- Email: [email protected]
Bacteriophage T3 DNA binding protein interaction with DNA
VIROLOGY
109, 148-156
(1981)
Bacteriophage MASAHIRO
T3 DNA Binding
YAMAGISHI,
Botanical
Department,
HISAO Faculty
Protein
FUJISAWA,’
of Science,
Kyoto
Interaction AND
with DNA
TEIICHI
University,
Kyoto
MINAGAWA 606, Japan
Single-stranded DNA binding protein (DBP), coded by T3 phage, is essential for concatemer formation (H. Fujisawa, M. Yamagishi, H. Matsuo-Kato, and T. Minagawa, 1980, Virology, 105, 480-489.) T3 DBP was purified to homogeneity and found to exist as a dimer. The structure of the T3 DBP-fd DNA complex appears as a condensed and beaded ring structure by electron microscopy. As judged by sucrose gradient centrifugation, DBP binds to single-stranded, but not to double-stranded DNA; at saturation, one protein monomer is bound per every 100 nucleotides. The T3 DBP has a strong ability to catalyze renaturation of DNA even in the absence of Mg2+. The results suggest that the T3 DBP acts in concatemer formation by stimulating pairing of the single-stranded redundant termini of T3 DNA.
5’ ends of the terminally redundant region of T3 DNA. The T3 DBP acts to limit the activity of the exonuclease and to stimulate intermolecular annealing of the exposed single-stranded regions of the DNA to form concatemers (Fujisawa et al., 1980a). We have also showed that the gene 32 product (gp32) of phage T4, one of the well known DBPs, could not substitute for T3 DBP in the packaging of T3 DNA in vitro, suggesting that there are some differences in functional property between these two proteins (Fujisawa et al, 1980a). We have isolated T3 DBP and studied its interaction with DNA. In this report, we demonstrate that T3 DBP catalyzes the annealing of denatured DNA even in the absence of Mg2+. In this respect, T3 DBP is quite different from T4 DBP (gp32). Additional properties of the protein and its possible role in T3 DNA packaging will be presented.
INTRODUCTION
We have developed in vitro DNA packaging system for studying morphogenesis of bacteriophage T3 (Fujisawa et al., 1978). In this system, mature T3 DNA, when incubated with extracts prepared from phageinfected cells, is converted to concatemers and then packaged into phage heads. T3 gene 6 exonuclease and T3 single-stranded DNA binding protein (DBP) are required for concatemer formation during the in vitro packaging reaction (Yamagishi et al., 1980; Fujisawa et al., 1980a, b). Proteins which bind to single-stranded (ss) DNA have been isolated from many organisms (see review by Champoux, 1978; Radding, 1978). The proteins have the ability to promote denaturation of doublestranded (ds) DNA (Alberts and Frey, 1970; Sigal et al., 1972). Interestingly, DBPs also can stimulate reannealing of DNA and, when incubated with ssDNA, can reduce the sensitivity of the ssDNA to single-strand nucleases (Alberts and Frey, 1970; Hotta and Stern, 1971; Banks and Spanos, 1975; Molineux and Gefter, 1975; Christiansen and Baldwin, 1977). We have proposed a model for T3 DNA concatemer formation involving T3 exonuclease (gene 6 product, or gp6) and T3 DBP. By this model, the exonuclease digests the I To whom
reprint
requests
should
0042-6822/81/030148-09$02.00/O Copyright All rights
0 1981 by Academic Press, Inc. of reproduction in any form reserved.
MATERIALS
AND
METHODS
The bacterial and phage strains and M9A medium have been described previously (Fujisawa et al., 1978). DNAs were prepared from purified phage according to Fujisawa et al. (1978). T4 DNA and Co1 El DNA were generous gifts from Mr. Nakasu; fd DNA and A DNA were generous gifts from Dr. H. Yamagishi; T4 gp32 was kindly provided by Dr. M. Takanami.
be addressed. 148
T3 DNA
BINDING
Preparation of T3 DBP. T3 DBP was purified by a modification of the procedure of Fujisawa et al. (1980a). The DBP fraction from ssDNA cellulose column chromatography was applied to a hydroxyapatite column equilibrated with 0.01 M potassium phosphate (pH 7.4)-0.2 mM dithiothreitol10% glycerol and eluted with linear gradient of 0.01 to 0.4 M potassium phosphate (pH 7.4) containing 0.2 mM dithiothreitol10% glycerol. The DBP eluted between 0.1 and 0.13 M potassium phosphate. The eluate was dialyzed against 10 mM potassium phosphate (pH 7.4)-0.2 mM dithiothreitol-50% glycerol and stored at -20°C. [3H]Leucine-labeled DBP was prepared from Escherichia coli cells infected with T3
PROTEIN
149
amber mutant defective in gene 5 (5extract) and labeled with [3H]leucine between 6 and 20 min after infection according to Fujisawa et al., (1980a). In vitro
DNA
packaging
experiments.
Preparation of extracts from phage-infected cells and the in vitro DNA packaging reaction were performed as described in a previous paper (Fujisawa et al., 1978). Assay for renaturation of DNA. T3 DBP and heat-denatured [“Hlthymidinelabeled T3 DNA were mixed in 0.1 M NaCl-Tris-HCl (pH 7.4)-0.2 m&I EDTA (EDTA buffer) and incubated at 30”. At the indicated times, 15~1 portions were poured into 47.5 ~1 of ice-chilled 0.02% SDS-O.3 M NaCl-80 mh4 sodium acetate
FIG. 1. Electron microscopy of complexes of fd DNA with T3 DBP. fd DNA (5 @g/ml) was incubated with various amounts of T3 DBP in 0.01 M potassium phosphate (pH 7.0) for 10 min at 30”. The samples were fixed with 0.01 M glutaraldehyde for 15 min at 30” and spread with formamide and cytochrome c. Free fd DNA was added before spreading. The relative contour length for free fd DNA was 1.98 f 0.08 pm. The amounts of T3 DBP were (a) 40 pgiml, (b) 50 pgiml, and (c) 100 pgiml. Bar represents 0.5 pm.
