Eur. J. Biochem. 86, 389-398 (,1978)
Complex Formation between the Adenovirus Type 5 DNA-Binding Protein and Single-Stranded DNA Peter C. van der VLIET, Wilko KEEGSTRA, and Hendrik S. JANSZ Laboratory for Physiological Chemistry and Institute for Molecular Biology, State University of Utrecht' (Received November 24, 1977)
The adenovirus-type-5-coded single-strand-specific DNA-binding protein was purified from infected human KB cells and characterized by sucrose gradient centrifugation and gel filtration. The protein has a sedimentation coefficient of 3.3 S and a diffusion coefficient of 4.4 x lo-' cm/s, which corresponds to a molecular weight of 68000 for the native protein. This is close to the peptide molecular weight of 72000 and indicates that the protein is a monomer. The frictional coefficient ratio is 1.82 which suggests that the DNA-binding protein is highly anisometric. The binding of the protein to various nucleic acids was studied by filtration through nitrocellulose filters and sucrose gradient centrifugation. The binding to single-stranded D N A is a f x t process which does not require bivalent cations or sulfhydryl groups and occurs over a broad pH range. The binding to synthetic polydeoxyribonucleotides is at least 10-fold less efficient than that to natural single-stranded DNA. At low protein-to-DNA ratios the binding to single-stranded DNA is cooperative. A maximal protein to DNA ratio of 33 (w/w) can be obtained which corresponds to 1 protein molecule for about 7 nucleotides. Sedimentation and electron microscopic studies indicate that the DNA-binding protein keeps single-stranded DNA in an extended configuration. The saturated nucleoprotein complex is slightly more resistant to nuclease digestion than single-stranded DNA. Human adenovirus type 5 is an oncogenic DNA virus that multiplies in the nucleus of permissive human cells. Early in infection a number of viruscoded or virus-induced proteins are synthesized, which are probably involved in the onset of adenovirus DNA replication and regulation of gene expression. One of these early proteins is a virus-coded single-strandspecific DNA-binding protein, which is synthesized in large quantities, up to 2 x lo7 molecules being present in adenovirus-2 or adenovirus-5-infected cells [1,2]. The DNA-binding protein, which has an apparent molecular weight of 72 000 in dodecylsulphate/ polyacrylamide gels, is phosphorylated [3 - 61 and can be converted into fragments of 44000-48000 M , which are presumably proteolytic breakdown products [ l , 71. Pulse-chase experiments have identified still another form of the adenovirus 2 DNA-binding protein, which has a slightly higher molecular weight [5], showing that several post-translational modifications can occur. The significance of these modifications is not clear. ~~
~~
Emyrnrs. Alkaline phosphatase (EC 3.1.3.1); catalase (EC 1.11.1.6); glyceraldehyde-3-phosphatedehydrogenase(EC 1.2.1.12); phosphoglycerate kinase (EC 2.7.2.3); ribonuclease (EC 3.3.4.22); endonuclease (EC 3.1.4.21).
The structural gene for the DNA binding protein has been mapped between 0.59 and 0.71 from the left molecular end of the DNA and a mutant in this gene, H5ts125, has been identified [8 - 111. This mutant produces a thermolabile DNA-binding protein [lo, 121 and is defective in the initiation of viral DNA replication [13,14]. Antibody against the DNA-binding protein inhibits the rate of DNA chain growth in isolated nuclei from adenovirus-infected cells [l5]. These data indicate that the DNA-binding protein is required for the first and subsequent rounds of adenovirus DNA replication and may serve during the initiation process as well as during elongation of nascent DNA chains. A function of the DNA-binding protein in viral DNA replication is also suggested by the finding that it constitutes the major protein in complexes that are capable of adenovirus DNA replication in vitro (C. Kedinger, J. Wilhelm and 0 . Brison, personal communication). We have studied some physicochemical properties of the purified DNA-binding protein and its interaction with single-stranded DNA. The experiments described here show that the protein is a monomer and does not behave as a globular protein. Its binding to single-stranded DNA is a fast, partly cooperative,
390
process which is only slowly reversible. When the DNA is saturated with protein, one protein molecule covers 7 nucleotides and forces the single-stranded DNA into an extended configuration.
Adenovirus DNA-Binding Protein
tein by dodecylsulphate/polyacrylamide electrophoresis have been described [15]. The purified protein which eluted from DNA cellulose with 1 M NaCl was stored at - 80 "C and was stable for at least 6 months. Spectral analysis showed that A280/A260 = 1.60 and the absorption coefficient E;& = 9.3.
MATERIALS AND METHODS Rabbit muscle glyceraldehyde-3-phosphate dehydrogenase, yeast 3-phosphoglycerate kinase, beef liver catalase and endonuclease from Neurospora crassa were purchased from Boehringer Mannheim. Pancreatic ribonuclease and Escherichia coli alkaline phosphatase were from Sigma chemicals. All synthetic polydeoxyribonucleotides were obtained from Miles Laboratories and all polyribonucleotides from Sigma Chemicals. [3 HILeucine (40 Ci/mmol), and [jHlthymidine (60 Ci/mmol) were from the Radiochemical Centre (Amersham, England). @X174 DNA was a kind gift from Dr P. D. Baas. Isolation of DNA-Binding Protein KB cells were grown in suspension in Eagle's minimal essential medium containing 5 calf serum. Adenovirus-5-infected cells were harvested 16- 20 h after infection when the amount of DNA-binding protein is maximal [1,2]. The cells were washed with phosphate-buffered saline and suspended in 20 mM Tris-HC1/0.01 M NaClI1.5 mM MgC12/2 mM dithiothreitol, pH 7.6, at a concentration of lo7 cells/ml. After 30 min at 0 "C the swollen cells were disrupted by freezing. In this stage the cells could be stored as a suspension at - 80 "C, for many months. All the buffers contained 2 mM phenylmethylsulfonyl fluoride and 20 pg/ml L-l-tosylamido-2-phenylethylchloromethyl ketone to inhibit proteolytic activity. For the isolation of H-labeled protein, the cells were infected in leucine-free medium to which [3H]leucine (1 pCi/ ml) was added 4 h post infection. The infection process was monitored by pulse-labeling the viral DNA with [3H]thymidine as described [13]. Frozen cells were thawed and cell dCbris was removed by centrifugation for 20 min at 10000 x g. To the supernatant EDTA was added to 10 mM, glycerol to 10% and NaCl to 0.15 M, final concentrations. The DNA-binding protein was precipitated by addition of ammonium sulfate (60 % saturation) and the precipitate was dissolved in 20 mM Tris-HC1, pH 8.1/1 mM EDTA/O.15 M NaCl/10% glycerol (buffer A) and dialysed against this buffer. A small precipitate, which formed during dialysis, was removed by centrifugation and the clear supernatant was applied to a DNA-cellulose column containing 0.8 mg single-stranded calf thymus DNA/ml. The procedures for DNA-cellulose chromatography, including elution with sodium dextrane sulfate and analysis of the pro-
Filter Binding Assay Nitrocellulose filters (Millipore HAWP, 0.45 pm) were boiled for 10 min in distilled water and soaked overnight in 20 mM Tris-HC1, pH 8.0jl mM EDTA/ 2 mM mercaptoethanol/50 mM NaC1/5 % (w/v) glycerol/l % dimethylsulfoxide (buffer B). Before use the filters were washed with 10 ml buffer B. DNA or synthetic polynucleotides were sonicated for 1 min at 50 W in a Branson sonifier before addition of protein. Routinely, DNA and protein were mixed in a total volume of 100 p1 and after 5 min at 20°C the samples were diluted with buffer B to 1 ml and filtered. The filters were washed with three portions of 3 ml buffer B. All filtrations and washings were done at a constant filtration rate of 5 ml/min, which was necessary to obtain reproducible results. When the stability of the DNA . protein complex to increasing NaCl concentrations was tested, the filters were soaked and washed with buffer B containing the indicated salt concentrations. After washing, the filters were dried, dissolved in 2 ml 1,2-dimethoxyethane and counted. Under these conditions single-stranded and doublestranded DNA pass through the filter while DNA . protein complexes are retained [24]. Electron Microscopy A saturated DNA . protein complex was prepared by mixing purified DNA-binding protein and QiX174 single-stranded DNA in a protein-to-DNA ratio of 33 (w/w). The components were mixed in bufferA containing 1 M NaCl and dialyzed against buffer A. Samples were spread according to a modified KleinSchmidt monolayer technique [40]. After dilution to 0.13 M ammonium acetate the complex was mixed with cytochrome c (0.02 % final concentration) and spread, without prior fixation, on a 0.01 M ammonium acetate hypophase without formamide. A low-salt hyperfase was required to prevent dissociation of the nucleoprotein complex. The DNA was picked up with carbon-coated grits stained with alcoholic uranyl acetate, rotary-shadowed, with platinum (7 - 10 "C) and examined in a Philips 301 electron microscope. Double-stranded @X174 replicative form DNA was treated in a similar way. Length measurements were done using a Hewlett Packard 9864 A digitizer connected to a 9820 A calculator.
