J. Mol. Biol. (1975) 93, 63-77
The Isolation and Properties of a DNA-unwinding from Ustilago maydis
Protein
G. R. BANKS AND A. SPANOS National Institute for Medical Mill Hill, London NW7 IAA,
Research Engkmd
(Received 17 September 1974) Mitotic cells of the eukaryote U&ago waaydis contain a DNA-unwinding protein. It has been purified to apparent homogeneity and it possesses many of the properties of the proksryotic DNA-unwinding proteins. It has a molecular weight of 20,000, binds tightly and specifically to single-stranded DNA and catalyses its renatumtion at physiological temperatures. It also denatures double-stranded DNA, reducing the melting temperature of poly[d(A-T)] * [d(A-T)] by almost 50 deg. C. One molecule of the protein apparently binds to not more than seven to ten single-stranded DNA nucleotides and there is evidence that the binding may be co-operative. It stimulates the initial rate of DNA synthesis by the Ustilago DNA polymerase when the latter repairs partially single-stranded DNA templates. Conversely, it inhibits the degradation of double-stranded DNA by Eschetichia coli exonuclease III and that of singlestranded DNA by U. naaydb DNAase I. One cell of U. maydis contains 2.5 to 3.0X IO6 molecules of the protein, a number that may suggest 8 structural rather than a catalytic role.
1. Introduction DNA-unwinding proteins have been isolated from several prokaryotic organisms (Albert8 et al., 1972; Oey & Knippers, 1972; Reuben & Gefter, 1973,1974; Scherzinger et al., 1973 ; Sigal et al., 1972). Their properties are similar to those of the product of bacteriophage T4 gene 32, the first of these proteins to be isolated and characterized (Alberts & Frey, 1970; Delius et al., 1972; Huberman et al., 1971). Because of the existence of strains of T4 carrying mutations in this gene, there is direct evidence that the protein is involved in DNA replication and recombination (Epstein et al., 1963; Tomizawa et al., 1966; Berger et al., 1969; Broker & Lehman, 1971; Sinha & Snustad, 1971). This evidence is lacking for the other proteins, but because their properties are so similar, they may also possesssimilar functions. It has been suggested that the proteins may be required at the DNA replication fork (Alberts & Prey, 1970; Alberts, 1970; Huberman et al., 1971; Ioannou, 1973; Pratt e.tal., 1974), and models for recombination invoke unwinding of the DNA strands of homologous chromosomes followed by reannealing to form heteroduplex molecules (Whitehouse, 1963; Holliday, 1964,1968; Meselson, 1972; Sigal & Alberts, 1972). The properties of the binding proteins may suggest a role for them in these processes, because they are able to bind tightly and specifically to single-stranded DNA, unwind double-stranded DNA, stimulate the rate of DNA synthesis by DNA polymerases in vitro, and in some cases catalyse the renaturation of DNA. 63
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Similar proteins have been isolated from eukaryotic sources. Hotta & Stern (1971ab) were able to detect them after partial purification of extracts of meiotic cells, but not of their mitotic counterparts, of Lilium and several mammals. They suggested that such proteins promote pairing and crossing-over of DNA strands of homologous chromosomes during meiosis. The P8 protein of cultured mammalian cells binds to single-stranded DNA, but possesses none of the other properties of the prokaryotic unwinding proteins (Tsai & Green, 1973). Recently, an unwinding protein has been isolated from calf thymus tissue (Herrick & Alberta, 1973). We describe the isolation protein from mitotic cells of the basidioand some properties of a DNA-unwinding mycete fungus Ustilugo may&s.