150
YAMAGISHI,
FUJISAWA,
(pH 4.3)-0.08 mil4 ZnSO, and heated at 70” for 2 min in a water bath. The mixtures were treated with 5 ~1 of Sl nuclease (5000 units) and incubated at 30” for 20 min. Fifty-microliter portions were spotted onto filter paper disks (Toyo Roshi No. 2) and, after washing the disks with trichloroacetic acid and acetone, the acid-insoluble radioactivity was measured in a liquid scintillation counter. Sedimentation analysis of DNA. ssDNA, treated with T3 DBP, was centrifuged through a 5 to 20% sucrose density gradient (5 ml) in EDTA buffer in a Hitachi RPS50-2 rotor at 32,000 rpm for 150 min at 15”. After centrifugation, fractions were collected from the bottom of the gradients onto filter paper disks and radioactivity was measured as described above. Electron microscopy. Samples were mixed with 0.1 M Tris-HCl (pH 8.5), 30% formamide, and 100 pg/ml of cytochrome c.
FIG.
AND
MINAGAWA
20
10 Time
( mln
1
FIG. 2. Kinetics of catalysis of DNA renaturation with T3 DBP. Single-stranded “H-labeled T3 DNA (20 pgiml) was incubated with T3 DBP or T4 gp32 in EDTA buffer and the mixture were incubated at 30”. At the times indicated, portions were taken and Sl nuclease resistant radioactivity was determined as described under Materials and Methods. No protein (x); T4 gp32 (40 pgiml) (0); T3 DBP (80 pgiml) (0); (40 &ml) (0); (20 &ml) (A); (10 pgiml) (W. One-half of the sample containing 80 pg/mI of T3 DBP was incubated for 6 min at 0” and shifted to 30 (0 - - - 0).
3a
T3 DNA
BINDING
PROTEIN
FIG. 3. Electron microscopy of dsDNA formed from ssDNA of T3 DNA by T3 DBP. ssT3 DNA (10 pgiml) was incubated with (a, b) or without (c) T3 DBP (6’7 Fgiml) in EDTA buffer for 10 min at 30”. after the addition of co1 El DNA as a dsDNA marker, the samples were spread with formamide and cytochrome e immediately after the reaction. (a) DNA aggregate; dsDNA with monomer length was arrowed. (b) Renatured product with single-stranded regions. Arrows indicate single-stranded regions. (c) ssDNA incubated without T3 DBP. Supertwisted co1 El molecules can be seen. Bar represents 2 pm.
Fifty microliters of this hyperphase was spread on 1 ml of hypophase containing 0.01 A4 Tris-HCl (pH 8.6) and 20% formamide. The samples were picked ux) on parlodion-coated grids, stained with 0.b5 M many1 acetate in 90% ethanol, washed in isopenteane, and rotatory shadowed with 80% Pt-20% Pd (Yamagishi et al., 1980).
The grids were examined in a Hitachi HU-11D electron microscope. RESULTS
Structure
of the T3 DBP-jd
DNA
Complex
As mentioned in the Introduction, T4 DBP (gp32) cannot substitute for T3 DBP
152
YAMAGISHI,
FUJISAWA.
in the in vitro DNA packaging system of T3. To compare the properties of these two proteins, complexes of fd DNA with T3 DBP or T4 gp32 were examined by electron microscopy. fd DNA complexed with T4 gp32 had an extended conformation as described by Delius et al. (1972). On the contrary, the T3 DBP-fd DNA complex had a condensed appearance like a beaded necklace (Fig. la). With increasing amounts of T3 DBP, the protein-DNA complex became a condensed form with knot-like structures (Fig. lc); their circumferences were 30 and 52% smaller than that measured for the free DNA at the protein:DNA ratios of 1O:l (Fig. lb) and 2O:l (Fig. lc), respectively. Renaturation
of ssDNA with T3 DBP
To examine the effect of T3 DBP on the renaturation of DNA, heat-denatured, “Hlabeled T3 DNA was incubated with T3 DBP or with T4 gp32 in EDTA buffer at 30”. Aliquots were withdrawn at various times and reannealing of DNA was measured by resistance to Sl nuclease as described under Materials and Methods (Fig. 2). ssDNA incubated with T4 gp32 was completely digested with Sl nuclease. On the other hand, most of the DNA became Sl resistant within 20 min of treatment with T3 DBP. These results indicate that T3 DBP catalyzes the renaturation of DNA. The rate of the renaturation of DNA was dependent on the amount of T3 DPB. The reaction proceeded at a very slow rate at 0” (Fig. 2). The addition of spermidine (1 mM) or Mg2+ (10 m&I) was slightly inhibitory for the reaction (data not shown). The optimal concentration of NaCl was between 0.1 M and 0.15 M (data not shown). Electron micrographs clearly show the formation of dsDNA in the presence of T3 DBP (Fig. 3). The DNA tended to form aggregates that contained single-stranded regions (Fig. 3b). Apart from the aggregates, complete dsDNA molecules with monomer size were observed (Fig. 3a). When denatured hDNA was treated with T3 DBP, the DNA was observed by electron microscopy to become double-stranded. However, the renaturation of T4 DNA was
AND
MINAGAWA
not catalyzed by T3 DBP, although the formation of DNA-protein complex was observed (data not shown). Sedimentation profiles of DNA treated with T3 DBP are shown in Fig. 4. ssT3 DNA, after incubation with T3 DBP, sedimented rapidly. Conversion of T3 DNA to the rapidly sedimenting material was dependent upon the concentrations of both the DNA and the DBP (Fig. 4; compare (b) to (c), (c) to (d) or (e), (e) to (f)). The presence of Mg*+ and ATP did not affect the conversion process (data not shown). Rapid sedimentation of DNA was not due to the association of DNA with T3 DBP because SDS treatment at 70” did not affect the sedimentation profile (data not shown). In fact, when a reaction mixture containing ‘IH-labeled DBP and 32P-labeled DNA was incubated and sedimented through gradient, most of the “H-labeled DBP was recovered at the top of the gradient, while “‘P-labeled DNA sedimented rapidly (Fig. 4e). DNA in the fractions of the gradient was resistant to Sl nuclease (Fig. 4f). Stoichiometry
of Binding of the T3 DBP
to ssDNA
The stoichiometry of the binding of T3 DBP to ssDNA was determined by sucrose gradient sedimentation of a fixed amount of fd DNA in the presence of various amounts of :$H-labeled T3 DBP. Two distinct peaks of the radioactivity were seen, one sedimenting rapidly with DNA and the other sedimenting slowly as the free protein (Fig. 5). The amount of radioactivity in the former peak was constant, but that in the latter peak increased with increasing amounts of the protein. From the fraction of protein bound, a weight ratio of protein to DNA of about 1 to 1 was determined. From the known molecular weight of fd DNA (2 x lo”, 6400 nucleotides) and of T3 DBP (average molecular weight, 31,500), we estimate that the DNA-DBP complex contains about one protein molecule for every 100 nucleotides of ssDNA. The Biological Activity DNA
of the Renatured
Previously (Fujisawa et al., 1978), we have shown that exogenous T3 DNA can
T3 DNA
BINDING
153
PROTEIN
packaging with 3--extract gave a single peak at the position corresponding to a dimer size. The relative amount of the two polypeptides was constant through the fractions (Fig. 7b). DISCUSSION
Properties
Fraction
no
FIG. 4. Sedimentation profiles of T3 DNA treated with T3 DBP. Single-stranded “Ylabeled T3 DNA was incubated with T3 DBP in various ratios of DNA to protein in EDTA buffer for 10 min at 30” and sedimented through sucrose gradients. The amounts of DNA and protein were expressed as micrograms per milliliter. (a) No protein; (b) 5 FgDNA to 25 pg protein; (c) 20 pg DNA to 25 pg protein; (d) 20 pg DNA to 180 pg protein; (e) 20 pg DNA to 75 kg protein; (f) 10 pg DNA to 38 pg protein. “‘P-Labeled T3 DNA (0). (e) ‘“P-Labeled DNA and :‘H-labeled DBP (0) were used for the reaction. (f) After centrifugaticJl1, fractions were spotted onto filter paper disks with (x) or without (0) Sl nuclease treatment as described under Materials and Methods.