P. C. van der W e t , W. Keegstrd, and H. S. Jansz
391
Sedimentation Analysis
Sedimentation of purified DNA-binding protein or DNA . protein complexes was performed in 5 27 % isokinetic sucrose gradients in 20 mM Tris-HC1 (pH S.l)/l mM EDTA/10% glycerol containing0.05 M NaCl or 1M NaCLThe nitrocellulose tubes were coated by incubation with 2 mg/ml bovine serum albumin for 30 min at 50 "C, washed with distilled water and dried at 80 "C. This prevented sticking of the protein to the wall of the tubes. Sedimentation markers were E. coli alkaline phosphatase (6.0 S [16]) glyceraldehyde-3phosphate dehydrogenase (7.7 S [17]) and 3-phosphoglycerate kinase (3.1 S [18]). Enzymatic activities were tested in appropriate samples according to the procedures described in these references. RESULTS Physicochemical Properties of the Purified DNA-Binding Protein
Selective extraction of infected KB cells followed by ammonium sulfate precipitation and DNA-cellulose chromatography provides a rapid and effective purification procedure in which a highly purified protein is obtained. Polyacrylamide gel electrophoresis in the presence of dodecylsulphate (Fig. 1) shows a predominant band at 72000 M , with some weak bands at 42 000 - 46 000 M,, presumably proteolytic products of the 72000-Mr protein [6]. As estimated from the radioactivity in gel slices 92% of the label is found in the DNA-binding protein region of the gel. Sedimentation of the native protein at high ionic strength (1 M NaC1) in the presence of marker enzymes showed a homogeneous peak at 3.3 S (Fig.2). This value did not change when the centrifugation was performed at 0.15 M NaCl at low concentrations (less than 20 pg/ml). At high concentrations (100 pg/ml or more), however, the protein aggregates in low-ionicstrength buffers. This aggregation is pH dependent. When H-labeled DNA-binding protein (100 pg/ml) was dialyzed against buffers of low ionic strength ( I = 0.06 M) with pH of 6.8,S.l or 9.0 and centrifuged for 20 min at 2000 rev./min, the pellet contained 83 %, 16% and 7 % of the radioactivity respectively. The pellets could be easily redissolved in a buffer containing 1 M NaCl. A value of 3.3 S is rather low for a protein of M , 72000. For globular proteins, 3.3 S would correspond to a molecular weight of about 40000. We considered the possibility that the protein was degraded to its 42000 - 48 000 proteolytic products during the sedimentation, giving rise to the low sedimentation value. However, when the 3.3-S peak obtained after sucrose gradient centrifugation was analyzed by electrophoresis, 80% of the protein was still present at the 72000-M, position in the gel.
Slice number Fig. 1. Dodecylsulphate gel electrophoresis of the DNA-binding protein. [3H]Leucine-labeied protein (10 pg) obtained after DNAcellulose chromatography was electrophoresed in 10 % polyacrylamide gels containing dodecylsulphate and stained with Coomassie brilliant blue. After destaining the gel was fractionated into 2-mm slices and the radioactivity was measured. The main peak had an apparent molecular weight of 72000 as determined from marker proteins run in parallel gels
73
0.4 &
400
0
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m
m
T
0.2
200
-0
n
0 20 10 Fraction number
30
Fig. 2. Sucrose grudient sedimentution of the DNA-binding protein. [3 HILeucine-labeled DNA-binding protein (2,2 pg, 8250 dis./min) was layered on a 5 - 27 isokinetic sucrose gradient in 20 mM TrisHC1 (pH 8.1)/1 M NaCl/I mM EDTA/10% glycerol. E . colt alkaline phosphatase (6.0 S) and 3-phosphoglycerate kinase (3.1 S) were added as sedimentation markers. Centrifugation was for 24 h at 50000 rev./min at 4°C in an SW 65 rotor. The recovery was 76%. Control experiments showed that the presence of these marker enzymes did not influence the sedimentation of the protein. 3H ( 0 ) ;alkaline phosphatase activity, A400 (0);3-phosphoglycerdte kinase activity, A340 (A)
Adenovirus DNA-Binding Protein
392
0
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-
C ._
-5 2000 L,j .-
U
v
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50
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t
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“i
Fraction
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Fig. 3. Gel filtration of the DNA-binding protein on u Sephuder- G-200 c.ol~orr/r.-’H-labeled binding protein (35 pg, 700000 dis./min was chromatographed on a 100 x 1.2-cm G-200 column in 20 m M Tris-HC1 (pH 8.1)/1 mM EDTAI0.5 M NaC1/10% glycerol. [‘4C]Leucine was added to determine V , and dextrane blue to measure Vo. Fractions of 1.9 ml were collected and appropriate samples were counted. The recovery of the protein was 55 %. In separate experiments the elution volume V , of inarker enzymes was determined and V,/ VOwas plotted against the diffusion coefficients D (see inset). G A P D H = glyceraldehyde-3-phosphate dehydrogenase
To obtain more information about the molecular weight and the shape of the native protein, the diffusion coefficient was determined by gel filtration on Sephadex G-200. At low ionic strength, even in the presence of 0.05 % Triton X-100 as detergent, the yields were very low and the results were irreproducible, presumably because the protein tends to aggregate. At high ionic strength a symmetrical elution profile was reproducibly obtained (Fig. 3). The DNA-binding protein eluted between the position of catalase (Dzo, = 4.17 x cmjs) and glyceraldehyde-3-phosphate dehydrogenase (Dzo,w = 5.0 x cmjs). A straight line was obtained for the marker proteins when V,/Vo was plotted against DZO, according to Andrews [25], and D20,w= 4 . 4 ~ cmjs was found by interpolation for the DNA-binding protein (Fig. 3). From the sedimentation coefficient and the diffusion coefficient the molecular weight of the native protein can be calculated using the Svedberg formula : M , = RTs/D(l - vze) = 68000, where M I = molecular weight, T = absolute temperature, vz = partial specific volume, Q = density of water at 20 “C. A value of 0.73 mljg was calculated for uz from the partial specific volumes of the amino acid residues and the amino acid composition [3]. From the diffusion coefficient the Stokes radius a = 4.9 nm was calculated. A similar result was obtained when the gel filtration data were plotted ac-
cording to Siege1 and Monty [19]. The frictional coefficient ratio f/ji was determined by substitution =~ 1.82 )~’~ in the classical equationfro = a/(3 O ~ M , / ~ N where M I is the calculated molecular weight of 68 000, a is the Stokes radius and NA is Avogadro’s number. Assuming 0.2 g HzO of hydration/g protein this corresponds, with a prolate ellipsoid, to an axial ratio of about 1 : 10 [29]. These data indicate that the protein is a highly anisometric monomer and does not behave as an typical globular protein.
Binding
of
the Protein to DNA
We used two methods to study the interaction between the DNA binding protein and various polynucleotides, i.e. sucrose gradient centrifugation and a filter-binding assay. For the filter-binding test [24] DNA and protein were mixed and filtered on nitrocellulose filters. The complex is retained while free DNA passes the filter readily. Fig. 4 shows the binding to single-stranded @XI74DNA. The binding increases continuously and about 30 times the amount of protein (on a weight basis) is required to retain all DNA on the filter. No binding to double-stranded adenovirus 5 DNA is observed, in agreement with previous results [l]. Binding to single-stranded DNA proceeds very rapidly. At 4°C the binding process was complete
P. C. van der Vliet, W. Keegstra, and H. S. Jansz
393
100
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_ _ _ _ __-- - - ---20
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40 50 Protein - t o - D N A ratio Fig. 4. Coniplex fornzation hetween DNA-binding pi'ofein and .singlestranded DNA. A constant amount of I4C-labeled single-stranded a x 1 7 4 DNA (0.15 pg, 7940 counts/min) (O---O) or 3ZP-labeled double-stranded adenovirus type 5 DNA (0.22 big, 492 countsimin. 0 --0) was mixed with increasing concentrations of binding protein and filtered on nitrocellulose filters. The percentage of input DNA retained on the filter was determined as described in Methods 10
30
within 10 s. This is comparable to results obtained with E. coli DNA-binding protein [22] and unlike that found with the fd gene-5 protein where binding requires 3-5 min [23].Binding occurred in a broad range of pH values (pH 4- 10). Addition of 5 mM EDTA or 10 mM N-ethylmaleimide did not prevent complex formation, indicating that bivalent cations or sulf'hydryl groups are not required. As expected, increasing the NaCl concentration results in a reduction with 50 % binding at 0.32 M NaCl (Fig. 5). Once formed, the complex appeared to be rather stable. To study the dissociation of protein from singlestranded DNA we added a 50-fold excess of unlabeled DNA to a preformed complex containing 32P-labeled DNA. The mixture was incubated at 4 ° C or 37°C and samples were filtered at various times. The rate of release of protein was measured by the decrease of label retained by the filter as a function of time (Fig. 6). A non-linear curve was obtained with a t l / z of' about 1 h at 37 "C and 5 h at 4 "C. This indicates that the complex is rather stable. The non-linearity of the dissociation curve is not easily explained. Interpretation of' the results niay be complicated by the cooperative nature of the binding, which tends to favour renewed binding of released protein to DNA molecules that are still complexes with many protein molecules. This would lead to an underestimation of the rate of release. The filter assay was also used to study binding to various synthetic polynucleotides. Single-stranded [14C]@X174DNA was first mixed with an excess of unlabeled polynucleotides followed by addition of
