VIROLOGY
69, 143-147 (1976)
Interaction
of Vaccinia BARRY
Department
of Molecular,
Cellular
DNA-binding
POLISKY’
AND
Proteins JOSEPH
and Developmental Biology, 8a302
with DNA in Vitro
KATES2
University
of Colorado,
Boulder,
Colorado
Accepted July 30,1975 The interaction between the components of the vaccinia DNA-protein complex isolated from infected cells has been studied. The rapid sedimentation of complex-associated DNA is destroyed by the addition of 2 M NaCl. However, the DNA is restored to a rapidly sedimenting state upon removal of the salt. This reconstitution is Pronase sensitive, indicating that conversion requires complex-associated protein. It was demonstrated that competitor DNA but not RNA from eucaryotic or bacterial sources inhibited restoration of rapid sedimentation of endogenous complex-associated DNA. The results suggest that complex-associated proteins responsible for DNA complexing are not specific for viral DNA. INTRODUCTION
Eucaryotic chromosomes contain protein molecules that presumably function to structurally organize long DNA molecules into configurations compatible with transcription, replication, and cell division. An understanding of the interaction between these structural elements and DNA has been hampered by the size and sequence heterogeneity of chromosomal DNA as well as the difficulty in determining which of the many proteins in isolated chromatin are native and which are artifacts of preparative methods. These difficulties have motivated an analysis of a viral DNA-protein complex that resembles a eucaryotic chromosome in many respects. Such a complex exists transiently in vaccinia-infected cells (Cairns, 1960; Joklik and Becker, 1964). Newly replicated vaccinia DNA can be isolated from cytoplasmic fractions of infected cells as a rapidly sedimenting DNA-protein complex capable of transcription of viral-specific mRNA in vitro (Dahl and Kates, 1970). The protein components of
11794. 143 0 1976 by Academic Press, Inc. of reproduction in any form reserved.
MATERIALS
AND
METHODS
Cells and virus. HeLa S3 cells were grown in suspension culture in Eagle’s minimal essential medium (Joklik’s modification, Grand Island Biological Company, Grand Island, N.Y.) supplemented with 5% calf serum. Vaccinia virus strain
I Present address: Department of Biochemistry and Biophysics, University of California Medical School, San Francisco, Calif. 94122. * Present address: Department of Microbiology, State University of New York, Stony Brook, N. Y.
Copyright All rights
the complex are limited in heterogeneity consisting primarily of two viral-specific, DNA-binding polypeptides designated A and B (Polisky and Kates, 1972; Sarov and Joklik, 1973). Host polypeptides appear to play no role in the structure of the complex (Polisky and Kates, 1975). Since treatment of the complex with Pronase or ionic detergents destroys the rapid sedimentation of complex-associated DNA, it has been inferred that associated protein plays a structural role in noncovalently complexing DNA molecules (Polisky and Kates, 1972). To gain insight into the basis and consequences of the interaction between complex-associated DNA and protein, we have developed procedures to dissociate and reconstitute the complex in vitro. Reconstitution experiments with exogenous, competing nucleic acids suggest that complex-associated proteins can bind to heterologous native or denatured DNA, but not virusspecific RNA.
144
POLISKY
WR was grown in HeLa cells and purified as described by Becker and Joklik (1964). In vitro reconstitution of the DNA-protein complex. The complex was prepared from infected cell cytoplasm by using 0.5% NP-40 (Nonidet P-40) as described previously (Polisky and Kates, 1975). Isolated complex in 0.01 M Tris-HCl (tris(hydroxymethyl)aminomethane), pH 8.0, 0.005 M EDTA (ethylenediaminetetraacetic acid), and 0.05 M NaCl was dissociated by the addition of solid NaCl to a final concentration of 2 M. As the salt dissolved, a large increase in the viscosity of the solution was apparent. To reduce the viscosity, the DNA was sheared by homogenization in a glass Dounce tissue homogenizer with a tight-fitting B pestle. After incubation for 10 min at 37”, the dissociated complex was centrifuged in an International B-20 centrifuge at 15,000 rpm for 15 min at 5”. This step removed complex-associated material that was insoluble in 2 M NaCl and any nondissociated complex. Usually 20-30% of the 13Hlthymidine-labeled viral DNA originally present in the isolated complex was insoluble under these conditions. To achieve reconstitution, the supernatant fluid was dialyzed overnight against 2 liters of 0.01 M Tris-HCl, pH 8.0, 0.05 M NaCl, 0.001 M EDTA. The dialysis was usually carried out at 5” but could also be done at room temperature with no change in the results. The amount of reconstitution was determined by centrifuging the dialysate for 15 min at 15,000 rpm at 5” in the International B-20 and measuring the amount of labeled viral DNA in the pelleted material. In reconstitution experiments involving addition of exogenous competing DNA’s, these DNA’s were added after addition of 2 M NaCl and prior to removal of insoluble material. Enzymatic digestions of the complex. (a) Pronase was obtained from Sigma. The enzyme (5 mg/ml) was heated at 80” in 0.05 M sodium acetate, pH 5.0, 0.005 M CaC12, 0.001 M P-mercaptoethanol for 20 min prior to use. The complex was treated with 100 pg of enzyme/ml for 1 hr at 37”. (b) Sl nuclease, purified by the method of Vogt (1973), was a gift of Gary Stormo. The