be packaged into phage particles in an system composed of a mixture of 3-- and 5--extracts (3--extract is an extract prepared from cells infected with a T3 mutant of gene 3). Denatured T3 DNA, when treated with T3 DBP, was active in the in vitro packaging system. As shown in Fig. 6, phage titers increased with increasing amounts of either DNA or DBP. With a particular amount of DNA, a plateau level of packaging was achieved with 50 pg/ml of DBP. The renatured DNA was more efficiently packaged than native DNA.
of the T3 DBP
Like other DBPs that have been described, T3 DBP binds preferentially to single-stranded DNA. However, T3 DBP has certain properties that distinguished it from the other DBPs. The complex of fd DNA with excess T3 DBP appears as a beaded and condensed ring structure; the E. coli DBP-fd DNA complex shows a similar but less folded structure (Sigal et al., 1972). The structure of fd DNA complex with fd gp5 is rod-like (Alberts et al., 1972). T4 gp32 holds fd DNA in an extended configuration (Delius et al., 1972). DNA reassociation catalyzed by T3 DBP does not require Mg’+. In contrast, DNA reassociation by both T4 and E. coli DBPs
in vitro
Evidence
that T3 DBP Aggregates
Purified T3 DBP, when analyzed by SDS-polyacrylamide gel electrophoresis, consists of two protein bands with molecular weights of 31,000 and 32,000 (Fujisawa et al., 1980a). Gel filtration experiment was performed to explore the native molecular weight of T3 DBP in the buffer used for the renaturation reaction (Fig. 7). The complementation activity for in vitro DNA
-
15-
Fractson
no.
FIG. 5. Stoichiometry of binding of T3 DBP to fd DNA. The reaction mixtures (0.2 ml) contained 2 pg fd DNA and 5 pg T3 DBP (a); 2 pg fd DNA and 10 pg T3 DBP (b); or 10 /*g T3 DBP (c). The mixtures were incubated at 1.i” for 30 min and centrifuged through 5 to 30% sucrose gradients in EDTA buffer containing 20 @g/ml of bovine serum albumin in a Hitachi RPS50-2 rotor at 48,000 rpm for 90 min at 4”. Fractions were collected onto filter paper disks and the radioactivity was measured as described under Materials and Methods. The recovery of the radioactivity was more than 90%. The arro\v indicates the position of fd DNA.
154
YAMAGISHI,
FUJISAWA,
require high concentrations of Mgz+. The Eco DBP requires spermidine in addition to Mg2+. Recently, Weinstock et al. (1979) reported that ret A protein has an ability to stimulate annealing of DNA; in this case, both Mg2+ and ATP are required for the reaction. The high rate of DNA reassociation occurs at a lower protein:DNA ratio for T3 DBP. A much higher ratio of protein to DNA is required for other DBPs (Alberts and Frey, 1970; Christiansen and Baldwin, 1977). The high efficiency of the T3 DBP for DNA reassociation may be related to the unique stoichiometric nature of binding of the protein to DNA. At saturation of the protein, the fd DNA-T3 DBP complex contains 100 nucleotides per protein monomer (Fig. 5). Complexes of DNA with other DBPs contain about 10 nucleotides per protein monomer (Alberts and Frey, 1970; Alberts et al., 1972; Oey and Knippers, 1972; Sigal et al., 1972; Reubin and Gefter, 1974). No evidence of the denaturation of dsDNA with T3 DBP was obtained under the conditions which are used for denaturation activity of T4 gp32, E. coli DBP, and fd DBP (data not shown). Although the facts do not exclude the possibility that T3 DBP destabilizes the double helix of DNA under some conditions, the result might be expected because T3 DBP has a strong ability to catalyze reannealing of DNA under the conditions. Our previous results indicate that T3 DBP and gp6 act together to form concatemeric T3 DNA (Fujisawa et al., 1980a). We have also shown that concatemeric DNA is the substrate for packaging DNA into phage particles. After concatemer formation, DBP and gp6 are no longer required for the in vitro packaging reaction. Denatured T3 DNA, after treatment with T3 DBP, is packaged more efficiently than native T3 DNA in the in vitro packaging system (Fig. 6). This is not due to concatemer formation, as DBP-treated DNA is not packaged in 3-- or 6-extract (data not shown). We suggest that the action of DBP on denatured T3 DNA forms structures that are converted to concatemers more efficiently than native T3 DNA.
AND
MINAGAWA
0
50 100 T3 DBP ( )-‘g/ml )
FIG. 6. Biological activity of dsDNA produced by T3 DBP. Heat-denatured T3 DNA was incubated with various amounts of T3 DBP in EDTA buffer for 15 min at 30”. After the incubation, the mixture was heated to 70” for 2 min and added to i?z vitro DNA packaging system using a mixture of Y--extract and Cextract. The concentrations of DNA were 60 pgiml (0); 18 pgiml (0); and 5 kg/ml (A). Phage yields in the in vitro system using native T3 DNA at the given concentrations are indicated on the ordinate as horizontal bars.