2. Experimental Procedure (a) Materials Calf thymus DNA, ovalbumin, pancreatic DNAase I and bacterial alkaline phosphatase inhibitor from the Sigma were products of the Worthington Biochemical Corp., trypsin and deoxyribonucleoside triphosphates Chemical Co., lysozyme, poly[d(A-T)].[d(A-T)] from PL Biochemicals Inc., bovine serum albumin from Pentex and Eeclte&chia co& exonuclease III from the Boehringer Corp. Munktell410 cellulose was supplied by Camlab and carboxymethylcellulose (CM32) by Whatman Biochemicals Ltd. Radiochemicals were from the Radiochemical Centre and nitrocellulose filters were type BA86 from Schleicher and Schiill. U. maydia DNAase I was a gift from Dr W. K. Holloman and U. lnuydis DNA polymerase from M. V. K&iris. (b) Method8 (i) Nucleic
a&d-s
T7 DNA, unlabelled and labelled with [3H]thymidine, was extracted from the purified phage particles essentially according to Richardson (1966). It was denatured by heating at 100°C for 10 min and rapidly cooled in ice, or by the alkali treatment of Studier (1965). Native T7 DNA was treated with exonuclease III until 5 to 10% of the total nucleotides were made acid-soluble. (ii) DNA-cellulose Single-stranded calf thymus DNA-cellulose was prepared by the method of Alberts & Herrick (1971). About I.5 mg DNA per g cellulose remained bound after extensive washing. (iii)
Polymylamide
gel electrophwesis
The procedure for native polyacrylamide gel electrophoresis was that of Davis (1964). After electrophoresis, the gels were stained with 0.25% Coomassie blue in 50% methanol, 10% acetic acid, destained and scanned at 640 nm using a Unicam SP1809 scanning densitometer. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate was carried out according to Weber & Osborn (1969), except that the gels contained 5 M-urea. Samples were prepared for electrophoresis by concentration to about 0.5 mg protein/ml by dialysis against 30% polyethylene glycol, followed by dialysis against 0.01 M-sodium phosphate (pH 7+2), 0.1% sodium dodecyl sulphate, O-1 y. 2-mercaptoethanol and 5 M-Urea, and the resulting solution heated at IOO’C for 2 min. (iv) Prot&n determinations Protein concentrations were determined bovine serum albumin as the standard.
by the
method
of Lowry
et al. (1951)
using
(v) Aesczys (1) DNA-binding PO&&. The sssay measures the amount of single-stranded T7 [3H]DNA bound to nitrocellulose filters in the presence of the protein and is essentially that developed by Tad & Green (1973). Denatured T7 [3H]DNA (1 to 5 pg) wss incubated
DNA-UNWINDING
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with the protein (0 to 30 pg) in 20 mu-Tris.HCl (pH 8-O), 1 mu-EDTA, 2 mb4-t-mercaptoethanol, 50 mm-N&l and 5% glycerol (total volume 1.0 ml) at 20°C for 10 min. The solution was then filtered at a flow-rate of 5 ml/min through a nitrocellulose titer prewashed with 10 ml buffer BD (20 ma6-Tris*HCl (pH 8-O), 1 mM-EDTA, 2 mna-2-mercaptoethanol, 50 mM-N&Cl, 5% glycerol and 1% dimethylsulphoxide). The filter was washed 3 times with 3 ml buffer BD, dried and the radioactivity determined in & toluene-bssed scintilla.tion fluid. The nitrocellulose filters were boiled in water and soaked overnight in buffer BD before use. In the absence of the binding protein, about 1% of the DNA in the assay was retained by the filter. Maximum binding activity w&s attained in less than 30 s at 25°C and remained constant for 20 min. It was not possible to assay the protein in crude cell-free extracts by this method because of the presence of endogenous DNA. Even after removal of the latter (fraction II), estimates of binding activity were unreliable, presumably because other proteins interfered, and it was not until fraction III in the purification procedure that the assay became reproducible, and proportionality between the amount of protein and DNA bound WEW achieved (see Fig. 4). The reaction mixtures (0.15 ml) contained 67 mM(2) U. maydis DNA poZymerme. 10 mM-dithiothreitol, 6.7 mM each of Tris.HCl (pH 7.5), 100 m&r-KCl, 6.7 mu-MgCl,, dATP, TTP, dGTP and dCTP, I.25 PCi r3H]TTP, U. rrsaydis DNA polymerase (10 to 15 units) and DNA template as indicated (Banks, Holloman, K&iris, Yarranton & Spanos, unpublished work). After incubation at 37”C, O.l-ml portions of the mixture were assayed by the method of Bollum (1968). (3) Deoxyribonucleases. E. coli exonuclease III was assayed by the method of Richard& Holliday (1973). Fraction son et al. (1964) and U. maydis DNAase I by that of Holloman V of the binding protein was assayed for DNA polymerase activity as described above, exonuclease activity by the method of Holloman & Holliday (1973), endonucleaae activity by that of Wright et al. (197 1) using ColEI [3H]DNA and RNAase H activity by the method of Banks (1974). (vi) Growth of cells U. may&a (wild-type haploid aab, strain) was grown in 300 1 medium containing 3 kg Oxoid yeast extract, 6 kg Oxoid bacteriological Peptone, 6 kg sucrose and 50 ml Silicone RD antifoam at 32°C. The cells (approx. 12 kg) were harvested at late log phase by centrifugation in a Sharples supercentrifuge model 6A at 15,000 revs/min and the packed cells stored at -20°C. (vii)
Purification
All operations
of
the binding protein
were conducted
at 4OC.