AND
KATE.3
complex was incubated with 1 unit/ml of Sl in 0.2 M sodium acetate, pH 4.7, 0.001 M ZnClz, 0.3 M NaCl for 30 min at 37”. At 5-min intervals, aliquots were removed and acid-insoluble radioactivity measured. Exogenous nucleic acids. Vaccinia, Escherichia coli, and HeLa DNA’s were purified essentially by the method of Marmur (1961). Vaccinia cores and core RNA synthesis was carried out as described by Kates and Beeson (1970). RESULTS
In Vitro Reconstitution tein Complex
of the DNA-Pro-
Experiments described previously suggested that the major complex-associated polypeptides, A and B, were apparently nonspecific DNA-binding proteins (Polisky and Kates, 1972). However, it remained possible that these proteins could exhibit preferential binding to vaccinia DNA, relative to other DNA’s presented simultaneously. To perform these experiments, an in vitro reconstitution system was developed. The assay for dissociation and reconstitution exploited the observation that isolated complex could be dissociated into component DNA and protein moieties by an increase in ionic strength. In high-salt sucrose gradients, it is possible to separate 90% of the protein from the DNA moiety (Fig. lb). Fig. la shows that both DNA and protein in isolated complex resediment to the bottom of the tube under low-salt conditions. When solutions containing dissociated complex in high salt were dialyzed to low ionic strength, over 90% of the DNA was restored to a rapid-sedimenting condition (Fig. 2a). Figure 2b shows that Pronase treatment eliminates the rapid sedimentation of the reconstituted complex, demonstrating that protein is required for conversion of DNA to a rapidly sedimenting state. The pronase used in this experiment contained no detectable DNase activity (data not shown). Reconstitution with Homologous erologous Components
and Het-
If the proteins responsible for maintaining complex-associated DNA in a rapid-
VACCINIA
5
10 FRACTION
DNA-BINDING
15
FIG. 1. Sedimentation of isolated 13H]leucine-, [“C]thymidine-labeled complex on low- and highsalt sucrose gradients. Double-labeled complex was prepared as described in Materials and Methods. The complex was then divided into two parts. One part was made 2 M NaCl, by the addition of solid NaCl. The remainder was made 0.05 M NaCl, and layered on a 38-50% sucrose gradient containing 0.05 M NaCl. The high-salt-containing sample was layered on an identical gradient containing 2 M NaCl. Both gradients were spun in the SW 27 rotor, 20,000 rpm, 17 hr, at 5”. Following centrifugation, fractions were collected and acid insoluble radioactivity measured. (a), Low-salt gradient; (b), highsalt gradient. Closed circles, [“Clthymidine counts per minute; open circles, ISH]leucine counts per minute. Sedimentation was from right to left.
sedimenting state were indeed dissociated from the DNA in high salt, then addition of purified vaccinia DNA (phenol extracted from the complex) might be expected to result in a replacement of the endogenous DNA with exogenous, homologous DNA. When this experiment was performed, it was found that addition of increasing amounts of vaccinia DNA to dissociated complex resulted in an inhibition ofreconstitution. Figure 3 shows that addition of 0.4 endogenous equivalents of unlabeled, purified vaccinia DNA, i.e., 40% of the amount of endogenous DNA, results in an 80% inhibition of entry of endogenous DNA into the reconstituted complex. Heterologous DNA’s were then tested for their ability to inhibit complex formation under these conditions. By comparing the amounts of competing DNA necessary to inhibit reconstitution, an indication of the relative “affinity” of these DNA’s for complex-associated protein was estab-
145
PROTEINS
=‘,
5
10 FRACTION
15
FIG. 2. Restoration of dissociated complex-associated DNA to a fast-sedimenting form after reconstitution. Isolated [JH]leucine, [14Clthymine-labeled complex was prepared. The complex was resuspended in 4 ml of 0.01 M Tris-HCl, pH 8.0, 0.05 M NaCl, 0.001 M EDTA, and solid NaCl was added to make the final NaCl concentration 2 M. The viscous solution was homogenized in a Dounce homogenizer and then centrifuged at 15,000 rpm for 15 min. The supernatant fluid was dialyzed overnight against the above buffer, except that the NaCl concentration was 0.05 M. The dialysate was removed and divided into two equal parts. One part was treated with 200 pg/ml heat-treated Pronase. The other part was untreated. Both samples were layered on separate 38 50% (w/v) sucrose gradients containing a 5-ml 84% (w/v) sucrose cushion. The gradients were centrifuged at 20,000 rpm for 30 min at 5”. Fractions were collected and acid-insoluble radioactivity was measured: (a), Untreated reconstituted complex; (b), Pronase-treated reconstituted complex. Closed circles, [‘Clthymidine counts; open circles, [JH]leucine counts. Sedimentation was from right to left.