NaCl concentration ( M ) Fig. 5. Elfecf of ionic. strength 0 1 7 cwnp/cJ.u foi'mafior7. ''C-labelcd @XI74 DNA (0.8 pg/ml) and DNA-binding protein (9 pgjml) were mixed in 20 m M Tris-HCI (pH 8.0)/1 mM EDTAi2 rnM mercaptoethanol/5 yd, glycerol containing increasing NaCl concentrations. The samples were filtered as described in Fig. 4 except that buffer ol'corresponding ionic strength were used for washings
37OC
0 0
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1
1
I
1
2
3
4
5
Time ( h )
Fig. 6 . Di.s.soc.iution of tlw c:on7plc~.xhetitwn bindingprotein and .singlestranded D N A . A complex between I4C-labeled @XI74 D N A (125411 counts/min) and unlabeled binding protein was I'ormed by mixing protein and DNA in a ratio of 11 : 1. After 5 min at 20 - C a 50-fold excess of unlabeled @XI74 D N A was added and the mixture was incubated at 4'C or 37°C. Samples were filtered at various times as described in Fig. 4
DNA-binding protein and the solution was filtered (Table 1). A reduction in label bound to the filter was taken as an indication for binding to the synthetic polynucleotides. In this test system, poly(dT), poly(dC) and poly(dG) showed some affinity for the DNAbinding protein but poly(dA) did not, even if present in a 100-fold excess (Table 1). Among the ribohomopolymer, poly(rG) and poly(r1) competed with ['"CI@X174 DNA but poly(rA), poly(rC) and poly(rU) did not. Single-stranded fd DNA competed as effec-
Adenovirus DNA-Binding Protein
394 Table 1. Competztion of polynucleotides for the udenovirus DNAbinding protein 14C-labeled @X174 DNA (0.25 pggimi) was mixed with a 10-fold or 100-fold excess of unlabeled polynucleotide followed by addition of 2.5 pg/ml DNA-binding protein. Binding assays were performed at 20 ‘C and the solution was filtered through nitrocellulose filters as described in Methods. Without competing polymer, 51 of the input [14C]@X174 DNA was bound to the filter. This value was set at 100% to calculate the amount of competition Competing polynucleotide
Ratio of competitor to [14C]DNA
[14C]DNA bound to the filter
74 None @X174DNA fd DNA (single stranded) Adenovirus 5 DNA (double stranded) Poly(dA)
10 10
100 15 13
Poly(dT) Poly(dC) Poly(dG)
100 10 100 10 10 10
98 94 106 58 36 34
Poly(rA) Poly(rU) Poly(rC) Poly(rG) Poly(r1)
100 100 10 10 10
109 108 111 35 39
-
tively as QX174 DNA and, as expected, doublestranded adenovirus 5 DNA was inactive. Stoichiometry of the D N A . Protein Complex To determine the maximal number of protein molecules that can be bound to the single-stranded DNA we added increasing amounts of 3H-labeled DNA-binding protein to a fixed amount of I4Clabeled QX174 DNA. The mixture was centrifuged in a neutral sucrose gradient to separate bound and unbound protein (Fig. 7). Addition of small amounts of protein (0.5 - 1 pg) to 1 pg DNA leads to two peaks in the sucrose gradient (Fig. 7A,B). Almost all [I4C]DNA sediments at the position of free DNA (25 S) while most 3H-labeled protein sediments slightly ahead of this peak. The 3H peak represents the DNA . protein complex, since free protein sediments at 3.3 S (see Fig. 2). An input ratio of 0.5 w/w corresponds to about 12 protein molecules/DNA molecule. When a random chance would exist for each protein molecule to bind DNA, the 3H and 14C radioactivity would coincide. The separation of the protein . DNA complex from the majority of the DNA indicates a preferential binding of protein to those DNA molecules that are already partly covered with DNA-binding protein, suggesting a cooperative binding. The cooperative effect is not very strong, however, and disappears when more protein is added to the DNA (Fig. 7 C, D).
When protein-to-DNA ratios of 30 or higher were applied the DNA appeared to be saturated, since increasing amounts of free protein remained at the top of the gradient (Fig. 8). Moreover, the sedimentation value did not increase anymore and the H : 14C ratio in the complex peak remained constant. From the specific activities of DNA and protein we could calculate that 1 pg DNA can bind maximally 33 pg protein. Taking into account a molecular weight of 68000 for the protein monomer and 310 for an average nucleotide in QX174 DNA this corresponds to one protein molecule for 6.7 nucleotides. At saturation, the nucleoprotein sediments as a broad peak, compared to non-complexed DNA sedimented to the same position in the gradient. This broadening reflects a change in hydrodynamic properties of the DNA rather than a breakdown of the DNA, which may also lead to broadening of the peak. That the DNA remains intact during interaction with the binding protein is revealed by centrifugation of the saturated complex in a sucrose gradient containing 1 M NaCl (not shown). Extended Configuration of the Saturated Complex Fig. 7 and 8 show that the sedimentation values of the complex increase with increasing mass and reach a maximum of 67 S at saturation, where the mass of the complex is 33-fold compared to protein-free DNA. The single-stranded QX174 DNA sediments at 25 S under these ionic conditions. The value of 67 S should be compared to 114 S for the QXl74virion [26], which has only one-eighth of the mass of the saturated binding-protein . DNA complex. This difference can be ascribed to an increased frictional coefficient of the complex, which suggests an extended configuration [21]. Independent evidence that the complex is extended was obtained from electron microscopic studies. A saturated DNA . protein complex was prepared and spread in 0.13 M ammonium acetate on a 0.01 M ammonium acetate hypophase. These low-salt conditions were essential to prevent dissociation of the nucleoprotein. Glutaraldehyde fixation was not required and the complex was spread directly in the absence of formamide. Under these conditions singlestranded DNA collapses and is visualized as bushlike structures. The saturated complex is present as open circles (Fig. 9a, b). Fig. 9 d shows a mixture of single-stranded DNA and the complex. The length of the single-stranded DNA complexed with DNA-binding protein was 1.44 pm f 0.16 ( n = 57). This should be compared to the length of double-stranded sPXl74 replicativeform DNA spread under identical conditions (Fig. 9 d) which was 1.65 pm 0.04 (n = 55). Histograms of the length distribution are presented in Fig. 10.
P. C. van der Vliet, W. Keegstra, and H . S. Jansz 800 1
I
395 900
I
.1
ID
400
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200
. E E ' 3
V
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1 1500
1000
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0 Fraction number Fig. 7. Sucrose gradient centrifugation ofsingle-stranded D N A and increasing concentrations of DNA-binding protein. I4C-labeled 0x174 DNA (10 pg/ml, 2100 dis. min-' pg-') was mixed with increasing concentrations of 3H-labeled DNA-binding protein (1670 dis. rnin-l pg-') and layered on a 5 - 27 isokinetic sucrose gradient in 20 mM Tris-HC1 (pH S.O)/l mM EDTAj50 m M NaCI/S glycerol. Centrifugation was for 16 h at 24000 rev./min at 4 "C in a SW 41 rotor. The arrow indicates the position of protein-free 0 x 3 7 4 D N A as determined in a parallel experiment. R = protein-to-DNA ratio (w/w); (A) R = 0.5; (B) R = 1; (C) R = 2; (D) R = 5. 3 H (e); I4C (A)
Digestion of the Nucleoprotein Complex with SingleStrand-Specgic Nuclease from Neurospora crassa One of the possible functions of the DNA-binding protein in vivo could be the stabilization of the singlestranded DNA in replicative intermediates against nuclease digestion. To test whether such a protection could be observed in vitro, a saturated complex between ['"C] 4jX174 DNA and DNA-binding protein was prepared. To this complex 3H-labeled 4jX174 DNA was added as an internal marker and the mixture was incubated with the endonuclease from Neurospora crassa [ZS]. The digestion pattern was analyzed by sucrose gradient centrifugation in the presence of 1 M NaCl to dissociate remaining protein . DNA complexes (Fig. 11). After a 1-min incubation period [3H]DNA was partly broken down while most of the [14C]DNA remained intact (Fig. 11A). During prolonged incubation both the complex and the proteinfree DNA were further digested, although the nucleoprotein complex was slightly more resistant than the naked DNA (Fig.11B). In a control experiment without DNA-binding protein (Fig. 11C) or without nuclease (not shown) both t3H]DNA and [14C]DNA coincided completely.
2000
--
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0 Fraction number
Fig. 8. Sucrose gradient cenirifugaiion of a saturcited complex between @ X I 7 4 D N A and D N A binding protein. 14C-labeled @XI74 D N A (A) and 3H-labeled binding protein (e)were mixed in a ratio of 72 (w/w) and centrifuged in a neutral sucrose gradient as described in Fig. 7
Adenovirus DNA-Binding Protein
396
of ( I .sutuwtcd coniidc.r hctnww crtkiwoi5ir.u.s 5 Dh‘A-hiritliiifi Iwoteiii triitl @ X I 74 D h f A . A complex of DNA and Fig. 9. Elccirori iiiri~ro~rupli protein was made and prepared for electron microscopy without prior fixation as described in Methods. (a) Saturated complex; (b,d) saturated complex + OX174 single-stranded D N A ( 1 : 1); (c) @XI74 replicative form 11 D N A + @X174 single-stranded DNA (1 : 1)
The results indicate that under certain conditions the protein can protect single-stranded DNA partly, but not completely, against nuclease action.