(1) Preparation of crude eztruct. Frozen cells (40 g) were suspended and washed twice in buffer A (20 m&f-Tris.HCl (pH &+O), 1 mM-EDTA, 1 mrvr-2-mercaptoethanol, 50 mu-N&l and 10% glycerol). The cells were then suspended in 80 ml buffer A containing 2.0 M-N&~ by sonic&ion (30 s at full power with a Dawe Soniprobe fitted with a microtip) and crushed by passage through a French pressure cell at 16,000 lb/in2. Cell debris w&s removed by centrifugation at 12,000 revs/min for 30 min to give fraction I. (2) Removal of nucleic a&da. Nucleic acids were removed by precipitation with polyethylene glycol (Carbowax 6000) (Alberts & Herrick, 1971). Polyethylene glycol (30% in 2.0 x-N&l) was added slowly with stirring to fraction I to a flnel concentration of lo%, and after stirring for a further 20 min, the suspension was centrifuged at 18,000 revs/min for 20 min to remove the nucleic acid precipitate. The resulting clear supernatant fraction W&S dialysod against two 5-1 changes in buffer A for 20 h. The precipitate formed during dialysis was removed by centrifugation to give fraction II. (3) DNA c*olumn washed 6
cellulose chromatography. Fraction II (95 ml) was loaded at 10 ml/h on a (3 cm x 12 cm) of single-stranded DNA cellulose that. had been extensively with buf%r A. The column w&8 washed with buffer A containing O-1 M-NaCl
G. R. BANKS
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(300 ml) and then eluted with a gradient of O-16 M to 2.0 ~-Nacl in buffer A (total 300 ml). The resulting fraotions were each dialysed overnight against buffer A and for DNA binding activity. The peak fractions were combined to give fraction III. preparations, the O-1 M-NaCI wash was followed by 0.6 u-NaCl (200 ml), and the activity eluted by 2-O M-NaCl (all in buffer A).
volume assayed In later binding
Fraction III (50 ml) was loaded on a column of CM(4) CM-ceZZ&se c!~onza~gqn~y. cellulose (2 cm x 20 cm), which was then washed with buffer A (100 ml) and then eluted with a 0.1 to 0.6 M-NaCl linear gradient in the same buffer system (total volume 300 ml). The single-stranded DNA binding activity was found in the fractions eluted during loading and washing. The peak fractions were concentrated by dialysis against 30% glycerol in buffer A or, when necessary, 6rst by dialysis against 30% polyethylene glycol in buffer A to give fraction IV. It wss stored at - 15°C at 0.1 to 0.15 mg/ml and was stable for 3 to 4
1
(a) I.6 -
I.2 -
0.6 -
0 Origin
I
2
3
4
5
6
7
Gel length (cm)
Fra. 1. Polyacrylamide gel eleotrophoresis of Ustilqo binding protein fraotions. After eleotrophorcsis, the bands on the gels were visualized as desoribed in Experimental Procedure. The traoes are of fraotion III (a) or IV (b) in the presence of sodium dodlecyl sulphate and uwa (20 ,qq protein for each). Migration was from left to right.
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months under these conditions. The recovery of the protein cannot be calculated precisely, because it was not possible to essay binding activity in crude extracts (seeabove). Fraction IV was purified I-fold over fraction III with a yield of 86%.
3. Results When a single-stranded DNA cellulose column was loaded with fraction II and then &ted with a 0.15 to 2.0 M-N&I gradient, binding activity appeared at 1.6 u-salt. Sometimes 5 to 10% of the total binding activity was not retained by the column, but was retained when applied to B second column. When about half the usual amount of protein was ohromatographed on a DNA-cellulose column of the same dimensions as those given above, the binding activity wa,s less strongly bound, being eluted at 04 to 1.0 M-N&L On analysis of fraction III by polyacrylamide gel electrophoresis either in the presence (Fig. l(a)) or absence of sodium dodecyl sulphete and urea, four protein bands were revealed by staining with Coomassie blue. Identical analysis of fraction IV revealed a single protein band (Fig. l(b)). The molecular weight of this protein, determined by comparison of its relative mobility during gel electrophoresis in the presence of sodium dodeoyl sulphate with those of proteins of known molecular weights, was about 20,000 (Fig. 2).
Pm. 2. Migration of the Ustilago binding protein during polyaorylamide gel eleotrophoresis. Fraction IV was run in gels containing sodium dodeoyl sulpbate and urea along with the marker proteins lysozyme (L), trypsin inhibitor (TI), panoreatio DNAI (D), ovalbumin (0), bovine serum elbumin (BSA) and its dimer (BSAs). Molecular weights were taken from the Handbook of Biochmniatry (ed. by H. A. Sober, the Chemical Rubber Co., Cleveland, 1970).