lished. Unlabeled native DNA from E. coli and HeLa cells was used in these competition experiments. The results (Fig. 3) show that both DNA preparations inhibit reconstitution at identical concentrations relative to that of endogenous complex DNA. The results suggest that complexing proteins cannot distinguish between native DNAs. It was possible that complexing proteins could bind but not crosslink heterologous DNA. To test this possibility, 3H-labeled HeLa DNA was added to dissociated 14Clabeled complex at various concentrations and the amount of labeled reconstituted DNA measured. Figure 4 shows that at subinhibiting concentrations of HeLa
146
POLISKY AND KATES
DNA a heterologous, rapidly sedimenting complex containing both vaccinia and HeLa DNA can be formed. This result demonstrates that complex-associated proteins can complex heterologous DNA. To determine if RNA could inhibit complex formation in vitro, salt-dissociated complex was incubated with increasing amounts of RNA trasncribed in. vitro by vaccinia cores. Figure 4 shows that RNA is not inhibitory at the concentrations tested. Denatured DNA is about 50% more effrcient as an inhibitor of reconstitution than EXOG. DNA EQ”l”A:ENTS native DNA (unpublished experiment). To FIG. 3. Effect of double-stranded exogenous determine whether the complex contained DNA’s on reconstitution of endogenous VH]thymiDNA, t3HlTdR-labeled single-stranded dine-labeled complex-associated DNA. VH]thymicomplex was treated with the singledine-labeled complex was prepared from 6 x 10” strand-specific nuclease Sl, isolated from infected cells. Complex was dissociated and divided Neurospora. Approximately 40% of cominto aliquots containing 55 pg endogenous DNA/ml. plex-associated DNA is degraded by the Purified DNA’s were added in increasing amounts to nuclease (data not shown). the dissociated complex. The abscissa indicates the
lzoL-J!$E%J
amount of exogenously added DNA relative to endogenous DNA where one equivalent is 55 pg. The samples were dialyzed as described in Fig. 2, and reconstitution was assayed as described in Materials and Methods. Purified DNA’s were prepared by the method of Marmur W61). Eighty-one percent reconstituted endogenous DNA corresponded to 72,100 cpm. Circles, vaccinia DNA (purified from complex); squares, HeLa DNA; triangles, E. coli DNA.
DISCUSSION
Data presented here have shown that proteins associated with newly replicated vaccinia DNA can associate with DNA in vitro, resulting in a rapidly sedimenting complex. The conversion of DNA from a slowly sedimenting to a rapidly sedimenting state depends on interaction with protein, since the conversion is Pronase sensiCI , 1 tive. The association between DNA and protein in vitro is sensitive to alterations in ionic strength as is resedimentation of the complex isolated from infected cells. Complex-associated proteins appear to be nonspecific DNA-binding proteins. This conclusion is based on the ability of various DNA’s at a given concentration to inhibit reconstitution of endogenous complex to similar extents. Figures 3 and 4 I show that addition of about 40% excess .6 5 4 .2 ~~06 DNA 0, RNA EQUIVALENTS exogenous DNA to the complex results in loss of rapid sedimentation of about 90% of FIG. 4. Effect of DNA or RNA on reconstitution. [Wlthymidine-labeled complex was dissociated and endogenous complex DNA. Since the proincreasing amounts of 3H-labeled HeLa DNA (4660 tein:DNA ratio of isolated complex is 6:l
cpm/pg) or unlabeled vaccinia core RNA was added. Procedures for core preparation and RNA synthesis in vitro were those of Kates and Beeson (1970). Endogenous complex contained 42 pg of DNA/aliquot. Eighty-six percent reconstitution of endogenous DNA corresponded to 41,000 cpm (square on y axis). The amount of endogenous (“C-labeled) and exogenous (SH-labeled) DNA present in a rapidly sedi-
menting form after dialysis to low ionic strength was measured. Cpen circles, 3H-labeled HeLa DNA counts present in reconstituted complex; triangles, endogenous DNA reconstituted in the presence of increasing amounts of HeLa DNA; closed circles, endogenous DNA reconstituted in the presence of increasing amounts of RNA.