E
1
L 1.2
1.4
1.6
Length (Fm)
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1.2
1 .4 1 .6 Length (Fm)
1.8
DISCUSSION The purified adenovirus type 5 DNA-binding protein has a relatively low sedimentation coefficient of 3.3 S and behaves as a large protein with a Stokes radius of 4.9 nm during gel filtration on Sephadex G-200. Similar results have been obtained for the closely related adenovirus 2 binding protein [20]. We have interpreted these data to mean that the DNAbinding protein has a non-spherical shape. Calculation of the molecular weight and the frictional coefficient ratio from these two parameters indicate that the binding protein is an anisometric monomer with
v/f;)
P. C. van der Vliet, W. Keegstra, and H. S. Jansz
391
Fraction number Fig. 11. Nuc/ea.Fr digestion of' the .~nturrrrrdconiplex. ['"C]Q,X174 DNA was mixed with DNA-binding protein in a ratio of 1 : 33 to obtain a saturated nucleoprotein complex. Then [3H]@X174DNA was added as an internal marker. The mixture was incubated for 1 min (A) or 15 (B) at 37 "C with 0.15 unit/ml of N . crussu endonuclease. Incubation was performed in 0.1 M Tris-HCI (pH 8.1)/12.5 mM MgClz containing 4 &ml denatured calf thymus DNA and 10 pg/ml native calf thymus DNA. The reaction was stopped by cooling and addition of 25 m M EDTA. NaCl was added to 1 M and the mixture was layered on a 5-27 yo sucrose gradient containing 20 mM Tris-HCI (pH 8.0)! 1 mM EDTA/I M NaCl and centrifuged for 16 h at 33000 rev./min at 4 ' C in a SW 41 rotor. Sedimentation is from right to left. (C) Incubation for 15 min at 37°C without DNA-binding protein. '"C (A); 3H ( 0 )
an axial ratio of about 1:10. A non-spherical shape has been found for other proteins that bind to singlestranded DNA like T4 gene-32 protein [21], E. coli DNA- binding protein [22] and DNA-dependent ATPase [27]. Implicit in our interpretation is the assumption that the DNA-binding protein does not contain carbohydrate, since glycoproteins behave aberrantly during gel filtration [25]. No glycosylation has been found in vivo for the 72000-M, DNA protein however [30]. Moreover, the 72000-M, DNA-binding protein is synthesized in a translation system in vitro which is not capable of glycosylation [31]. Whether the phosphorylation of the binding protein contributes to its aberrant shape is not clear. To obtain reproducible results measurements have been performed in high salt and under these conditions the protein behaves as a monomer. In low salt the protein aggregates, which suggests a strong protein - protein interaction. Such an interaction may also explain the cooperative nature of the binding to single-stranded DNA observed at low protein-to-DNA input ratios. Our studies on the stoichiometry of the complex show that at saturation 1 pg single-stranded DNA can bind 33 yg protein, which is more than twice the amount observed for other single-strand-specific DNAbinding proteins. Owing t o the high molecular weight of the protein monomer the calculated number of nucleotides bound by a single protein is about 7, which is within the range of 5- 10 nucleotides observed in other systems [21- 23,32,33]. The length of a stretch of 7 nucleotides (2.3 nm) is well below the observed Stokes radius of 4.9 nm. This suggests that in the saturated complex either extensive protein -protein overlap occurs or that the D N A binding site is located very asymmetrically on the protein molecule. The latter hypothesis is in agreement with the non-spherical shape of the native molecule.
Sedimentation studies as well as electron microscopy show that the adenovirus 5 DNA-binding protein holds single-stranded DNA in an extended complex. This property, together with the fast rate of complex formation, places this protein in the class of T4 gene-32 protein [21], E. coli DNA-binding protein [22], T7 DNA-binding protein [35] and the 24000 M , calf thymus DNA-binding protein [33]. The protein differs in this respect from the gene-5 protein from filamentous phages, which also binds specifically to single-stranded DNA but forms a collapsed rodlike structure [36]. The latter protein is involved in single-strand DNA synthesis rather than in DNA replication [37]. Several lines of evidence indicate that the adenovirus 5 DNA-binding protein is required for viral DNA replication. Studies on the mechanism of adenovirus DNA synthesis have shown that replication starts at one or both molecular ends and proceeds by a displacement mechanism, producing a single-stranded DNA of genome length. The displaced single strand is next converted into a duplex molecule [34]. The large amount of single-stranded DNA thus produced is presumably covered in vivo with the DNA-binding protein, since replicative intermediates isolated from infected nuclei as a D NA . protein complex contain the DNA-binding protein as a major constituent ([38] and C. Kedinger, J. Wilhelm and 0. Brison, personal communication). The protein might function to stabilize the single-stranded DNA against nucleases or to keep the DNA in a configuration which is favourable for replication by preventing the formation of extensive secondary structure. Our data show that a partial' resistance against N . crassu nuclease can be detected but at longer incubation time or higher enzyme concentration the complex is easily digested.
398
P. C. van der Vliet, W. Keegstra, and H. S. Jansz: Adenovirus DNA-Binding Protein
Although complexed protein does not exchange readily with the same DNA added to the complex (see Fig. 6) we can not exclude the possibility that gaps arise during incubation which can be attacked by the nuclease. We do not know whether the observed resistance also occurs in vivo. Studies with the mutant HSts125 have indicated that late after a shift up to the non-permissive temperature some breakdown of adenovirus DNA occurs [13], which may indicate destabilization of the single-stranded DNA in the absence of functional DNA-binding protein. Apart from possible stabilization the protein is required in initiation of viral DNA replication as well as in the elongation of nascent DNA chains [13- 151. Such multifunctional properties have also been observed for T4 gene-32 protein [39]. The mechanism of initiation and the role of the protein in this process is not well understood. No enzymatic activities have been found for the DNA-binding protein. The possibility of partial destabilization of the double helix at the origin has been raised [15]. We have tested whether the protein is able to destabilize DNA using the unwinding of poly[d(A-T)] as a model system. Measurements at various pH values (7.4 or 9.0) and various protein-to-DNA ratios (3 - 50) all gave negative results. Under these conditions we could easily detect melting of poly[d(A-T)] by bacterial DNA-binding proteins like gene-5 protein of fd phage. The absence of unwinding activity on poly[d(A-T)] might be explained by the weak interaction of the adenovirus type 5 DNA-binding protein with poly(dA) and poly(dT) (see Table 1). Another possible function, the interaction of the binding protein with other proteins required for initiation like DNA polymerases, is currently under investigation. We thank Dr P. D. Baas for a gift of @XI74 DNA and L. G . de Jong-van Dijk for expert technical assistance. Stimulating discussions with Dr J. S. Sussenbach are gratefully acknowledged. This study was supported in part by the Netherlands Foundation for Chemical Research (S.O.N.) with financial aid from the Netherlands Organization for the Advancement of Pure Research (Z.W.O.).
REFERENCES 1. van der Vliet, P. C. & Levine, A. J. (1973) Nat. New Biol. 246, 170-174. 2. Gilead, Z., Sugawara, K., Shanmugam, G. & Green, M. (1976) J . Virol. 18, 454-460. 3. Linnk, T., Jornvall, H. & Philipson, L. (1977) Eur. J. Biochem. 76, 481 -490. 4. Levinson, A. D., Postel, E. H. & Levine, A. J. (1977) Virology, 79, 144-159. 5. Jeng, Y., Wold, W. S. M., Sugawara, K., Gilead, Z. & Green, M. (1977) J . Virol. 22, 402-411.
6. Russell, W. C. & Blair, G . E. (1977) J . Gen. Virol. 34, 19-35. 7. Rosenwirth, B., Anderson,C. W. & Levine,A. J. (1976) Virology, 69, 617-625. 8. Lewis, J . B., Atkins, J. F., Baum, P. R., Solem, R., Gesteland, R. F. & Anderson, C. W. (1976) Cell, 7, 141- 151. 9. Ensinger, M. J. &Ginsberg, H. S. (1972)J. Virol. 10,328-339. 10. van der Vliet, P. C., Levine, A. J., Ensinger, M. S. & Ginsberg, H. S. (1975) J . Virol. 15, 348-354. 11. Grodzicker, T., Anderson, C. W., Sambrook, J. & Mathews, M. B. (1977) Virology, 80, 111- 126. 12. Ginsberg, H. S., Lundholm, U. & Linne, T. (1977) J . Virol. 23, 142-152. 13. van der Vliet, P. C. & Sussenbach, J. S. (1975) Virology, 67, 415-426. 14. Ginsberg, H. S., Ensinger, M. S., Kauffman, R. S., Mayer, A. J. & Lundholm, U. (1974) Cold Spring Harbor Symp. Quant. Biol. 39,419-426. 15. van der Vliet,P. C., Zandberg, J. & Jansz, H. S. (1977) Virology, 80, 98-110. 16. Applebury, M . L. & Coleman, J. E. (1969) J . B i d . Chem. 244, 308- 318. 17. Jaenicke, R., Schmid, D. & Knof, S. (1968). Biochemistry, 7 , 919-926. 18. Larsson-Raznikiewicz, M. (1970) Eur. J . Biochem. 15, 574580. 19. Siegel, L. M. & Monty, K. J. (1966) Biochim. Biophys. Acta, 112, 346-362. 20. Sugawara, K., Gilead, Z. & Green, M. (1977) J . Virol. 21, 338- 346. 21 Alberts, B. M. & Frey, L. (1970) Nature (Lond.) 227, 13131318. 22 Weiner, J. H., Bertsch, L. L. & Kornberg, A. (1975) J , Biol. Chem. 250, 1972- 1980. 23 Oey, J. L. & Knippers, R. (1972) J . Mol. B i d . 68, 125- 138. 24 Yarus, M. & Berg, P. (1967) J . Mol. B i d . 28, 479-490. 25 Andrews, P. (1970) Methods Biochem. Anal. 18, 1-49. 26 Sinsheimer, R. L. (1968) Prog. Nucleic Acid Res. Mol. Biol. 8, 115- 170. 27 Abdel-Monem, M. & Hoffmann-Berling, H. (1976) Eur. J . Biochem. 65,431 -440. 28 Linn, S . & Lehman, J. R. (1965) J . Biol. Chem. 240, 1287. 29 Haschemeyer, R . H. & Haschemeyer, A. E. V. (1973) Proteins: A Guide to Study by Physical and Chemical Methods, p. 173. Wiley, New York. 30 Ishibashi, M. & Maizel, J. V. (1974) Virology, 58, 345-361. 31 Lewis, J . B., Atkins, J. F., Baum, P. R., Solem, R., Gesteland, R. F. & Anderson, C. W. (1976) Cell, 7, 141-151. 32 Banks, G . R. & Spanos, A. (1975) J . Mol. Biol. Y3, 63-77. 33 Herrick, G . & Alberts, B. M. (1976) J . Biol. Chem. 251, 21332141. 34 Levine, A. J., van der Vliet, P. C. & Sussenbach, J. S. (1976) Curr. Top. Microbiol. Immunol. 73, 67- 124. 35. Reuben, R. C. & Gefter, M. L. (1974) J . B i d . Chem. 249, 3843 - 3850. 36. Alberts, B. M., Frey, L. & Delius, H. (1972) J. Mol. B i d . 68, 139- 152. 37. Salstrom, J. S. & Pratt, D. (1971) J . Mol. Bid. 61, 489-501. 38. Shanmugam, G., Bhaduri, S., Arens, M. & Green, M. (1975) Biochemistry, 14, 332- 337. 39. Breschkin, A. M. & Mosig, G . (1977) J . Mol. B i d . 112, 279309. 40. Kleinschmidt, A. K., Lang, D., Jackerts, D. & Zahn, R. K. (1962) Biochim. Biophys. Acta, 61, 857- 864.