Role of the T3 DBP in in Vitro DNA Packaging
In a previous paper (Fujisawa et al., 1980a), we postulated two possible roles for the T3 DBP in the in vitro packaging reaction. One is that DBP binds to singlestranded regions at the termini of T3 DNA generated by gp6 exonuclease. By binding at these regions, the DBP would prevent excessive digestion by gp6 exonuclease. However, T3 DBP does not affect the exonucleolytic activity of gp6 (data not shown). The second possibility is that DBP stimulates intermolecular pairing of single-stranded regions produced by gp6 at the redundant termini of T3 DNA. The present results are more consistent with the second possibility than the first. The T3 DBP may be involved in genetic recombination because the pairing of homologous strands is an important early step in recombination (see a review by Radding, 1978). In fact, in vitro recombination is greatly reduced in 3--extract (Fujisawa et al., 1978). It is possible that the T3 DBP may have additional functions. The T7 DBP is thought to be involved in DNA replication (Scherzinger et al., 1973; Reubin and Gefter, 1974; Richardson et al., 1979). The availability of a host mutant defec-
T3 DNA
Hemoglobtn
Cytochrome
BINDING
no
1s
c
14 Fraction
PROTEIN
b
15
16
17 18 Fraction
19 no.
20
21
22
FIG. 7. Gel filtration of T3 DBP. T3 DBP (80 pgiml) was loaded onto a Sephadex G-200 column (1.5 x 75 cm) elquilibrated with EDTA buffer with cytochrome c (MW = 13,000) and hemoglobin (MW = 67,000) as molecular weight standards. The eluates were assayed for ilc vitro complementing activity with S--extract (a) and were subjected to SDS-polyacrylamide gel electrophoresis (b) as described elsewhere (Fujisawa et rti., 1980b). B.D., blue dextran.
tive in E. coli DBP (ssb-1 (Meyer et al., 1979)) makes it possible to examine the participation of phage DBP in various DNA metabolic processes. ACKNOWLEDGMENTS We are grateful to Dr. F. Fujimura, La Jolla Cancer Research Foundation, for invaluable help with the manuscript, and Dr. Ryo for his helpful discussion. This study was in part supported by grantsin-aid for Scientific Research to H.F. from the Ministry of Education of Japan (348386).
REFERENCES ALBERTS, B., FREY, D., and DELIUS, H. (1972). Isolation and characterization of gene 5 proteins of filamentous bacterial viruses. J. Mol. Biol. 68, 139- 152. ALIIERTS, B. M., and FREY, L. (1970). T4 bacteriophage gene 32: A structural protein in the
replication and 227, 1313-1318. ALBERTS, B. M., cent excitement Nnfxre (London)
recombination.
Nature
(Lo~dorrl
and STERNGI.ANZ, R. (1977). Rein the DNA replication problem. 269, 655-661.
BANKS, G. R., and SPANOS, A. (1975). The isolation and properties of a DNA-unwinding protein from Ustilngo maydis. J. Mol. Biol. 93, 63-77. CHAMPOUX, J. J. (1978). Proteins that affect DNA conformation. Annu. Rev. Biochem. 47, 449-480. CHRISTIANSEN, C., and BALDWIN. R. L. (1977). Catalysis of DNA association by the Escherichin coli DNA binding protein. Polyamine dependent reaction. J. Mol. Biol. 115, 441-454. DELIUS, H., MANTELL, N. ,J.. and ALBERTS. B. (1972). Characterization by electron microscopy of the complex formed between T4 bacteriophage gene 32-protein and DNA. J. Mol. Biol. 67, 341350. FUJISAU’A. H.. MIYAZAKI, J., and MINAGAWA. T. (1978). In vitro packaging of phage T3 DNA. Virology 87, 394-400.
156
YAMAGISHI,
FUJISAWA,
FUJISAWA, H., YAMAGISHI, M., MATSUO-KATO, H., and MINAGAU~A, T. (1980a). Purification of DNAbinding proteins of bacteriophage T3 and their role in i)~ vitro packaging of phage T3 DNA. Virology 105, 480-489. FU.JISAWA, H., YAMAGISHI, M.. and MIXAGAB’A, T. (1980b). II/ csifro formation of the concatemeric DNA of bacteriophage T3 and its biological role in the in z’jtro packaging reaction. Virology 101, 327-334. HOTTA, Y., and STERN, H. (1971). A DNA-binding protein in meiotic cells of Lilium, Develop. Biol. 26, 87-99. MEYER, R. R., GLASSBERG, J.. and KORNBERG, A. (1979). An Escherichia coli mutant defective in single-stranded binding protein is defective in DNA replication. Proc. b’at. Acad. Sri. USA. 76, 17021705. MOLINEUX, I. J., and GEFTE:R, M. L. (1975). Properties of the Eschetichia coli DNA-binding (unwinding) protein interaction with nucleolytic enzymes and DNA. d. Mol. Biol. 98, 811-825. OXY. J. L., and KNIPPERS, R. (1972). Properties of the isolated gene 5 protein of bacteriophage fd. d. Mol. Biol. 68, 125-138. RADDING, C. M. (1978). Genetic recombination: Strand transfer and mismatch repair. Au1174. RPU. Biochen~. 17, 847-80.
AND MINAGAWA REUBIP~, R. C., and GEFTER, M. L. (1974). A deoxyribonucleic acid binding protein induced by bacteriophage T’7. Purification and properties of the protein. J. Biol. Chem. 249, 3843-3850. RICHARDSON, C. C.. ROMANO, L. J., KOLODNER. J. E., TAMAAVI, F., ENGLER, M. J., DEAN. F. B., and RICHARDSON. D. S. (1979). Replication of bacteriophage T7 by purified proteins. Cold Spll’ng Harbor
Symp.
Quart.
Bid.
43, 427-440.
SCHERZIXGER, E.. LITFIN, F.. and .JOST, E. (1973). Stimulation of T7 DNA polgmearse by a new phagr-coded protein. Mol. Gen. Gewf. 123, 247262. SIG& and tein with
N., DELIUS, H., KORNBERG, T.. GF.FTE:R.M., ALBERTS, B. (1972). A DNA-unwinding proisolated from Eschetichia coli: Its interaction DNA and DNA polymerases. Proc. Nat. Accrd. SC;. USA 69, 3537-3541.
WEXSTOCK, G. M.. MCENTEE, K., and I,F,HILIAN. I. R. (1979). ATP-dependent renaturation of DNA catalyzed by the ret A protein of Eschwichia co/i. Proc. Nnf. Acad. Sci. USA 76, 126-130. YAYAGISHI, M., FUJISA~VA, H., YAMAGISHI. H., and MINAGAU’A. T. (1980). Purification of gene 6 product of bacteriophage T3 and its role in in vitro DNA packaging. Virology 100, 382-389.