VACCINIA
DNA-BINDING
PROTEINS
147
(unpublished experiment), these data sug cess DNA concentration is compatible gest that when the protein:DNA ratio in with the second model. vitro is approximately 4:1, DNA is no ACKNOWLEDGMENTS longer rapidly sedimenting. Experiments using a filter assay to detect DNA-protein This work was supported by Public Health Serinteractions (Yarus and Berg, 1970) have vice Grant No. 1 ROl AI-08413 from the National demonstrated that complex-associated pro- Institute of Allergy and Infectious Diseases. teins exchange to exogenous DNA during REFERENCES reconstitution (unpublished experiment). Thus, the rapid sedimentation of vaccinia BECKER, Y., and JOKLIK, W. K. (19641. Messenger RNA in cells infected with vaccinia virus. Proc. DNA in uiuo probably is dependent on a Nat. Acad. Sci. USA 51, 577-585. high stoichiometric ratio of complex-associCAIRNS, J. (1960). The initiation of vaccinia infecated proteins. tion. Virology 11,603-618. About 40% of complex-associated DNA DAHL, R., and KATE~, J. (1970). Intracellular strucwas degraded by the single-strand-specific tures containing vaccinia DNA: Isolation and nuclease Sl, demonstrating the existence characterization. Virology 42,453-462. of single-stranded DNA in the isolated JOKLIK, W. K. and BECKER, Y. (1964). The replicacomplex. It is of interest that in adenovition and coating of vaccinia DNA. J. Mol. Biol. rus-infected KB cells, 25-30% of replicat10.452-474. ing viral DNA is sensitive to Sl (Peters- KATEB, J. R., and BEESON, J. (1970). Ribonucleic acid synthesis in vaccinia virus. I. The mechason, 1973), presumably due to asynchronism of synthesis and release of RNA in vaccinia nous replication of the complementary cores. J. Mol. Biol. 50, 1-18. DNA strands (Sussenbach et aZ., 1972). MARMUR, J. (1961). A procedure for the isolation of These experiments clearly suggest that DNA from microorganisms. J. Mol. Biol. 3, 206complex-associated proteins function to 218. hold newly replicated viral DNA mole- Pzrrzasfi~n, U. (1973). Some unusual properties of cules together in uiuo. We can consider two replicating adenovirus type 2 DNA. J. Mol. Biol. general models for the interaction between 81, 521-527. complexing proteins and DNA. In the first POLISKY,B., and KATES, J. R. (19721.Vaccinia virus intracellular DNA-protein complex: Biochemical model, DNA is complexed by proteins that characteristics of associated protein. Virology 49, contain two DNA-binding sites on each 68-79. monomer, the protein behaving analogously to a DNA antibody. In the second POLISKY, B., and KATES, J. R. (1975). Viral-specific polypeptides associated with newly replicated vacmodel, DNA is complexed via protein-procinia DNA. Virology 66,128-139. tein interactions, mediated by monomers SAROV, I., and JOXLIX, W. K. (19731. Isolation and that contain one DNA-binding site and characterization of intermediates in vaccinia vione protein-binding site. The antibody rus morphogenesis. Virology 52.223-233. model clearly predicts a complex at excess SAROV, I., and BECKER, Y. (1967). Studies on vacciDNA concentration, because the probabilnia DNA. Virology 33,369-375. ity of both DNA-binding sites being occu- SUMENBACH, J. S., VAN DER VLIET, P. C., Et LENS, D. J., and JANSZ, H. S. (1972). Linear interpied by the same DNA molecule should mediates in the replication of adenovirus DNA. decrease as a function of DNA concentraNature New Biol. 239, 47-49. tion. On the other hand, the secod model VOGT, V. (1973). Purification and further characterpredicts that protein-protein interactions istics of a single-strand specific nuclease from Asand consequent complexing should become pergillus orysae. Eur. J. Biochem. 33,192-200. increasingly rare as the concentration of YARUS, M., and BERG, P. (1970). On the properties binding sites is raised, resulting in nonand utility of a membrane filter assay for associacomplexed protein-bound DNA. The obtion of trRNA with protein. Anal. B&hem. 35, served dissociation of the complex at ex450-465.