A sample of fraction IV was sedimented through a glycerol gradient and fractions from the gradient assayed for binding activity (Fig. 3). A single peak of activity was found with a sedimentation coefficient of 26 S, which agrees well with a molecular weight of about 20,000, assuming an essentially spherical molecule and a partial specific volume similar to that of the marker protein (Martin & Ames, 1901). Fraction IV possessed no detectable exo- or endodeoxyribonuclease, DNA poly merase or RNAase H activity when assayed m described in Experimental Procedure.
88
G. R. BANKS
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Fraction no
Pm. 3. Glycerol gradient sedimentation of the U&ago binding protein. Fraction IV (60 pg) was dialysed into 60 mnn-Tris.HCl (pH ‘7.6), 10 m&r-2-mercaptoethanol, 10% glycerol and layered on a preformed 20% to 40% glycerol gradient in the same buffer. Centrifugation was for 21 h at 46,000 revs/mm and 4°C in a SW66 rotor of a Beckman L266B ultracentrifuge. Fractions were collected after puncturing the bottom of the tube and a portion of each assayed in the standard binding assay. Bacterial alkaline phosphatase (6.3 S) was included as a marker and was assayed by the method of Garen & Levinthal (1960).
Although fraction IV, eluted from the CM-cellulose column before the salt gradient, was unable to bind to double-stranded T7 DNA (see Fig. 4), two proteins that could bind to this DNA were eluted by the subsequent gradient. Both bound less efficiently to single-stranded T7 DNA, in that an amount of each su&ient to bind 5 pg doublestranded DNA to nitrocellulose filters, bound only 0.6 pg single-stranded DNA in the standard assay. The properties of these proteins are now under investigation. (b) Binding of the protein to DNA The Ustikqo protein bound to single, but not to double-stranded T7 [3H]DNA (with and without 5 x 10v4 J/mm2 U.V. irradiation), when assayed by the standard binding reaction (Fig. 4). Single-stranded E. coli [3H]DNA was also equally e0icient in the binding assay. When the binding activity with single-stranded DNA was plotted as a function of binding protein concentration, a pronounced sigmoidal curve was generated, which was not altered by decreasing the degree of washing of the nitrocellulose Glters (Fig. 4), in contrast to the binding found for E. coli RNA polymerase to DNA (Hinkle & Chamberlin, 1972). To determine if the protein itself was able to bind to the filters, 50 pg was incubated in a standard assay mixture but lacking DNA. The solution was filtered through a nitrocellulose fdter and the filtrate collected. To this was then added single-stranded T7 [3H]DNA and, after further incubation, filtered through a second alter. No DNA (less than 1% of the input) was bound to the latter, suggesting that the binding protein itself had bound to the first filter in the absence of DNA. It has been observed that other proteins will also bind to nitrocellulose filters (Hinkle t
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,
0 Binding protein (rg)
Fro. 4. DNA binding aativity of the U&&go protein as a function of its oonoentration. The atanderd binding assay included inareclsing amounts of protein using 6 erg denatured T7 [3H]DNA u&radiated or irradiated with 6 x 10 - l J/mm” (-a-e-) or native T7 [3H]DNA (-O-O-), U.V. irradiation. The in& shows the initial portion of the ourve. Duplicate aaasys were run and after filtration of the reaction mixtures, one membrane was wsahed es usual (--a-@-) and the other was dried and oounted without washing (-+-+-). Control ~sseys contained no binding protein, and the radioaotivity thet was ret.sined wea subtracted from the experimental V8lU08.