109, 148-156
(1981)
Bacteriophage MASAHIRO
T3 DNA Binding
YAMAGISHI,
Botanical
Department,
HISAO Faculty
Protein
FUJISAWA,’
of Science,
Kyoto
Interaction AND
with DNA
TEIICHI
University,
Kyoto
MINAGAWA 606, Japan
Single-stranded DNA binding protein (DBP), coded by T3 phage, is essential for concatemer formation (H. Fujisawa, M. Yamagishi, H. Matsuo-Kato, and T. Minagawa, 1980, Virology, 105, 480-489.) T3 DBP was purified to homogeneity and found to exist as a dimer. The structure of the T3 DBP-fd DNA complex appears as a condensed and beaded ring structure by electron microscopy. As judged by sucrose gradient centrifugation, DBP binds to single-stranded, but not to double-stranded DNA; at saturation, one protein monomer is bound per every 100 nucleotides. The T3 DBP has a strong ability to catalyze renaturation of DNA even in the absence of Mg2+. The results suggest that the T3 DBP acts in concatemer formation by stimulating pairing of the single-stranded redundant termini of T3 DNA.
5’ ends of the terminally redundant region of T3 DNA. The T3 DBP acts to limit the activity of the exonuclease and to stimulate intermolecular annealing of the exposed single-stranded regions of the DNA to form concatemers (Fujisawa et al., 1980a). We have also showed that the gene 32 product (gp32) of phage T4, one of the well known DBPs, could not substitute for T3 DBP in the packaging of T3 DNA in vitro, suggesting that there are some differences in functional property between these two proteins (Fujisawa et al, 1980a). We have isolated T3 DBP and studied its interaction with DNA. In this report, we demonstrate that T3 DBP catalyzes the annealing of denatured DNA even in the absence of Mg2+. In this respect, T3 DBP is quite different from T4 DBP (gp32). Additional properties of the protein and its possible role in T3 DNA packaging will be presented.
INTRODUCTION
We have developed in vitro DNA packaging system for studying morphogenesis of bacteriophage T3 (Fujisawa et al., 1978). In this system, mature T3 DNA, when incubated with extracts prepared from phageinfected cells, is converted to concatemers and then packaged into phage heads. T3 gene 6 exonuclease and T3 single-stranded DNA binding protein (DBP) are required for concatemer formation during the in vitro packaging reaction (Yamagishi et al., 1980; Fujisawa et al., 1980a, b). Proteins which bind to single-stranded (ss) DNA have been isolated from many organisms (see review by Champoux, 1978; Radding, 1978). The proteins have the ability to promote denaturation of doublestranded (ds) DNA (Alberts and Frey, 1970; Sigal et al., 1972). Interestingly, DBPs also can stimulate reannealing of DNA and, when incubated with ssDNA, can reduce the sensitivity of the ssDNA to single-strand nucleases (Alberts and Frey, 1970; Hotta and Stern, 1971; Banks and Spanos, 1975; Molineux and Gefter, 1975; Christiansen and Baldwin, 1977). We have proposed a model for T3 DNA concatemer formation involving T3 exonuclease (gene 6 product, or gp6) and T3 DBP. By this model, the exonuclease digests the I To whom
reprint
requests
should
0042-6822/81/030148-09$02.00/O Copyright All rights
0 1981 by Academic Press, Inc. of reproduction in any form reserved.
MATERIALS
AND
METHODS
The bacterial and phage strains and M9A medium have been described previously (Fujisawa et al., 1978). DNAs were prepared from purified phage according to Fujisawa et al. (1978). T4 DNA and Co1 El DNA were generous gifts from Mr. Nakasu; fd DNA and A DNA were generous gifts from Dr. H. Yamagishi; T4 gp32 was kindly provided by Dr. M. Takanami.
be addressed. 148
T3 DNA
BINDING
Preparation of T3 DBP. T3 DBP was purified by a modification of the procedure of Fujisawa et al. (1980a). The DBP fraction from ssDNA cellulose column chromatography was applied to a hydroxyapatite column equilibrated with 0.01 M potassium phosphate (pH 7.4)-0.2 mM dithiothreitol10% glycerol and eluted with linear gradient of 0.01 to 0.4 M potassium phosphate (pH 7.4) containing 0.2 mM dithiothreitol10% glycerol. The DBP eluted between 0.1 and 0.13 M potassium phosphate. The eluate was dialyzed against 10 mM potassium phosphate (pH 7.4)-0.2 mM dithiothreitol-50% glycerol and stored at -20°C. [3H]Leucine-labeled DBP was prepared from Escherichia coli cells infected with T3
PROTEIN
149
amber mutant defective in gene 5 (5extract) and labeled with [3H]leucine between 6 and 20 min after infection according to Fujisawa et al., (1980a). In vitro
DNA
packaging
experiments.
Preparation of extracts from phage-infected cells and the in vitro DNA packaging reaction were performed as described in a previous paper (Fujisawa et al., 1978). Assay for renaturation of DNA. T3 DBP and heat-denatured [“Hlthymidinelabeled T3 DNA were mixed in 0.1 M NaCl-Tris-HCl (pH 7.4)-0.2 m&I EDTA (EDTA buffer) and incubated at 30”. At the indicated times, 15~1 portions were poured into 47.5 ~1 of ice-chilled 0.02% SDS-O.3 M NaCl-80 mh4 sodium acetate
FIG. 1. Electron microscopy of complexes of fd DNA with T3 DBP. fd DNA (5 @g/ml) was incubated with various amounts of T3 DBP in 0.01 M potassium phosphate (pH 7.0) for 10 min at 30”. The samples were fixed with 0.01 M glutaraldehyde for 15 min at 30” and spread with formamide and cytochrome c. Free fd DNA was added before spreading. The relative contour length for free fd DNA was 1.98 f 0.08 pm. The amounts of T3 DBP were (a) 40 pgiml, (b) 50 pgiml, and (c) 100 pgiml. Bar represents 0.5 pm.