69, 143-147 (1976)
Interaction
of Vaccinia BARRY
Department
of Molecular,
Cellular
DNA-binding
POLISKY’
AND
Proteins JOSEPH
and Developmental Biology, 8a302
with DNA in Vitro
KATES2
University
of Colorado,
Boulder,
Colorado
Accepted July 30,1975 The interaction between the components of the vaccinia DNA-protein complex isolated from infected cells has been studied. The rapid sedimentation of complex-associated DNA is destroyed by the addition of 2 M NaCl. However, the DNA is restored to a rapidly sedimenting state upon removal of the salt. This reconstitution is Pronase sensitive, indicating that conversion requires complex-associated protein. It was demonstrated that competitor DNA but not RNA from eucaryotic or bacterial sources inhibited restoration of rapid sedimentation of endogenous complex-associated DNA. The results suggest that complex-associated proteins responsible for DNA complexing are not specific for viral DNA. INTRODUCTION
Eucaryotic chromosomes contain protein molecules that presumably function to structurally organize long DNA molecules into configurations compatible with transcription, replication, and cell division. An understanding of the interaction between these structural elements and DNA has been hampered by the size and sequence heterogeneity of chromosomal DNA as well as the difficulty in determining which of the many proteins in isolated chromatin are native and which are artifacts of preparative methods. These difficulties have motivated an analysis of a viral DNA-protein complex that resembles a eucaryotic chromosome in many respects. Such a complex exists transiently in vaccinia-infected cells (Cairns, 1960; Joklik and Becker, 1964). Newly replicated vaccinia DNA can be isolated from cytoplasmic fractions of infected cells as a rapidly sedimenting DNA-protein complex capable of transcription of viral-specific mRNA in vitro (Dahl and Kates, 1970). The protein components of
11794. 143 0 1976 by Academic Press, Inc. of reproduction in any form reserved.
MATERIALS
AND
METHODS
Cells and virus. HeLa S3 cells were grown in suspension culture in Eagle’s minimal essential medium (Joklik’s modification, Grand Island Biological Company, Grand Island, N.Y.) supplemented with 5% calf serum. Vaccinia virus strain
I Present address: Department of Biochemistry and Biophysics, University of California Medical School, San Francisco, Calif. 94122. * Present address: Department of Microbiology, State University of New York, Stony Brook, N. Y.
Copyright All rights
the complex are limited in heterogeneity consisting primarily of two viral-specific, DNA-binding polypeptides designated A and B (Polisky and Kates, 1972; Sarov and Joklik, 1973). Host polypeptides appear to play no role in the structure of the complex (Polisky and Kates, 1975). Since treatment of the complex with Pronase or ionic detergents destroys the rapid sedimentation of complex-associated DNA, it has been inferred that associated protein plays a structural role in noncovalently complexing DNA molecules (Polisky and Kates, 1972). To gain insight into the basis and consequences of the interaction between complex-associated DNA and protein, we have developed procedures to dissociate and reconstitute the complex in vitro. Reconstitution experiments with exogenous, competing nucleic acids suggest that complex-associated proteins can bind to heterologous native or denatured DNA, but not virusspecific RNA.
144
POLISKY
WR was grown in HeLa cells and purified as described by Becker and Joklik (1964). In vitro reconstitution of the DNA-protein complex. The complex was prepared from infected cell cytoplasm by using 0.5% NP-40 (Nonidet P-40) as described previously (Polisky and Kates, 1975). Isolated complex in 0.01 M Tris-HCl (tris(hydroxymethyl)aminomethane), pH 8.0, 0.005 M EDTA (ethylenediaminetetraacetic acid), and 0.05 M NaCl was dissociated by the addition of solid NaCl to a final concentration of 2 M. As the salt dissolved, a large increase in the viscosity of the solution was apparent. To reduce the viscosity, the DNA was sheared by homogenization in a glass Dounce tissue homogenizer with a tight-fitting B pestle. After incubation for 10 min at 37”, the dissociated complex was centrifuged in an International B-20 centrifuge at 15,000 rpm for 15 min at 5”. This step removed complex-associated material that was insoluble in 2 M NaCl and any nondissociated complex. Usually 20-30% of the 13Hlthymidine-labeled viral DNA originally present in the isolated complex was insoluble under these conditions. To achieve reconstitution, the supernatant fluid was dialyzed overnight against 2 liters of 0.01 M Tris-HCl, pH 8.0, 0.05 M NaCl, 0.001 M EDTA. The dialysis was usually carried out at 5” but could also be done at room temperature with no change in the results. The amount of reconstitution was determined by centrifuging the dialysate for 15 min at 15,000 rpm at 5” in the International B-20 and measuring the amount of labeled viral DNA in the pelleted material. In reconstitution experiments involving addition of exogenous competing DNA’s, these DNA’s were added after addition of 2 M NaCl and prior to removal of insoluble material. Enzymatic digestions of the complex. (a) Pronase was obtained from Sigma. The enzyme (5 mg/ml) was heated at 80” in 0.05 M sodium acetate, pH 5.0, 0.005 M CaC12, 0.001 M P-mercaptoethanol for 20 min prior to use. The complex was treated with 100 pg of enzyme/ml for 1 hr at 37”. (b) Sl nuclease, purified by the method of Vogt (1973), was a gift of Gary Stormo. The