P. C. van der Vliet and H. S. Jansz, Laboratorium voor Fysiologische Chemie, Rijksuniversiteit te Utrecht. Vondellaan 24a, NL-3521 GG Utrecht, The Netherlands W. Keegstra, Institut voor Moleculaire Biologie, Rijksuniversiteit te Utrecht, Transitorium 3, Universiteit’s Centrum ‘De Uithof, Pddualaan 8, NL-3584 CH Utrecht, The Netherlands
Complex Formation between the Adenovirus Type 5 DNA-Binding Protein and Single-Stranded DNA Peter C. van der VLIET, Wilko KEEGSTRA, and Hendrik S. JANSZ Laboratory for Physiological Chemistry and Institute for Molecular Biology, State University of Utrecht' (Received November 24, 1977)
The adenovirus-type-5-coded single-strand-specific DNA-binding protein was purified from infected human KB cells and characterized by sucrose gradient centrifugation and gel filtration. The protein has a sedimentation coefficient of 3.3 S and a diffusion coefficient of 4.4 x lo-' cm/s, which corresponds to a molecular weight of 68000 for the native protein. This is close to the peptide molecular weight of 72000 and indicates that the protein is a monomer. The frictional coefficient ratio is 1.82 which suggests that the DNA-binding protein is highly anisometric. The binding of the protein to various nucleic acids was studied by filtration through nitrocellulose filters and sucrose gradient centrifugation. The binding to single-stranded D N A is a f x t process which does not require bivalent cations or sulfhydryl groups and occurs over a broad pH range. The binding to synthetic polydeoxyribonucleotides is at least 10-fold less efficient than that to natural single-stranded DNA. At low protein-to-DNA ratios the binding to single-stranded DNA is cooperative. A maximal protein to DNA ratio of 33 (w/w) can be obtained which corresponds to 1 protein molecule for about 7 nucleotides. Sedimentation and electron microscopic studies indicate that the DNA-binding protein keeps single-stranded DNA in an extended configuration. The saturated nucleoprotein complex is slightly more resistant to nuclease digestion than single-stranded DNA. Human adenovirus type 5 is an oncogenic DNA virus that multiplies in the nucleus of permissive human cells. Early in infection a number of viruscoded or virus-induced proteins are synthesized, which are probably involved in the onset of adenovirus DNA replication and regulation of gene expression. One of these early proteins is a virus-coded single-strandspecific DNA-binding protein, which is synthesized in large quantities, up to 2 x lo7 molecules being present in adenovirus-2 or adenovirus-5-infected cells [1,2]. The DNA-binding protein, which has an apparent molecular weight of 72 000 in dodecylsulphate/ polyacrylamide gels, is phosphorylated [3 - 61 and can be converted into fragments of 44000-48000 M , which are presumably proteolytic breakdown products [ l , 71. Pulse-chase experiments have identified still another form of the adenovirus 2 DNA-binding protein, which has a slightly higher molecular weight [5], showing that several post-translational modifications can occur. The significance of these modifications is not clear. ~~
~~
Emyrnrs. Alkaline phosphatase (EC 3.1.3.1); catalase (EC 1.11.1.6); glyceraldehyde-3-phosphatedehydrogenase(EC 1.2.1.12); phosphoglycerate kinase (EC 2.7.2.3); ribonuclease (EC 3.3.4.22); endonuclease (EC 3.1.4.21).
The structural gene for the DNA binding protein has been mapped between 0.59 and 0.71 from the left molecular end of the DNA and a mutant in this gene, H5ts125, has been identified [8 - 111. This mutant produces a thermolabile DNA-binding protein [lo, 121 and is defective in the initiation of viral DNA replication [13,14]. Antibody against the DNA-binding protein inhibits the rate of DNA chain growth in isolated nuclei from adenovirus-infected cells [l5]. These data indicate that the DNA-binding protein is required for the first and subsequent rounds of adenovirus DNA replication and may serve during the initiation process as well as during elongation of nascent DNA chains. A function of the DNA-binding protein in viral DNA replication is also suggested by the finding that it constitutes the major protein in complexes that are capable of adenovirus DNA replication in vitro (C. Kedinger, J. Wilhelm and 0 . Brison, personal communication). We have studied some physicochemical properties of the purified DNA-binding protein and its interaction with single-stranded DNA. The experiments described here show that the protein is a monomer and does not behave as a globular protein. Its binding to single-stranded DNA is a fast, partly cooperative,
390
process which is only slowly reversible. When the DNA is saturated with protein, one protein molecule covers 7 nucleotides and forces the single-stranded DNA into an extended configuration.
Adenovirus DNA-Binding Protein
tein by dodecylsulphate/polyacrylamide electrophoresis have been described [15]. The purified protein which eluted from DNA cellulose with 1 M NaCl was stored at - 80 "C and was stable for at least 6 months. Spectral analysis showed that A280/A260 = 1.60 and the absorption coefficient E;& = 9.3.
MATERIALS AND METHODS Rabbit muscle glyceraldehyde-3-phosphate dehydrogenase, yeast 3-phosphoglycerate kinase, beef liver catalase and endonuclease from Neurospora crassa were purchased from Boehringer Mannheim. Pancreatic ribonuclease and Escherichia coli alkaline phosphatase were from Sigma chemicals. All synthetic polydeoxyribonucleotides were obtained from Miles Laboratories and all polyribonucleotides from Sigma Chemicals. [3 HILeucine (40 Ci/mmol), and [jHlthymidine (60 Ci/mmol) were from the Radiochemical Centre (Amersham, England). @X174 DNA was a kind gift from Dr P. D. Baas. Isolation of DNA-Binding Protein KB cells were grown in suspension in Eagle's minimal essential medium containing 5 calf serum. Adenovirus-5-infected cells were harvested 16- 20 h after infection when the amount of DNA-binding protein is maximal [1,2]. The cells were washed with phosphate-buffered saline and suspended in 20 mM Tris-HC1/0.01 M NaClI1.5 mM MgC12/2 mM dithiothreitol, pH 7.6, at a concentration of lo7 cells/ml. After 30 min at 0 "C the swollen cells were disrupted by freezing. In this stage the cells could be stored as a suspension at - 80 "C, for many months. All the buffers contained 2 mM phenylmethylsulfonyl fluoride and 20 pg/ml L-l-tosylamido-2-phenylethylchloromethyl ketone to inhibit proteolytic activity. For the isolation of H-labeled protein, the cells were infected in leucine-free medium to which [3H]leucine (1 pCi/ ml) was added 4 h post infection. The infection process was monitored by pulse-labeling the viral DNA with [3H]thymidine as described [13]. Frozen cells were thawed and cell dCbris was removed by centrifugation for 20 min at 10000 x g. To the supernatant EDTA was added to 10 mM, glycerol to 10% and NaCl to 0.15 M, final concentrations. The DNA-binding protein was precipitated by addition of ammonium sulfate (60 % saturation) and the precipitate was dissolved in 20 mM Tris-HC1, pH 8.1/1 mM EDTA/O.15 M NaCl/10% glycerol (buffer A) and dialysed against this buffer. A small precipitate, which formed during dialysis, was removed by centrifugation and the clear supernatant was applied to a DNA-cellulose column containing 0.8 mg single-stranded calf thymus DNA/ml. The procedures for DNA-cellulose chromatography, including elution with sodium dextrane sulfate and analysis of the pro-
Filter Binding Assay Nitrocellulose filters (Millipore HAWP, 0.45 pm) were boiled for 10 min in distilled water and soaked overnight in 20 mM Tris-HC1, pH 8.0jl mM EDTA/ 2 mM mercaptoethanol/50 mM NaC1/5 % (w/v) glycerol/l % dimethylsulfoxide (buffer B). Before use the filters were washed with 10 ml buffer B. DNA or synthetic polynucleotides were sonicated for 1 min at 50 W in a Branson sonifier before addition of protein. Routinely, DNA and protein were mixed in a total volume of 100 p1 and after 5 min at 20°C the samples were diluted with buffer B to 1 ml and filtered. The filters were washed with three portions of 3 ml buffer B. All filtrations and washings were done at a constant filtration rate of 5 ml/min, which was necessary to obtain reproducible results. When the stability of the DNA . protein complex to increasing NaCl concentrations was tested, the filters were soaked and washed with buffer B containing the indicated salt concentrations. After washing, the filters were dried, dissolved in 2 ml 1,2-dimethoxyethane and counted. Under these conditions single-stranded and doublestranded DNA pass through the filter while DNA . protein complexes are retained [24]. Electron Microscopy A saturated DNA . protein complex was prepared by mixing purified DNA-binding protein and QiX174 single-stranded DNA in a protein-to-DNA ratio of 33 (w/w). The components were mixed in bufferA containing 1 M NaCl and dialyzed against buffer A. Samples were spread according to a modified KleinSchmidt monolayer technique [40]. After dilution to 0.13 M ammonium acetate the complex was mixed with cytochrome c (0.02 % final concentration) and spread, without prior fixation, on a 0.01 M ammonium acetate hypophase without formamide. A low-salt hyperfase was required to prevent dissociation of the nucleoprotein complex. The DNA was picked up with carbon-coated grits stained with alcoholic uranyl acetate, rotary-shadowed, with platinum (7 - 10 "C) and examined in a Philips 301 electron microscope. Double-stranded @X174 replicative form DNA was treated in a similar way. Length measurements were done using a Hewlett Packard 9864 A digitizer connected to a 9820 A calculator.