150
YAMAGISHI,
FUJISAWA,
(pH 4.3)-0.08 mil4 ZnSO, and heated at 70” for 2 min in a water bath. The mixtures were treated with 5 ~1 of Sl nuclease (5000 units) and incubated at 30” for 20 min. Fifty-microliter portions were spotted onto filter paper disks (Toyo Roshi No. 2) and, after washing the disks with trichloroacetic acid and acetone, the acid-insoluble radioactivity was measured in a liquid scintillation counter. Sedimentation analysis of DNA. ssDNA, treated with T3 DBP, was centrifuged through a 5 to 20% sucrose density gradient (5 ml) in EDTA buffer in a Hitachi RPS50-2 rotor at 32,000 rpm for 150 min at 15”. After centrifugation, fractions were collected from the bottom of the gradients onto filter paper disks and radioactivity was measured as described above. Electron microscopy. Samples were mixed with 0.1 M Tris-HCl (pH 8.5), 30% formamide, and 100 pg/ml of cytochrome c.
FIG.
AND
MINAGAWA
20
10 Time
( mln
1
FIG. 2. Kinetics of catalysis of DNA renaturation with T3 DBP. Single-stranded “H-labeled T3 DNA (20 pgiml) was incubated with T3 DBP or T4 gp32 in EDTA buffer and the mixture were incubated at 30”. At the times indicated, portions were taken and Sl nuclease resistant radioactivity was determined as described under Materials and Methods. No protein (x); T4 gp32 (40 pgiml) (0); T3 DBP (80 pgiml) (0); (40 &ml) (0); (20 &ml) (A); (10 pgiml) (W. One-half of the sample containing 80 pg/mI of T3 DBP was incubated for 6 min at 0” and shifted to 30 (0 - - - 0).
3a
T3 DNA
BINDING
PROTEIN
FIG. 3. Electron microscopy of dsDNA formed from ssDNA of T3 DNA by T3 DBP. ssT3 DNA (10 pgiml) was incubated with (a, b) or without (c) T3 DBP (6’7 Fgiml) in EDTA buffer for 10 min at 30”. after the addition of co1 El DNA as a dsDNA marker, the samples were spread with formamide and cytochrome e immediately after the reaction. (a) DNA aggregate; dsDNA with monomer length was arrowed. (b) Renatured product with single-stranded regions. Arrows indicate single-stranded regions. (c) ssDNA incubated without T3 DBP. Supertwisted co1 El molecules can be seen. Bar represents 2 pm.
Fifty microliters of this hyperphase was spread on 1 ml of hypophase containing 0.01 A4 Tris-HCl (pH 8.6) and 20% formamide. The samples were picked ux) on parlodion-coated grids, stained with 0.b5 M many1 acetate in 90% ethanol, washed in isopenteane, and rotatory shadowed with 80% Pt-20% Pd (Yamagishi et al., 1980).
The grids were examined in a Hitachi HU-11D electron microscope. RESULTS
Structure
of the T3 DBP-jd
DNA
Complex
As mentioned in the Introduction, T4 DBP (gp32) cannot substitute for T3 DBP
152
YAMAGISHI,
FUJISAWA.
in the in vitro DNA packaging system of T3. To compare the properties of these two proteins, complexes of fd DNA with T3 DBP or T4 gp32 were examined by electron microscopy. fd DNA complexed with T4 gp32 had an extended conformation as described by Delius et al. (1972). On the contrary, the T3 DBP-fd DNA complex had a condensed appearance like a beaded necklace (Fig. la). With increasing amounts of T3 DBP, the protein-DNA complex became a condensed form with knot-like structures (Fig. lc); their circumferences were 30 and 52% smaller than that measured for the free DNA at the protein:DNA ratios of 1O:l (Fig. lb) and 2O:l (Fig. lc), respectively. Renaturation
of ssDNA with T3 DBP
To examine the effect of T3 DBP on the renaturation of DNA, heat-denatured, “Hlabeled T3 DNA was incubated with T3 DBP or with T4 gp32 in EDTA buffer at 30”. Aliquots were withdrawn at various times and reannealing of DNA was measured by resistance to Sl nuclease as described under Materials and Methods (Fig. 2). ssDNA incubated with T4 gp32 was completely digested with Sl nuclease. On the other hand, most of the DNA became Sl resistant within 20 min of treatment with T3 DBP. These results indicate that T3 DBP catalyzes the renaturation of DNA. The rate of the renaturation of DNA was dependent on the amount of T3 DPB. The reaction proceeded at a very slow rate at 0” (Fig. 2). The addition of spermidine (1 mM) or Mg2+ (10 m&I) was slightly inhibitory for the reaction (data not shown). The optimal concentration of NaCl was between 0.1 M and 0.15 M (data not shown). Electron micrographs clearly show the formation of dsDNA in the presence of T3 DBP (Fig. 3). The DNA tended to form aggregates that contained single-stranded regions (Fig. 3b). Apart from the aggregates, complete dsDNA molecules with monomer size were observed (Fig. 3a). When denatured hDNA was treated with T3 DBP, the DNA was observed by electron microscopy to become double-stranded. However, the renaturation of T4 DNA was
AND
MINAGAWA
not catalyzed by T3 DBP, although the formation of DNA-protein complex was observed (data not shown). Sedimentation profiles of DNA treated with T3 DBP are shown in Fig. 4. ssT3 DNA, after incubation with T3 DBP, sedimented rapidly. Conversion of T3 DNA to the rapidly sedimenting material was dependent upon the concentrations of both the DNA and the DBP (Fig. 4; compare (b) to (c), (c) to (d) or (e), (e) to (f)). The presence of Mg*+ and ATP did not affect the conversion process (data not shown). Rapid sedimentation of DNA was not due to the association of DNA with T3 DBP because SDS treatment at 70” did not affect the sedimentation profile (data not shown). In fact, when a reaction mixture containing ‘IH-labeled DBP and 32P-labeled DNA was incubated and sedimented through gradient, most of the “H-labeled DBP was recovered at the top of the gradient, while “‘P-labeled DNA sedimented rapidly (Fig. 4e). DNA in the fractions of the gradient was resistant to Sl nuclease (Fig. 4f). Stoichiometry
of Binding of the T3 DBP
to ssDNA
The stoichiometry of the binding of T3 DBP to ssDNA was determined by sucrose gradient sedimentation of a fixed amount of fd DNA in the presence of various amounts of :$H-labeled T3 DBP. Two distinct peaks of the radioactivity were seen, one sedimenting rapidly with DNA and the other sedimenting slowly as the free protein (Fig. 5). The amount of radioactivity in the former peak was constant, but that in the latter peak increased with increasing amounts of the protein. From the fraction of protein bound, a weight ratio of protein to DNA of about 1 to 1 was determined. From the known molecular weight of fd DNA (2 x lo”, 6400 nucleotides) and of T3 DBP (average molecular weight, 31,500), we estimate that the DNA-DBP complex contains about one protein molecule for every 100 nucleotides of ssDNA. The Biological Activity DNA
of the Renatured
Previously (Fujisawa et al., 1978), we have shown that exogenous T3 DNA can
T3 DNA
BINDING
153
PROTEIN
packaging with 3--extract gave a single peak at the position corresponding to a dimer size. The relative amount of the two polypeptides was constant through the fractions (Fig. 7b). DISCUSSION
Properties
Fraction
no
FIG. 4. Sedimentation profiles of T3 DNA treated with T3 DBP. Single-stranded “Ylabeled T3 DNA was incubated with T3 DBP in various ratios of DNA to protein in EDTA buffer for 10 min at 30” and sedimented through sucrose gradients. The amounts of DNA and protein were expressed as micrograms per milliliter. (a) No protein; (b) 5 FgDNA to 25 pg protein; (c) 20 pg DNA to 25 pg protein; (d) 20 pg DNA to 180 pg protein; (e) 20 pg DNA to 75 kg protein; (f) 10 pg DNA to 38 pg protein. “‘P-Labeled T3 DNA (0). (e) ‘“P-Labeled DNA and :‘H-labeled DBP (0) were used for the reaction. (f) After centrifugaticJl1, fractions were spotted onto filter paper disks with (x) or without (0) Sl nuclease treatment as described under Materials and Methods.