AND
KATE.3
complex was incubated with 1 unit/ml of Sl in 0.2 M sodium acetate, pH 4.7, 0.001 M ZnClz, 0.3 M NaCl for 30 min at 37”. At 5-min intervals, aliquots were removed and acid-insoluble radioactivity measured. Exogenous nucleic acids. Vaccinia, Escherichia coli, and HeLa DNA’s were purified essentially by the method of Marmur (1961). Vaccinia cores and core RNA synthesis was carried out as described by Kates and Beeson (1970). RESULTS
In Vitro Reconstitution tein Complex
of the DNA-Pro-
Experiments described previously suggested that the major complex-associated polypeptides, A and B, were apparently nonspecific DNA-binding proteins (Polisky and Kates, 1972). However, it remained possible that these proteins could exhibit preferential binding to vaccinia DNA, relative to other DNA’s presented simultaneously. To perform these experiments, an in vitro reconstitution system was developed. The assay for dissociation and reconstitution exploited the observation that isolated complex could be dissociated into component DNA and protein moieties by an increase in ionic strength. In high-salt sucrose gradients, it is possible to separate 90% of the protein from the DNA moiety (Fig. lb). Fig. la shows that both DNA and protein in isolated complex resediment to the bottom of the tube under low-salt conditions. When solutions containing dissociated complex in high salt were dialyzed to low ionic strength, over 90% of the DNA was restored to a rapid-sedimenting condition (Fig. 2a). Figure 2b shows that Pronase treatment eliminates the rapid sedimentation of the reconstituted complex, demonstrating that protein is required for conversion of DNA to a rapidly sedimenting state. The pronase used in this experiment contained no detectable DNase activity (data not shown). Reconstitution with Homologous erologous Components
and Het-
If the proteins responsible for maintaining complex-associated DNA in a rapid-
VACCINIA
5
10 FRACTION
DNA-BINDING
15
FIG. 1. Sedimentation of isolated 13H]leucine-, [“C]thymidine-labeled complex on low- and highsalt sucrose gradients. Double-labeled complex was prepared as described in Materials and Methods. The complex was then divided into two parts. One part was made 2 M NaCl, by the addition of solid NaCl. The remainder was made 0.05 M NaCl, and layered on a 38-50% sucrose gradient containing 0.05 M NaCl. The high-salt-containing sample was layered on an identical gradient containing 2 M NaCl. Both gradients were spun in the SW 27 rotor, 20,000 rpm, 17 hr, at 5”. Following centrifugation, fractions were collected and acid insoluble radioactivity measured. (a), Low-salt gradient; (b), highsalt gradient. Closed circles, [“Clthymidine counts per minute; open circles, ISH]leucine counts per minute. Sedimentation was from right to left.
sedimenting state were indeed dissociated from the DNA in high salt, then addition of purified vaccinia DNA (phenol extracted from the complex) might be expected to result in a replacement of the endogenous DNA with exogenous, homologous DNA. When this experiment was performed, it was found that addition of increasing amounts of vaccinia DNA to dissociated complex resulted in an inhibition ofreconstitution. Figure 3 shows that addition of 0.4 endogenous equivalents of unlabeled, purified vaccinia DNA, i.e., 40% of the amount of endogenous DNA, results in an 80% inhibition of entry of endogenous DNA into the reconstituted complex. Heterologous DNA’s were then tested for their ability to inhibit complex formation under these conditions. By comparing the amounts of competing DNA necessary to inhibit reconstitution, an indication of the relative “affinity” of these DNA’s for complex-associated protein was estab-
145
PROTEINS
=‘,
5
10 FRACTION
15
FIG. 2. Restoration of dissociated complex-associated DNA to a fast-sedimenting form after reconstitution. Isolated [JH]leucine, [14Clthymine-labeled complex was prepared. The complex was resuspended in 4 ml of 0.01 M Tris-HCl, pH 8.0, 0.05 M NaCl, 0.001 M EDTA, and solid NaCl was added to make the final NaCl concentration 2 M. The viscous solution was homogenized in a Dounce homogenizer and then centrifuged at 15,000 rpm for 15 min. The supernatant fluid was dialyzed overnight against the above buffer, except that the NaCl concentration was 0.05 M. The dialysate was removed and divided into two equal parts. One part was treated with 200 pg/ml heat-treated Pronase. The other part was untreated. Both samples were layered on separate 38 50% (w/v) sucrose gradients containing a 5-ml 84% (w/v) sucrose cushion. The gradients were centrifuged at 20,000 rpm for 30 min at 5”. Fractions were collected and acid-insoluble radioactivity was measured: (a), Untreated reconstituted complex; (b), Pronase-treated reconstituted complex. Closed circles, [‘Clthymidine counts; open circles, [JH]leucine counts. Sedimentation was from right to left.