Chamberlin, 1972). The sigmoidal nature of the binding activity versus protein concentration curve may indicate co-operative binding, or that more than one protein molecule per DNA strand is required to bind the latter to the filter (see Discul3sion). (c) Denuturatima alad rencduration of poly[d(A-T)] in the presence of the protein
*[d(A-T)]
When an excess of the U&ago protein (see Discussion for the method of calculation) was incubated with poly[d(A-T)].[d(A-T)] at 25°C for 30 minutes, a l-3-fold increase in the optical density of the solution at 260 nm was observed. This hyperchromic shift is characteristic of the helix-coil transition of DNA, and could be eliminated by increasing the NaCl concentration to 0.6 Y or decreasing the temperature to 4°C. The temperature at which this transition occurred was determined by the experiments shown in Figure 5. The optical density at 260 nm of a mixture of an exceaa of the protein and poly[d(A-T)]-[d(A-T)] was determined at increasing temperatures from 5 to 16°C. The results of these experimenta show that, in the absence of magnesium ions, the transition occurred at 10*7”C!. This, however, is not an equilibrium value, because when the optical densities were determined after the temperature cycle wag reversed, the lower melting temperature of 9*5”C! was found. In the presence of
70
C4. R. BANKS
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AND
I 3
A. SPANOS
I II
Temperature
I
13
AL.-..I!5
I
(“C)
FIG. 6. The absorbance versus temperature profile of poly[d(A-T)]+[d(A-T)] in the presenoe of the U&lag0 binding protein. The reaotion mixtures contained the alternating copolymer (6.3 pg) and binding protein (60 pg) in 10 maa-potassium phosphate (pH 76), 33 m&r-KC& 0.3 maa-EDTA, 1 mn-2meroeptoethanol, 6% glyoerol with and without 10 mna-MgCls at 6% (total volume 1-O ml). The blank oontained all oomponents except the alternating oopolymer. The mixtures were incubated in 1 am p&h-length ouvettes in 8 thermostatted auvette holder of a Zeiss PMQll speotrophotometer. The temperature, aontrolled by a water bath containing a cooling ooil, was increased by about 1 deg. C, and 16 mm later the temperature in B seoond blank ouvette determined by a Mettler TM16 temperature probe and the absorbanoe at 260 nm of the reaction mixtures determined. When the temperature reached 16°C (maximum hyperohromiaity), the temperature cycle was reversed and readings taken as described above. Reaction mixtures without (-a--e-, -O-O-) or with (-A-A-, --A-A-) magnesium ions, increasing (-a--e--, -A-A-) or deoreesing (-O---O-, -A-A-) the temperature. The absorbance values are uucorreoted for volume ohanges.
magnesium ions the transition on increasing the temperature occurred at 126°C and there was no hysteresis on decreasing the temperature again. The melting temperetures for poly[d(A-T)]+[d(A-T)] under identical conditions but in the absence of the protein, were 54°C and 62°C in the absence and presence of magnesium ions, respeotively. Thus the melting temperature of this alternating copolymer appears to be decreased by some 50 deg. C by the Ustilczgo protein. The transition is sharp, occurring over a range of only 2 to 3 deg. C ; and magnesium ions increase the rate of renaturation induced by the protein. No turbidity of the above reaction mixtures was observed, but when attempts were made to measure the melting temperature of T7 DNA in the presence of the protein, the solutions rapidly became turbid at the higher temperatures required. (d) Renaturation of single-stranded T7 DNA cutalysed by the protein To demonstrate the renaturation of single strands of T7 DNA by the Ustilago protein, use was made of the different buoyant densities of the single and doublestranded DNAs. The protein was incubated in the presence of magnesium ions with alkali-denatured T7 DNA as described in the legend to Figure 6. After treatment with
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U. MAYDIS
71
D
FIG. 6. Analytiaal ultreoentrifugation in E oaesium ohloride gradient of renatured T7 DNA. The reaction mixtures (total volume 0.56 ml) oontained alkali-denatured DNA (7.6 pg) with (a) no, (b) 11.6 clg (O-28 x saturation) or (0) 40 ~lg (1.12 x saturation) binding protein in 2 rnMTria.HCl (pH 8*0), 1 m-2.meroeptoethenol, 0.1 ma6-EDTA, 120 -.-Kc1 and 10 mxu-MgCl,. After incubation at 37°C for 30 min, 100 pg sodium dodeayl eulphate was added to disrupt proteinDNA oomplexes, and a sample of eaoh diluted and C&l added to 1.26 g/ams. Yeast mitoohondrial DNA waa added aa a marker (ref., p = 1.683 g/am3). The solutions were centrifuged to equilibrium in a Be&man model E analytical ultracentrifuge at 44,000 reva/min for 20 h at 20°C. The photographed gradients were scanned by a Joyae-Loebl densitometer. Both native (arrow N) and alkali-denatured (arrow D) were also run with the marker DNA.