be packaged into phage particles in an system composed of a mixture of 3-- and 5--extracts (3--extract is an extract prepared from cells infected with a T3 mutant of gene 3). Denatured T3 DNA, when treated with T3 DBP, was active in the in vitro packaging system. As shown in Fig. 6, phage titers increased with increasing amounts of either DNA or DBP. With a particular amount of DNA, a plateau level of packaging was achieved with 50 pg/ml of DBP. The renatured DNA was more efficiently packaged than native DNA.
of the T3 DBP
Like other DBPs that have been described, T3 DBP binds preferentially to single-stranded DNA. However, T3 DBP has certain properties that distinguished it from the other DBPs. The complex of fd DNA with excess T3 DBP appears as a beaded and condensed ring structure; the E. coli DBP-fd DNA complex shows a similar but less folded structure (Sigal et al., 1972). The structure of fd DNA complex with fd gp5 is rod-like (Alberts et al., 1972). T4 gp32 holds fd DNA in an extended configuration (Delius et al., 1972). DNA reassociation catalyzed by T3 DBP does not require Mg’+. In contrast, DNA reassociation by both T4 and E. coli DBPs
in vitro
Evidence
that T3 DBP Aggregates
Purified T3 DBP, when analyzed by SDS-polyacrylamide gel electrophoresis, consists of two protein bands with molecular weights of 31,000 and 32,000 (Fujisawa et al., 1980a). Gel filtration experiment was performed to explore the native molecular weight of T3 DBP in the buffer used for the renaturation reaction (Fig. 7). The complementation activity for in vitro DNA
-
15-
Fractson
no.
FIG. 5. Stoichiometry of binding of T3 DBP to fd DNA. The reaction mixtures (0.2 ml) contained 2 pg fd DNA and 5 pg T3 DBP (a); 2 pg fd DNA and 10 pg T3 DBP (b); or 10 /*g T3 DBP (c). The mixtures were incubated at 1.i” for 30 min and centrifuged through 5 to 30% sucrose gradients in EDTA buffer containing 20 @g/ml of bovine serum albumin in a Hitachi RPS50-2 rotor at 48,000 rpm for 90 min at 4”. Fractions were collected onto filter paper disks and the radioactivity was measured as described under Materials and Methods. The recovery of the radioactivity was more than 90%. The arro\v indicates the position of fd DNA.
154
YAMAGISHI,
FUJISAWA,
require high concentrations of Mgz+. The Eco DBP requires spermidine in addition to Mg2+. Recently, Weinstock et al. (1979) reported that ret A protein has an ability to stimulate annealing of DNA; in this case, both Mg2+ and ATP are required for the reaction. The high rate of DNA reassociation occurs at a lower protein:DNA ratio for T3 DBP. A much higher ratio of protein to DNA is required for other DBPs (Alberts and Frey, 1970; Christiansen and Baldwin, 1977). The high efficiency of the T3 DBP for DNA reassociation may be related to the unique stoichiometric nature of binding of the protein to DNA. At saturation of the protein, the fd DNA-T3 DBP complex contains 100 nucleotides per protein monomer (Fig. 5). Complexes of DNA with other DBPs contain about 10 nucleotides per protein monomer (Alberts and Frey, 1970; Alberts et al., 1972; Oey and Knippers, 1972; Sigal et al., 1972; Reubin and Gefter, 1974). No evidence of the denaturation of dsDNA with T3 DBP was obtained under the conditions which are used for denaturation activity of T4 gp32, E. coli DBP, and fd DBP (data not shown). Although the facts do not exclude the possibility that T3 DBP destabilizes the double helix of DNA under some conditions, the result might be expected because T3 DBP has a strong ability to catalyze reannealing of DNA under the conditions. Our previous results indicate that T3 DBP and gp6 act together to form concatemeric T3 DNA (Fujisawa et al., 1980a). We have also shown that concatemeric DNA is the substrate for packaging DNA into phage particles. After concatemer formation, DBP and gp6 are no longer required for the in vitro packaging reaction. Denatured T3 DNA, after treatment with T3 DBP, is packaged more efficiently than native T3 DNA in the in vitro packaging system (Fig. 6). This is not due to concatemer formation, as DBP-treated DNA is not packaged in 3-- or 6-extract (data not shown). We suggest that the action of DBP on denatured T3 DNA forms structures that are converted to concatemers more efficiently than native T3 DNA.
AND
MINAGAWA
0
50 100 T3 DBP ( )-‘g/ml )
FIG. 6. Biological activity of dsDNA produced by T3 DBP. Heat-denatured T3 DNA was incubated with various amounts of T3 DBP in EDTA buffer for 15 min at 30”. After the incubation, the mixture was heated to 70” for 2 min and added to i?z vitro DNA packaging system using a mixture of Y--extract and Cextract. The concentrations of DNA were 60 pgiml (0); 18 pgiml (0); and 5 kg/ml (A). Phage yields in the in vitro system using native T3 DNA at the given concentrations are indicated on the ordinate as horizontal bars.