lished. Unlabeled native DNA from E. coli and HeLa cells was used in these competition experiments. The results (Fig. 3) show that both DNA preparations inhibit reconstitution at identical concentrations relative to that of endogenous complex DNA. The results suggest that complexing proteins cannot distinguish between native DNAs. It was possible that complexing proteins could bind but not crosslink heterologous DNA. To test this possibility, 3H-labeled HeLa DNA was added to dissociated 14Clabeled complex at various concentrations and the amount of labeled reconstituted DNA measured. Figure 4 shows that at subinhibiting concentrations of HeLa
146
POLISKY AND KATES
DNA a heterologous, rapidly sedimenting complex containing both vaccinia and HeLa DNA can be formed. This result demonstrates that complex-associated proteins can complex heterologous DNA. To determine if RNA could inhibit complex formation in vitro, salt-dissociated complex was incubated with increasing amounts of RNA trasncribed in. vitro by vaccinia cores. Figure 4 shows that RNA is not inhibitory at the concentrations tested. Denatured DNA is about 50% more effrcient as an inhibitor of reconstitution than EXOG. DNA EQ”l”A:ENTS native DNA (unpublished experiment). To FIG. 3. Effect of double-stranded exogenous determine whether the complex contained DNA’s on reconstitution of endogenous VH]thymiDNA, t3HlTdR-labeled single-stranded dine-labeled complex-associated DNA. VH]thymicomplex was treated with the singledine-labeled complex was prepared from 6 x 10” strand-specific nuclease Sl, isolated from infected cells. Complex was dissociated and divided Neurospora. Approximately 40% of cominto aliquots containing 55 pg endogenous DNA/ml. plex-associated DNA is degraded by the Purified DNA’s were added in increasing amounts to nuclease (data not shown). the dissociated complex. The abscissa indicates the
lzoL-J!$E%J
amount of exogenously added DNA relative to endogenous DNA where one equivalent is 55 pg. The samples were dialyzed as described in Fig. 2, and reconstitution was assayed as described in Materials and Methods. Purified DNA’s were prepared by the method of Marmur W61). Eighty-one percent reconstituted endogenous DNA corresponded to 72,100 cpm. Circles, vaccinia DNA (purified from complex); squares, HeLa DNA; triangles, E. coli DNA.
DISCUSSION
Data presented here have shown that proteins associated with newly replicated vaccinia DNA can associate with DNA in vitro, resulting in a rapidly sedimenting complex. The conversion of DNA from a slowly sedimenting to a rapidly sedimenting state depends on interaction with protein, since the conversion is Pronase sensiCI , 1 tive. The association between DNA and protein in vitro is sensitive to alterations in ionic strength as is resedimentation of the complex isolated from infected cells. Complex-associated proteins appear to be nonspecific DNA-binding proteins. This conclusion is based on the ability of various DNA’s at a given concentration to inhibit reconstitution of endogenous complex to similar extents. Figures 3 and 4 I show that addition of about 40% excess .6 5 4 .2 ~~06 DNA 0, RNA EQUIVALENTS exogenous DNA to the complex results in loss of rapid sedimentation of about 90% of FIG. 4. Effect of DNA or RNA on reconstitution. [Wlthymidine-labeled complex was dissociated and endogenous complex DNA. Since the proincreasing amounts of 3H-labeled HeLa DNA (4660 tein:DNA ratio of isolated complex is 6:l
cpm/pg) or unlabeled vaccinia core RNA was added. Procedures for core preparation and RNA synthesis in vitro were those of Kates and Beeson (1970). Endogenous complex contained 42 pg of DNA/aliquot. Eighty-six percent reconstitution of endogenous DNA corresponded to 41,000 cpm (square on y axis). The amount of endogenous (“C-labeled) and exogenous (SH-labeled) DNA present in a rapidly sedi-
menting form after dialysis to low ionic strength was measured. Cpen circles, 3H-labeled HeLa DNA counts present in reconstituted complex; triangles, endogenous DNA reconstituted in the presence of increasing amounts of HeLa DNA; closed circles, endogenous DNA reconstituted in the presence of increasing amounts of RNA.