sodium dodecyl sulphate to destroy protein-DNA complexes, the buoyant density of the DNA was determined by analytical ultracentrifugation in a caesium chloride gradient. After incubation with subsaturating levels of protein (Fig. 6(b)), a relatively broad band of DNA was observed, possessing a mean density between those for double and single-stranded T7 DNAs. However, when saturating levels of the protein were used (Fig. 6(c)), almost all of the DNA was in a sharp band with a density of that for
U. R. BANKS
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native T7 DNA. The U&ago protein is therefore able to catalyse the conversion of single-stranded DNA molecules to their fully double-stranded state. Little, if any, renaturation was observed in the absence of the protein (Fig. 6(a)). (e) Effect of the binding protein on in vitro DNA q/rztbis by the Ustilago DNA polymerase Only one DNA polymerase has been detected so far in cell-free extracts of U. maya% (Jeggo, 1973), and there is some evidence that it is responsible for DNA replication in viva (Jeggo et al., 1973). The purified enzyme preferentially uses native DNA templates that have been extensively nicked with pancreatic DNAase I or partially degraded with E. coli exonuclease III, is also active to a limited extent with heatdenatured calfthymus DNA, but almost inactive on denatured T7 DNA (Jeggo, 1973; Banks et al., unpublished work). When the polymerase activity using an exonuclease III-treated native DNA template was determined in the presence of increasing amounts of the Ustdugo binding protein, a twofold stimulation was found (Fig. 7(a)). If the amount of binding protein present in the assay was increased above that necessary for this stimulation, the latter was gradually inhibited. DNA synthesis by the Ustilugo polymerase with exonuclease III-treated templates appears to be of the repair type, because enough deoxyribonucleotides are incorporated into acid-insoluble DNA to make the terminal single-stranded regions, generated by the exonuclease III, double-stranded. No more nucleotides are then incorporated. Other evidence using homopolydeoxyribonucleotide templates with complementary homooligodeoxyribonucleotide primers also lead to this conclusion (Banks et al., unpublished work). Addition of the binding protein does not influence the final extent of synthesis, but does increase the initial rate (Fig. 7(b)). Very little, if any, stimulation occurred if DNAase I-nicked calf thymus DNA templates were used, but a twofold 200
100*
Binding protein (pg/ossoy) (a)
lncubotion
time IminI ib)
FIG. 7. Stimulation of DNA synthesis by the U.&&go binding protein. Assays with the U.&ago DNA polymer&se were oar&d out as described in Experimental Proooedure and oontained 2.26 nmol nuoleotide equivalent of native T7 DNA, of whioh 170 pmol (7.6%) had been mede single&ended by treatment with exonuolease III. In (a) assays were for 6 min and inoluded increasing @mounts of the binding protein, end in (b) 0.2 pg binding protein was present (--O-O-) or ebeent (-e-a--) and the reaction mixtures inoubated for the times indioeted.
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stimulation was also obtained with heat-denatured calf thymus DNA (results not shown). The protein did not stimulate the rate of DNA synthesis by either E. coli DNA polymerase I or the Micrococcus luteus polymerase when repairing partially single-stranded templates. (f) Effect of the binding protein on deoxyribonuclease activities (i) E. coli exonucleme III The degradation of native T7 [3H]DNA by exonuclease III was assayed in the presence of increasing amounts of the binding protein. A complete inhibition of activity could be obtained, 5076 inhibition occurring when enough protein was added to saturate the DNA present (Fig. 8). This inhibition presumably reflects the ability of the protein to denature the double-stranded DNA, the specific substrate for exonuclease III. The fd gene 5 and T7 unwinding proteins also inhibit this exonuclease (Oey & Knippers, 1972; Scherzinger et al., 1973). (ii) U. maydis DNAase I Figure 8 also shows that the binding protein could inhibit completely the degradation of single-stranded T7 [3H]DNA by Us&go DNAase I. This enzyme exhibits a high specificity for single-stranded substrates (Rolloman t Holliday, 1973 ; Holloman, 1973), but it can nick double-stranded DNA containing mismatched bases (Holloman & Ahmad, personal communication) and is implicated in recombination (Holloman & Holliday, 1973). Its inhibition may result from the protein binding to the singlestranded DNA, which is then inaccessible to the nucleolytic action of the enzyme. About a threefold excess of protein necessary to saturate the DNA was required to generate 50% inhibition of the DNAase I, however.
FIO. 8. Inhibition of deoxyribonuclease activity by the u8tdago binding protein. Assays were oarried out aa desoribed in Experimental Prooedure and contained in 0.16 ml, native T7 [sH]DNA (0.18 H) and exonuolease III (0.19 units), incubated for 30 min at 37°C (-•-a-), or heatdenatured T7 [sH]DNA (0.2 pg) and Uatila~o DNAaae T (0.7 units), incubated for 16 min at 37°C (-o-o-).
