majerus, P. W., Alberts, A. W. & Vagelos, P. R. (1965). Proc. Nat. Acad. Sci., U.S.A. 53, 41 O-417. Markham, R. & Smith, J. D. (1952). B&hem. J. 52, 55&565. Maxam, A. M. & Gilbert, W. (1977). Proc. Nut. Acad. Sci., U.S.A. 74, 566564. Morrison, A. & Cozzarelli, N. R. (1979). Cell, 17, 175184. Nishimura, S. (1972). Prog. Nucl. Acids Res. Mol. Biol. 12, 49-85. Nomoto, A., Detjen, B., Pozzatti, R. & Wimmer, E. (1977). Nature (London), 268,208213. Ortin, J., Vinuela, E., Sales, M. $ Vasquez, C. (1971). Nature New Btil. 234, 276277.
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