Role of the T3 DBP in in Vitro DNA Packaging
In a previous paper (Fujisawa et al., 1980a), we postulated two possible roles for the T3 DBP in the in vitro packaging reaction. One is that DBP binds to singlestranded regions at the termini of T3 DNA generated by gp6 exonuclease. By binding at these regions, the DBP would prevent excessive digestion by gp6 exonuclease. However, T3 DBP does not affect the exonucleolytic activity of gp6 (data not shown). The second possibility is that DBP stimulates intermolecular pairing of single-stranded regions produced by gp6 at the redundant termini of T3 DNA. The present results are more consistent with the second possibility than the first. The T3 DBP may be involved in genetic recombination because the pairing of homologous strands is an important early step in recombination (see a review by Radding, 1978). In fact, in vitro recombination is greatly reduced in 3--extract (Fujisawa et al., 1978). It is possible that the T3 DBP may have additional functions. The T7 DBP is thought to be involved in DNA replication (Scherzinger et al., 1973; Reubin and Gefter, 1974; Richardson et al., 1979). The availability of a host mutant defec-
T3 DNA
Hemoglobtn
Cytochrome
BINDING
no
1s
c
14 Fraction
PROTEIN
b
15
16
17 18 Fraction
19 no.
20
21
22
FIG. 7. Gel filtration of T3 DBP. T3 DBP (80 pgiml) was loaded onto a Sephadex G-200 column (1.5 x 75 cm) elquilibrated with EDTA buffer with cytochrome c (MW = 13,000) and hemoglobin (MW = 67,000) as molecular weight standards. The eluates were assayed for ilc vitro complementing activity with S--extract (a) and were subjected to SDS-polyacrylamide gel electrophoresis (b) as described elsewhere (Fujisawa et rti., 1980b). B.D., blue dextran.
tive in E. coli DBP (ssb-1 (Meyer et al., 1979)) makes it possible to examine the participation of phage DBP in various DNA metabolic processes. ACKNOWLEDGMENTS We are grateful to Dr. F. Fujimura, La Jolla Cancer Research Foundation, for invaluable help with the manuscript, and Dr. Ryo for his helpful discussion. This study was in part supported by grantsin-aid for Scientific Research to H.F. from the Ministry of Education of Japan (348386).
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replication and 227, 1313-1318. ALBERTS, B. M., cent excitement Nnfxre (London)
recombination.
Nature
(Lo~dorrl
and STERNGI.ANZ, R. (1977). Rein the DNA replication problem. 269, 655-661.
BANKS, G. R., and SPANOS, A. (1975). The isolation and properties of a DNA-unwinding protein from Ustilngo maydis. J. Mol. Biol. 93, 63-77. CHAMPOUX, J. J. (1978). Proteins that affect DNA conformation. Annu. Rev. Biochem. 47, 449-480. CHRISTIANSEN, C., and BALDWIN. R. L. (1977). Catalysis of DNA association by the Escherichin coli DNA binding protein. Polyamine dependent reaction. J. Mol. Biol. 115, 441-454. DELIUS, H., MANTELL, N. ,J.. and ALBERTS. B. (1972). Characterization by electron microscopy of the complex formed between T4 bacteriophage gene 32-protein and DNA. J. Mol. Biol. 67, 341350. FUJISAU’A. H.. MIYAZAKI, J., and MINAGAWA. T. (1978). In vitro packaging of phage T3 DNA. Virology 87, 394-400.
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YAMAGISHI,
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FUJISAWA, H., YAMAGISHI, M., MATSUO-KATO, H., and MINAGAU~A, T. (1980a). Purification of DNAbinding proteins of bacteriophage T3 and their role in i)~ vitro packaging of phage T3 DNA. Virology 105, 480-489. FU.JISAWA, H., YAMAGISHI, M.. and MIXAGAB’A, T. (1980b). II/ csifro formation of the concatemeric DNA of bacteriophage T3 and its biological role in the in z’jtro packaging reaction. Virology 101, 327-334. HOTTA, Y., and STERN, H. (1971). A DNA-binding protein in meiotic cells of Lilium, Develop. Biol. 26, 87-99. MEYER, R. R., GLASSBERG, J.. and KORNBERG, A. (1979). An Escherichia coli mutant defective in single-stranded binding protein is defective in DNA replication. Proc. b’at. Acad. Sri. USA. 76, 17021705. MOLINEUX, I. J., and GEFTE:R, M. L. (1975). Properties of the Eschetichia coli DNA-binding (unwinding) protein interaction with nucleolytic enzymes and DNA. d. Mol. Biol. 98, 811-825. OXY. J. L., and KNIPPERS, R. (1972). Properties of the isolated gene 5 protein of bacteriophage fd. d. Mol. Biol. 68, 125-138. RADDING, C. M. (1978). Genetic recombination: Strand transfer and mismatch repair. Au1174. RPU. Biochen~. 17, 847-80.
AND MINAGAWA REUBIP~, R. C., and GEFTER, M. L. (1974). A deoxyribonucleic acid binding protein induced by bacteriophage T’7. Purification and properties of the protein. J. Biol. Chem. 249, 3843-3850. RICHARDSON, C. C.. ROMANO, L. J., KOLODNER. J. E., TAMAAVI, F., ENGLER, M. J., DEAN. F. B., and RICHARDSON. D. S. (1979). Replication of bacteriophage T7 by purified proteins. Cold Spll’ng Harbor
Symp.
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43, 427-440.
SCHERZIXGER, E.. LITFIN, F.. and .JOST, E. (1973). Stimulation of T7 DNA polgmearse by a new phagr-coded protein. Mol. Gen. Gewf. 123, 247262. SIG& and tein with
N., DELIUS, H., KORNBERG, T.. GF.FTE:R.M., ALBERTS, B. (1972). A DNA-unwinding proisolated from Eschetichia coli: Its interaction DNA and DNA polymerases. Proc. Nat. Accrd. SC;. USA 69, 3537-3541.
WEXSTOCK, G. M.. MCENTEE, K., and I,F,HILIAN. I. R. (1979). ATP-dependent renaturation of DNA catalyzed by the ret A protein of Eschwichia co/i. Proc. Nnf. Acad. Sci. USA 76, 126-130. YAYAGISHI, M., FUJISA~VA, H., YAMAGISHI. H., and MINAGAU’A. T. (1980). Purification of gene 6 product of bacteriophage T3 and its role in in vitro DNA packaging. Virology 100, 382-389.