74
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4. Discussion A DNA-binding protein has been purified to apparent homogeneity from mitotic cells of the simple eukaryote U. may&s. It is not known if it is nuclear and/or cytoplasmic in origin. Its properties are very similar to those of the unwinding proteins isolated from several prokaryotic sources (Alberts & Frey, 1970; Huberman et al., 1971; Alberts et al., 1972; Oey & Knippers, 1972; Reuben & Gefter, 1973,1974; Sigal et al., 1972; Scherzinger et al., 1973). The single protein band has a molecular weight of about 20,000 when determined by polyacrylamide gel electrophoresis under protein denaturing conditions. This value was also found for the binding activity from glycerol gradient sedimentation, which suggests that self-association, found with the gene 32 and fd gene 5 products, and the E. mli protein (Carroll et al., 1972 ; Oey & Knippers, 1972; Molineux et al., 1974) does not occur to a significant extent under these conditions. The T4 protein forms stable dimers under a variety of conditions, although higher aggregates of the monomer may predominate under others (Carroll et al., 1972), while the E. coli protein exists as a stable tetramer (Molineux et al., 1974). The degree of self-association of the former depends on salt and protein concentrations, pH and the age of the preparation. In particular, it has been shown to be unstable under the low ionic strength used for the glycerol gradient sedimentation of the Ustilago protein. Further molecular weight determinations under a variety of conditions should determine if this protein is also capable of self-association. The binding activity is specific for single-stranded DNA and a sigmoidal curve is found when binding activity versus protein concentration is plotted. The nature of this curve was not perturbed by the degree of washing of the nitrocellulose filters, and because the protein was apparently bound to the latter in the absence of any DNA, it may indicate co-operative binding by the protein to single-stranded DNA or a requirement for more than one protein molecule per single strand to bind the latter to the filter. McGhee & von Hippel (1974) have recently analysed in detail the problem in interpreting binding curves for the non-specific (with respect to the base sequence in the DNA) class of DNA-binding proteins. These arise because potential binding sites overlap one another and so, at any degree of binding saturation, the remaining number of sites depends on the distribution of bound protein molecules as well as on their number. The fact that the strength of binding of the Ustilago protein to singlestranded DNA-cellulose is related to its concentration is also circumstantial evidence for binding involving protein-protein interactions (see Results, section (a)). Sucrose gradient sedimentation of [3H]protein-DNA complexes with varying protein-DNA ratios, as done for other DNA-binding proteins, should resolve this question. Experiments are in progress to examine protein-protein interactions using [“Hlleucinelabelled material and to visualize the protein-DNA complexes in the electron microscope. If the linear part of the binding curve (Fig. 4) reflects the stoichiometry between DNA and the protein bound to it, a simple calculation reveals that 1 pg single-stranded DNA binds 6 to 7 pg protein. This gives a figure of one protein molecule per 7 to 10 single-stranded nucleotides. The calculation rests on the assumption that at each point on the curve, the DNA bound to the filter is saturated with protein. Thus when the minimum amount of protein that binds all the DNA in the assay to the filter is present, no more protein is able to bind to that DNA. Further binding would not be detected because the assay measures only DNA bound to the filter. The above figure for the number of nucleotides bound by a single protein
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molecule is, therefore, only a maximum. If the DNA bound is only half saturated with protein, then one molecule would bind to 3 to 6 nucleotides. The proteins investigated by other workers bind to 3 to 11 nucleotides, depending on the size of the protein monomer. A calculation of the number of protein molecules per cell is difhcult, because it was found not to be possible to assay accurately the binding protein in crude extracts, but assuming 100% recovery of activity from the DNA-cellulose chromatography, 1 g of cells yields 25 to 30 pugprotein. This gives a figure of 2.5 to 3.0~ IO6 molecules of binding protein per cell. The Ustilago binding protein stimulates the initial rate of in vitro DNA synthesis by the Ustilago DNA polymerase when it repairs partially single-stranded DNA templates, but does not influence the final amount of synthesis. The maximum stimulation is observed when about enough protein is present to saturate the singlestranded regions (calculated from the above figures) end is independent of the amount of polymerase. This suggests that its action is mediated principally via interaction with the template rather than the enzyme. The stimulation of in vitro DNA synthesis by binding proteins from other organisms does show specificity, however, because it is found only when both the polymerase and binding