P. C. van der W e t , W. Keegstrd, and H. S. Jansz
391
Sedimentation Analysis
Sedimentation of purified DNA-binding protein or DNA . protein complexes was performed in 5 27 % isokinetic sucrose gradients in 20 mM Tris-HC1 (pH S.l)/l mM EDTA/10% glycerol containing0.05 M NaCl or 1M NaCLThe nitrocellulose tubes were coated by incubation with 2 mg/ml bovine serum albumin for 30 min at 50 "C, washed with distilled water and dried at 80 "C. This prevented sticking of the protein to the wall of the tubes. Sedimentation markers were E. coli alkaline phosphatase (6.0 S [16]) glyceraldehyde-3phosphate dehydrogenase (7.7 S [17]) and 3-phosphoglycerate kinase (3.1 S [18]). Enzymatic activities were tested in appropriate samples according to the procedures described in these references. RESULTS Physicochemical Properties of the Purified DNA-Binding Protein
Selective extraction of infected KB cells followed by ammonium sulfate precipitation and DNA-cellulose chromatography provides a rapid and effective purification procedure in which a highly purified protein is obtained. Polyacrylamide gel electrophoresis in the presence of dodecylsulphate (Fig. 1) shows a predominant band at 72000 M , with some weak bands at 42 000 - 46 000 M,, presumably proteolytic products of the 72000-Mr protein [6]. As estimated from the radioactivity in gel slices 92% of the label is found in the DNA-binding protein region of the gel. Sedimentation of the native protein at high ionic strength (1 M NaC1) in the presence of marker enzymes showed a homogeneous peak at 3.3 S (Fig.2). This value did not change when the centrifugation was performed at 0.15 M NaCl at low concentrations (less than 20 pg/ml). At high concentrations (100 pg/ml or more), however, the protein aggregates in low-ionicstrength buffers. This aggregation is pH dependent. When H-labeled DNA-binding protein (100 pg/ml) was dialyzed against buffers of low ionic strength ( I = 0.06 M) with pH of 6.8,S.l or 9.0 and centrifuged for 20 min at 2000 rev./min, the pellet contained 83 %, 16% and 7 % of the radioactivity respectively. The pellets could be easily redissolved in a buffer containing 1 M NaCl. A value of 3.3 S is rather low for a protein of M , 72000. For globular proteins, 3.3 S would correspond to a molecular weight of about 40000. We considered the possibility that the protein was degraded to its 42000 - 48 000 proteolytic products during the sedimentation, giving rise to the low sedimentation value. However, when the 3.3-S peak obtained after sucrose gradient centrifugation was analyzed by electrophoresis, 80% of the protein was still present at the 72000-M, position in the gel.
Slice number Fig. 1. Dodecylsulphate gel electrophoresis of the DNA-binding protein. [3H]Leucine-labeied protein (10 pg) obtained after DNAcellulose chromatography was electrophoresed in 10 % polyacrylamide gels containing dodecylsulphate and stained with Coomassie brilliant blue. After destaining the gel was fractionated into 2-mm slices and the radioactivity was measured. The main peak had an apparent molecular weight of 72000 as determined from marker proteins run in parallel gels
73
0.4 &
400
0
I
m
m
T
0.2
200
-0
n
0 20 10 Fraction number
30
Fig. 2. Sucrose grudient sedimentution of the DNA-binding protein. [3 HILeucine-labeled DNA-binding protein (2,2 pg, 8250 dis./min) was layered on a 5 - 27 isokinetic sucrose gradient in 20 mM TrisHC1 (pH 8.1)/1 M NaCl/I mM EDTA/10% glycerol. E . colt alkaline phosphatase (6.0 S) and 3-phosphoglycerate kinase (3.1 S) were added as sedimentation markers. Centrifugation was for 24 h at 50000 rev./min at 4°C in an SW 65 rotor. The recovery was 76%. Control experiments showed that the presence of these marker enzymes did not influence the sedimentation of the protein. 3H ( 0 ) ;alkaline phosphatase activity, A400 (0);3-phosphoglycerdte kinase activity, A340 (A)
Adenovirus DNA-Binding Protein
392
0
3000
-
-
C ._
-5 2000 L,j .-
U
v
I
c9
1000
-
40
60
50
t
t
“0
“i
Fraction
number
Fig. 3. Gel filtration of the DNA-binding protein on u Sephuder- G-200 c.ol~orr/r.-’H-labeled binding protein (35 pg, 700000 dis./min was chromatographed on a 100 x 1.2-cm G-200 column in 20 m M Tris-HC1 (pH 8.1)/1 mM EDTAI0.5 M NaC1/10% glycerol. [‘4C]Leucine was added to determine V , and dextrane blue to measure Vo. Fractions of 1.9 ml were collected and appropriate samples were counted. The recovery of the protein was 55 %. In separate experiments the elution volume V , of inarker enzymes was determined and V,/ VOwas plotted against the diffusion coefficients D (see inset). G A P D H = glyceraldehyde-3-phosphate dehydrogenase
To obtain more information about the molecular weight and the shape of the native protein, the diffusion coefficient was determined by gel filtration on Sephadex G-200. At low ionic strength, even in the presence of 0.05 % Triton X-100 as detergent, the yields were very low and the results were irreproducible, presumably because the protein tends to aggregate. At high ionic strength a symmetrical elution profile was reproducibly obtained (Fig. 3). The DNA-binding protein eluted between the position of catalase (Dzo, = 4.17 x cmjs) and glyceraldehyde-3-phosphate dehydrogenase (Dzo,w = 5.0 x cmjs). A straight line was obtained for the marker proteins when V,/Vo was plotted against DZO, according to Andrews [25], and D20,w= 4 . 4 ~ cmjs was found by interpolation for the DNA-binding protein (Fig. 3). From the sedimentation coefficient and the diffusion coefficient the molecular weight of the native protein can be calculated using the Svedberg formula : M , = RTs/D(l - vze) = 68000, where M I = molecular weight, T = absolute temperature, vz = partial specific volume, Q = density of water at 20 “C. A value of 0.73 mljg was calculated for uz from the partial specific volumes of the amino acid residues and the amino acid composition [3]. From the diffusion coefficient the Stokes radius a = 4.9 nm was calculated. A similar result was obtained when the gel filtration data were plotted ac-
cording to Siege1 and Monty [19]. The frictional coefficient ratio f/ji was determined by substitution =~ 1.82 )~’~ in the classical equationfro = a/(3 O ~ M , / ~ N where M I is the calculated molecular weight of 68 000, a is the Stokes radius and NA is Avogadro’s number. Assuming 0.2 g HzO of hydration/g protein this corresponds, with a prolate ellipsoid, to an axial ratio of about 1 : 10 [29]. These data indicate that the protein is a highly anisometric monomer and does not behave as an typical globular protein.