VACCINIA
DNA-BINDING
PROTEINS
147
(unpublished experiment), these data sug cess DNA concentration is compatible gest that when the protein:DNA ratio in with the second model. vitro is approximately 4:1, DNA is no ACKNOWLEDGMENTS longer rapidly sedimenting. Experiments using a filter assay to detect DNA-protein This work was supported by Public Health Serinteractions (Yarus and Berg, 1970) have vice Grant No. 1 ROl AI-08413 from the National demonstrated that complex-associated pro- Institute of Allergy and Infectious Diseases. teins exchange to exogenous DNA during REFERENCES reconstitution (unpublished experiment). Thus, the rapid sedimentation of vaccinia BECKER, Y., and JOKLIK, W. K. (19641. Messenger RNA in cells infected with vaccinia virus. Proc. DNA in uiuo probably is dependent on a Nat. Acad. Sci. USA 51, 577-585. high stoichiometric ratio of complex-associCAIRNS, J. (1960). The initiation of vaccinia infecated proteins. tion. Virology 11,603-618. About 40% of complex-associated DNA DAHL, R., and KATE~, J. (1970). Intracellular strucwas degraded by the single-strand-specific tures containing vaccinia DNA: Isolation and nuclease Sl, demonstrating the existence characterization. Virology 42,453-462. of single-stranded DNA in the isolated JOKLIK, W. K. and BECKER, Y. (1964). The replicacomplex. It is of interest that in adenovition and coating of vaccinia DNA. J. Mol. Biol. rus-infected KB cells, 25-30% of replicat10.452-474. ing viral DNA is sensitive to Sl (Peters- KATEB, J. R., and BEESON, J. (1970). Ribonucleic acid synthesis in vaccinia virus. I. The mechason, 1973), presumably due to asynchronism of synthesis and release of RNA in vaccinia nous replication of the complementary cores. J. Mol. Biol. 50, 1-18. DNA strands (Sussenbach et aZ., 1972). MARMUR, J. (1961). A procedure for the isolation of These experiments clearly suggest that DNA from microorganisms. J. Mol. Biol. 3, 206complex-associated proteins function to 218. hold newly replicated viral DNA mole- Pzrrzasfi~n, U. (1973). Some unusual properties of cules together in uiuo. We can consider two replicating adenovirus type 2 DNA. J. Mol. Biol. general models for the interaction between 81, 521-527. complexing proteins and DNA. In the first POLISKY,B., and KATES, J. R. (19721.Vaccinia virus intracellular DNA-protein complex: Biochemical model, DNA is complexed by proteins that characteristics of associated protein. Virology 49, contain two DNA-binding sites on each 68-79. monomer, the protein behaving analogously to a DNA antibody. In the second POLISKY, B., and KATES, J. R. (1975). Viral-specific polypeptides associated with newly replicated vacmodel, DNA is complexed via protein-procinia DNA. Virology 66,128-139. tein interactions, mediated by monomers SAROV, I., and JOXLIX, W. K. (19731. Isolation and that contain one DNA-binding site and characterization of intermediates in vaccinia vione protein-binding site. The antibody rus morphogenesis. Virology 52.223-233. model clearly predicts a complex at excess SAROV, I., and BECKER, Y. (1967). Studies on vacciDNA concentration, because the probabilnia DNA. Virology 33,369-375. ity of both DNA-binding sites being occu- SUMENBACH, J. S., VAN DER VLIET, P. C., Et LENS, D. J., and JANSZ, H. S. (1972). Linear interpied by the same DNA molecule should mediates in the replication of adenovirus DNA. decrease as a function of DNA concentraNature New Biol. 239, 47-49. tion. On the other hand, the secod model VOGT, V. (1973). Purification and further characterpredicts that protein-protein interactions istics of a single-strand specific nuclease from Asand consequent complexing should become pergillus orysae. Eur. J. Biochem. 33,192-200. increasingly rare as the concentration of YARUS, M., and BERG, P. (1970). On the properties binding sites is raised, resulting in nonand utility of a membrane filter assay for associacomplexed protein-bound DNA. The obtion of trRNA with protein. Anal. B&hem. 35, served dissociation of the complex at ex450-465.