Binding
of
the Protein to DNA
We used two methods to study the interaction between the DNA binding protein and various polynucleotides, i.e. sucrose gradient centrifugation and a filter-binding assay. For the filter-binding test [24] DNA and protein were mixed and filtered on nitrocellulose filters. The complex is retained while free DNA passes the filter readily. Fig. 4 shows the binding to single-stranded @XI74DNA. The binding increases continuously and about 30 times the amount of protein (on a weight basis) is required to retain all DNA on the filter. No binding to double-stranded adenovirus 5 DNA is observed, in agreement with previous results [l]. Binding to single-stranded DNA proceeds very rapidly. At 4°C the binding process was complete
P. C. van der Vliet, W. Keegstra, and H. S. Jansz
393
100
80
-
c 1
80
." 60 3
n
40
20
0
_ _ _ _ __-- - - ---20
I
I
40 50 Protein - t o - D N A ratio Fig. 4. Coniplex fornzation hetween DNA-binding pi'ofein and .singlestranded DNA. A constant amount of I4C-labeled single-stranded a x 1 7 4 DNA (0.15 pg, 7940 counts/min) (O---O) or 3ZP-labeled double-stranded adenovirus type 5 DNA (0.22 big, 492 countsimin. 0 --0) was mixed with increasing concentrations of binding protein and filtered on nitrocellulose filters. The percentage of input DNA retained on the filter was determined as described in Methods 10
30
within 10 s. This is comparable to results obtained with E. coli DNA-binding protein [22] and unlike that found with the fd gene-5 protein where binding requires 3-5 min [23].Binding occurred in a broad range of pH values (pH 4- 10). Addition of 5 mM EDTA or 10 mM N-ethylmaleimide did not prevent complex formation, indicating that bivalent cations or sulf'hydryl groups are not required. As expected, increasing the NaCl concentration results in a reduction with 50 % binding at 0.32 M NaCl (Fig. 5). Once formed, the complex appeared to be rather stable. To study the dissociation of protein from singlestranded DNA we added a 50-fold excess of unlabeled DNA to a preformed complex containing 32P-labeled DNA. The mixture was incubated at 4 ° C or 37°C and samples were filtered at various times. The rate of release of protein was measured by the decrease of label retained by the filter as a function of time (Fig. 6). A non-linear curve was obtained with a t l / z of' about 1 h at 37 "C and 5 h at 4 "C. This indicates that the complex is rather stable. The non-linearity of the dissociation curve is not easily explained. Interpretation of' the results niay be complicated by the cooperative nature of the binding, which tends to favour renewed binding of released protein to DNA molecules that are still complexes with many protein molecules. This would lead to an underestimation of the rate of release. The filter assay was also used to study binding to various synthetic polynucleotides. Single-stranded [14C]@X174DNA was first mixed with an excess of unlabeled polynucleotides followed by addition of
NaCl concentration ( M ) Fig. 5. Elfecf of ionic. strength 0 1 7 cwnp/cJ.u foi'mafior7. ''C-labelcd @XI74 DNA (0.8 pg/ml) and DNA-binding protein (9 pgjml) were mixed in 20 m M Tris-HCI (pH 8.0)/1 mM EDTAi2 rnM mercaptoethanol/5 yd, glycerol containing increasing NaCl concentrations. The samples were filtered as described in Fig. 4 except that buffer ol'corresponding ionic strength were used for washings
37OC
0 0
I
I
1
1
I
1
2
3
4
5
Time ( h )
Fig. 6 . Di.s.soc.iution of tlw c:on7plc~.xhetitwn bindingprotein and .singlestranded D N A . A complex between I4C-labeled @XI74 D N A (125411 counts/min) and unlabeled binding protein was I'ormed by mixing protein and DNA in a ratio of 11 : 1. After 5 min at 20 - C a 50-fold excess of unlabeled @XI74 D N A was added and the mixture was incubated at 4'C or 37°C. Samples were filtered at various times as described in Fig. 4
DNA-binding protein and the solution was filtered (Table 1). A reduction in label bound to the filter was taken as an indication for binding to the synthetic polynucleotides. In this test system, poly(dT), poly(dC) and poly(dG) showed some affinity for the DNAbinding protein but poly(dA) did not, even if present in a 100-fold excess (Table 1). Among the ribohomopolymer, poly(rG) and poly(r1) competed with ['"CI@X174 DNA but poly(rA), poly(rC) and poly(rU) did not. Single-stranded fd DNA competed as effec-
Adenovirus DNA-Binding Protein
394 Table 1. Competztion of polynucleotides for the udenovirus DNAbinding protein 14C-labeled @X174 DNA (0.25 pggimi) was mixed with a 10-fold or 100-fold excess of unlabeled polynucleotide followed by addition of 2.5 pg/ml DNA-binding protein. Binding assays were performed at 20 ‘C and the solution was filtered through nitrocellulose filters as described in Methods. Without competing polymer, 51 of the input [14C]@X174 DNA was bound to the filter. This value was set at 100% to calculate the amount of competition Competing polynucleotide
Ratio of competitor to [14C]DNA
[14C]DNA bound to the filter
74 None @X174DNA fd DNA (single stranded) Adenovirus 5 DNA (double stranded) Poly(dA)
10 10
100 15 13
Poly(dT) Poly(dC) Poly(dG)
100 10 100 10 10 10
98 94 106 58 36 34
Poly(rA) Poly(rU) Poly(rC) Poly(rG) Poly(r1)
100 100 10 10 10
109 108 111 35 39
-
tively as QX174 DNA and, as expected, doublestranded adenovirus 5 DNA was inactive. Stoichiometry of the D N A . Protein Complex To determine the maximal number of protein molecules that can be bound to the single-stranded DNA we added increasing amounts of 3H-labeled DNA-binding protein to a fixed amount of I4Clabeled QX174 DNA. The mixture was centrifuged in a neutral sucrose gradient to separate bound and unbound protein (Fig. 7). Addition of small amounts of protein (0.5 - 1 pg) to 1 pg DNA leads to two peaks in the sucrose gradient (Fig. 7A,B). Almost all [I4C]DNA sediments at the position of free DNA (25 S) while most 3H-labeled protein sediments slightly ahead of this peak. The 3H peak represents the DNA . protein complex, since free protein sediments at 3.3 S (see Fig. 2). An input ratio of 0.5 w/w corresponds to about 12 protein molecules/DNA molecule. When a random chance would exist for each protein molecule to bind DNA, the 3H and 14C radioactivity would coincide. The separation of the protein . DNA complex from the majority of the DNA indicates a preferential binding of protein to those DNA molecules that are already partly covered with DNA-binding protein, suggesting a cooperative binding. The cooperative effect is not very strong, however, and disappears when more protein is added to the DNA (Fig. 7 C, D).
When protein-to-DNA ratios of 30 or higher were applied the DNA appeared to be saturated, since increasing amounts of free protein remained at the top of the gradient (Fig. 8). Moreover, the sedimentation value did not increase anymore and the H : 14C ratio in the complex peak remained constant. From the specific activities of DNA and protein we could calculate that 1 pg DNA can bind maximally 33 pg protein. Taking into account a molecular weight of 68000 for the protein monomer and 310 for an average nucleotide in QX174 DNA this corresponds to one protein molecule for 6.7 nucleotides. At saturation, the nucleoprotein sediments as a broad peak, compared to non-complexed DNA sedimented to the same position in the gradient. This broadening reflects a change in hydrodynamic properties of the DNA rather than a breakdown of the DNA, which may also lead to broadening of the peak. That the DNA remains intact during interaction with the binding protein is revealed by centrifugation of the saturated complex in a sucrose gradient containing 1 M NaCl (not shown). Extended Configuration of the Saturated Complex Fig. 7 and 8 show that the sedimentation values of the complex increase with increasing mass and reach a maximum of 67 S at saturation, where the mass of the complex is 33-fold compared to protein-free DNA. The single-stranded QX174 DNA sediments at 25 S under these ionic conditions. The value of 67 S should be compared to 114 S for the QXl74virion [26], which has only one-eighth of the mass of the saturated binding-protein . DNA complex. This difference can be ascribed to an increased frictional coefficient of the complex, which suggests an extended configuration [21]. Independent evidence that the complex is extended was obtained from electron microscopic studies. A saturated DNA . protein complex was prepared and spread in 0.13 M ammonium acetate on a 0.01 M ammonium acetate hypophase. These low-salt conditions were essential to prevent dissociation of the nucleoprotein. Glutaraldehyde fixation was not required and the complex was spread directly in the absence of formamide. Under these conditions singlestranded DNA collapses and is visualized as bushlike structures. The saturated complex is present as open circles (Fig. 9a, b). Fig. 9 d shows a mixture of single-stranded DNA and the complex. The length of the single-stranded DNA complexed with DNA-binding protein was 1.44 pm f 0.16 ( n = 57). This should be compared to the length of double-stranded sPXl74 replicativeform DNA spread under identical conditions (Fig. 9 d) which was 1.65 pm 0.04 (n = 55). Histograms of the length distribution are presented in Fig. 10.
P. C. van der Vliet, W. Keegstra, and H . S. Jansz 800 1
I
395 900
I
.1
ID
400
600
400
200
200
. E E ' 3
V
1
m
0
0
I c
,1
I D
1 1500
1000
500
0 Fraction number Fig. 7. Sucrose gradient centrifugation ofsingle-stranded D N A and increasing concentrations of DNA-binding protein. I4C-labeled 0x174 DNA (10 pg/ml, 2100 dis. min-' pg-') was mixed with increasing concentrations of 3H-labeled DNA-binding protein (1670 dis. rnin-l pg-') and layered on a 5 - 27 isokinetic sucrose gradient in 20 mM Tris-HC1 (pH S.O)/l mM EDTAj50 m M NaCI/S glycerol. Centrifugation was for 16 h at 24000 rev./min at 4 "C in a SW 41 rotor. The arrow indicates the position of protein-free 0 x 3 7 4 D N A as determined in a parallel experiment. R = protein-to-DNA ratio (w/w); (A) R = 0.5; (B) R = 1; (C) R = 2; (D) R = 5. 3 H (e); I4C (A)
Digestion of the Nucleoprotein Complex with SingleStrand-Specgic Nuclease from Neurospora crassa One of the possible functions of the DNA-binding protein in vivo could be the stabilization of the singlestranded DNA in replicative intermediates against nuclease digestion. To test whether such a protection could be observed in vitro, a saturated complex between ['"C] 4jX174 DNA and DNA-binding protein was prepared. To this complex 3H-labeled 4jX174 DNA was added as an internal marker and the mixture was incubated with the endonuclease from Neurospora crassa [ZS]. The digestion pattern was analyzed by sucrose gradient centrifugation in the presence of 1 M NaCl to dissociate remaining protein . DNA complexes (Fig. 11). After a 1-min incubation period [3H]DNA was partly broken down while most of the [14C]DNA remained intact (Fig. 11A). During prolonged incubation both the complex and the proteinfree DNA were further digested, although the nucleoprotein complex was slightly more resistant than the naked DNA (Fig.11B). In a control experiment without DNA-binding protein (Fig. 11C) or without nuclease (not shown) both t3H]DNA and [14C]DNA coincided completely.
2000
--
1600
. c
E
:1200
v
mI L
0
zv 800
400
0 Fraction number
Fig. 8. Sucrose gradient cenirifugaiion of a saturcited complex between @ X I 7 4 D N A and D N A binding protein. 14C-labeled @XI74 D N A (A) and 3H-labeled binding protein (e)were mixed in a ratio of 72 (w/w) and centrifuged in a neutral sucrose gradient as described in Fig. 7
Adenovirus DNA-Binding Protein
396
of ( I .sutuwtcd coniidc.r hctnww crtkiwoi5ir.u.s 5 Dh‘A-hiritliiifi Iwoteiii triitl @ X I 74 D h f A . A complex of DNA and Fig. 9. Elccirori iiiri~ro~rupli protein was made and prepared for electron microscopy without prior fixation as described in Methods. (a) Saturated complex; (b,d) saturated complex + OX174 single-stranded D N A ( 1 : 1); (c) @XI74 replicative form 11 D N A + @X174 single-stranded DNA (1 : 1)
The results indicate that under certain conditions the protein can protect single-stranded DNA partly, but not completely, against nuclease action.
E
1
L 1.2
1.4
1.6
Length (Fm)
1.8
1.2
1 .4 1 .6 Length (Fm)
1.8
DISCUSSION The purified adenovirus type 5 DNA-binding protein has a relatively low sedimentation coefficient of 3.3 S and behaves as a large protein with a Stokes radius of 4.9 nm during gel filtration on Sephadex G-200. Similar results have been obtained for the closely related adenovirus 2 binding protein [20]. We have interpreted these data to mean that the DNAbinding protein has a non-spherical shape. Calculation of the molecular weight and the frictional coefficient ratio from these two parameters indicate that the binding protein is an anisometric monomer with
v/f;)
P. C. van der Vliet, W. Keegstra, and H. S. Jansz
391
Fraction number Fig. 11. Nuc/ea.Fr digestion of' the .~nturrrrrdconiplex. ['"C]Q,X174 DNA was mixed with DNA-binding protein in a ratio of 1 : 33 to obtain a saturated nucleoprotein complex. Then [3H]@X174DNA was added as an internal marker. The mixture was incubated for 1 min (A) or 15 (B) at 37 "C with 0.15 unit/ml of N . crussu endonuclease. Incubation was performed in 0.1 M Tris-HCI (pH 8.1)/12.5 mM MgClz containing 4 &ml denatured calf thymus DNA and 10 pg/ml native calf thymus DNA. The reaction was stopped by cooling and addition of 25 m M EDTA. NaCl was added to 1 M and the mixture was layered on a 5-27 yo sucrose gradient containing 20 mM Tris-HCI (pH 8.0)! 1 mM EDTA/I M NaCl and centrifuged for 16 h at 33000 rev./min at 4 ' C in a SW 41 rotor. Sedimentation is from right to left. (C) Incubation for 15 min at 37°C without DNA-binding protein. '"C (A); 3H ( 0 )
an axial ratio of about 1:10. A non-spherical shape has been found for other proteins that bind to singlestranded DNA like T4 gene-32 protein [21], E. coli DNA- binding protein [22] and DNA-dependent ATPase [27]. Implicit in our interpretation is the assumption that the DNA-binding protein does not contain carbohydrate, since glycoproteins behave aberrantly during gel filtration [25]. No glycosylation has been found in vivo for the 72000-M, DNA protein however [30]. Moreover, the 72000-M, DNA-binding protein is synthesized in a translation system in vitro which is not capable of glycosylation [31]. Whether the phosphorylation of the binding protein contributes to its aberrant shape is not clear. To obtain reproducible results measurements have been performed in high salt and under these conditions the protein behaves as a monomer. In low salt the protein aggregates, which suggests a strong protein - protein interaction. Such an interaction may also explain the cooperative nature of the binding to single-stranded DNA observed at low protein-to-DNA input ratios. Our studies on the stoichiometry of the complex show that at saturation 1 pg single-stranded DNA can bind 33 yg protein, which is more than twice the amount observed for other single-strand-specific DNAbinding proteins. Owing t o the high molecular weight of the protein monomer the calculated number of nucleotides bound by a single protein is about 7, which is within the range of 5- 10 nucleotides observed in other systems [21- 23,32,33]. The length of a stretch of 7 nucleotides (2.3 nm) is well below the observed Stokes radius of 4.9 nm. This suggests that in the saturated complex either extensive protein -protein overlap occurs or that the D N A binding site is located very asymmetrically on the protein molecule. The latter hypothesis is in agreement with the non-spherical shape of the native molecule.
Sedimentation studies as well as electron microscopy show that the adenovirus 5 DNA-binding protein holds single-stranded DNA in an extended complex. This property, together with the fast rate of complex formation, places this protein in the class of T4 gene-32 protein [21], E. coli DNA-binding protein [22], T7 DNA-binding protein [35] and the 24000 M , calf thymus DNA-binding protein [33]. The protein differs in this respect from the gene-5 protein from filamentous phages, which also binds specifically to single-stranded DNA but forms a collapsed rodlike structure [36]. The latter protein is involved in single-strand DNA synthesis rather than in DNA replication [37]. Several lines of evidence indicate that the adenovirus 5 DNA-binding protein is required for viral DNA replication. Studies on the mechanism of adenovirus DNA synthesis have shown that replication starts at one or both molecular ends and proceeds by a displacement mechanism, producing a single-stranded DNA of genome length. The displaced single strand is next converted into a duplex molecule [34]. The large amount of single-stranded DNA thus produced is presumably covered in vivo with the DNA-binding protein, since replicative intermediates isolated from infected nuclei as a D NA . protein complex contain the DNA-binding protein as a major constituent ([38] and C. Kedinger, J. Wilhelm and 0. Brison, personal communication). The protein might function to stabilize the single-stranded DNA against nucleases or to keep the DNA in a configuration which is favourable for replication by preventing the formation of extensive secondary structure. Our data show that a partial' resistance against N . crassu nuclease can be detected but at longer incubation time or higher enzyme concentration the complex is easily digested.
398
P. C. van der Vliet, W. Keegstra, and H. S. Jansz: Adenovirus DNA-Binding Protein
Although complexed protein does not exchange readily with the same DNA added to the complex (see Fig. 6) we can not exclude the possibility that gaps arise during incubation which can be attacked by the nuclease. We do not know whether the observed resistance also occurs in vivo. Studies with the mutant HSts125 have indicated that late after a shift up to the non-permissive temperature some breakdown of adenovirus DNA occurs [13], which may indicate destabilization of the single-stranded DNA in the absence of functional DNA-binding protein. Apart from possible stabilization the protein is required in initiation of viral DNA replication as well as in the elongation of nascent DNA chains [13- 151. Such multifunctional properties have also been observed for T4 gene-32 protein [39]. The mechanism of initiation and the role of the protein in this process is not well understood. No enzymatic activities have been found for the DNA-binding protein. The possibility of partial destabilization of the double helix at the origin has been raised [15]. We have tested whether the protein is able to destabilize DNA using the unwinding of poly[d(A-T)] as a model system. Measurements at various pH values (7.4 or 9.0) and various protein-to-DNA ratios (3 - 50) all gave negative results. Under these conditions we could easily detect melting of poly[d(A-T)] by bacterial DNA-binding proteins like gene-5 protein of fd phage. The absence of unwinding activity on poly[d(A-T)] might be explained by the weak interaction of the adenovirus type 5 DNA-binding protein with poly(dA) and poly(dT) (see Table 1). Another possible function, the interaction of the binding protein with other proteins required for initiation like DNA polymerases, is currently under investigation. We thank Dr P. D. Baas for a gift of @XI74 DNA and L. G . de Jong-van Dijk for expert technical assistance. Stimulating discussions with Dr J. S. Sussenbach are gratefully acknowledged. This study was supported in part by the Netherlands Foundation for Chemical Research (S.O.N.) with financial aid from the Netherlands Organization for the Advancement of Pure Research (Z.W.O.).
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P. C. van der Vliet and H. S. Jansz, Laboratorium voor Fysiologische Chemie, Rijksuniversiteit te Utrecht. Vondellaan 24a, NL-3521 GG Utrecht, The Netherlands W. Keegstra, Institut voor Moleculaire Biologie, Rijksuniversiteit te Utrecht, Transitorium 3, Universiteit’s Centrum ‘De Uithof, Pddualaan 8, NL-3584 CH Utrecht, The Netherlands