protein originate from the same organism, although the E. coli and T7-induced binding proteins both stimulate synthesis by the T7 polymerase (Huberman et al., 1971; Herrick t Alberts, 1973; Reuben & Gefter, 1973,1974; Scherzinger et al., 1973). We have not investigated the specificity of the Ustihzgo binding protein fully, but no stimulation of DNA synthesis by the E. coli polymerase I and M. lute%9 polymerase was detected using partially single-stranded DNA templates. Reuben & Gefter (1974) have concluded that unwinding of the DNA template is not a sufficient explanation for the stimulation of DNA synthesis, but suggest that in addition, each polymerase may require the template to be aligned in a very specific configuration, which is induced only by the unwinding protein from the same organism as the polymerase. Our results support this suggestion, but it is perhaps surprising if the template for replication were aligned in a different way for each organism. We have attempted to detect an association of the binding protein with the Ustilago DNA polymerase by sedimentation of a mixture of the two through a glycerol gradient as described for the binding protein alone. No shift in the peaks of the binding (2.6 S) or polymerase (8.3 S) activities from those of the one in the absence of the other was detected, but it is possible that the method would not detect a weak association. It is clear from our investigations that, in contrast to the conclusions of Hotta & Stern (1971a,b), DNA-unwinding proteins can occur in mitotic cells of a eukaryotic organism. These results must weaken their evidence for the involvement of these proteins in meiotic recombination only. We now hope to determine if the same or a different protein is present in meiotio cells of U. may&s. We have assessed the levels of the binding protein in three mutant strains. Two of these, 1~011-1and tsd432, are temperature sensitive for DNA replication. There is evidence that the former possesses a temperature sensitive replicative DNA polymerase (Jeggo et al., 1973) and tsd432 is also defective in mitotic allelic recombination (Hollidey et al., 1975). Both were grown at the permissive temperature (22°C) and after switching to the restrictive temperature (32°C) for five hours, the binding protein was purified to fraction III. Wild-type levels were found in poll-l, but tsd432 contained two to three times the binding activity of the wild-type strain. Both
76
G. 33. BANKS
AND
A. SPANOS
proteins
were able to denature poly[d(A-T)]*[d(A-T)] and stimulate the wild-type DNA polymerase. The third mutant, reel-1, is sensitive to ultraviolet and ionising radiation and defective in spontaneous and radiation-induced mitotic allelic recombination (Holliday, 1967 ; Holliday et al., 1975). This also possessed a wild-type binding protein level. Thus the function of the binding protein in U. may&s is not yet known, but since its properties are so similar to those of the T4 gene 32 product, its role in DNA replication and recombination may also be similar. Furthermore, its ability to inhibit deoxyribonucleases might also be important in protecting single-stranded DNA within the cell from these enzymes, which might otherwise be lethal. U&ago
We thank Dr D. H. Williamson for carrying out the analytical ultraoentrifugation runs, Dr P. Unrau for providing the strains of U. maydis and Drs R. Ho&day and W. K. Holloman for many valuable discussions. REFERENCES Alberts, B. M. (1970). Fed. Proc. Fed. Amer. Sot. Eqn. Biol. 29, 11541163. Alberts, B. M. & Frey, L. (1970). Nature (London), 227, 1313-1318. Alberts, B. & Herrick, G. (1971). Methods in Enzymology (Grossman, L. & Moldave, K., eds), vol. 21D, pp. 198217, Academic Press, New York. Alberts, B., Frey, L. & Delius, H. (1972). J. ilfol. Biol. 68, 139-152. Banks, G. R. (1974). Eur. J. Biochem. 47, 499-507. Berger, H., Warren, A. J. & Fry, K. E. (1969). J. ViroZ. 3, 171-175. Bollum, F. J. (1968). Methods in Enzymology (Grossman, L. & Moldave, K., eds), vol. 21B, pp. 591-611, Academic Press, New York. Broker, T. R. & Lehman, I. R. (1971). J. MoZ. BioZ. 60, 131-149. Carroll, R. B., Neet, K. E. & Goldthwait, D. A. (1972). Proc. Nat. Acad. Sci., U.S.A. 69, 2741-2744. Davis, B. J. (1964). Ann. N. Y. Acad. Sci. 121, 404-427. Delius, H., Mantell, N. J. & Alberta, B. (1972). J. MoZ. BioZ. 67, 341-360. Epstein, R. H., Bolle, A., Steinberg, C. M., Kellenberger, E., Boy de la Tour, E., Chevalley, R., Edgar, R. S., Susman, M., Denhardt, G. H. & Lielausis, H. (1963). CoZo!Spring Harbor Symp. Quant. BioZ. 28, 375-392. Garen, A. & Levinthal, C. (1960). Biochim. Biophys. Acta, 38, 470-483. Herrick, G. & Alberts, B. (1973). Fed. Proc. Fed. Amer. Sot. Exp. BioZ. 32, 497. Hinkle, D. C. & Chamberlin, M. J. (1972). J. Mol. Biol. 70, 157-185. Holliday, R. (1964). Genet. Re.s. 5, 282-304. Holliday, R. (1967). Mutat. Rea. 4, 275-288. Holliday, R. (1968). In RepZicution and Recombination of Genetic Materiul (Peacock, W. J. & Brock, R. D., eds), pp. 157-174, Australian Academy of Science, Canberra. Holliday, R., Holloman, W. K., Banks, G. R., Unrau, P. & Pugh, J. E. (1975). Oak Ridge Symposium on Mechaniems in Recombination. Holloman, W. K. (1973). J. BioZ. Chem. 248, 8114-8119. Holloman W. K. & Holliday, R. (1973). J. BioZ. Chem. 248, 8107-8113. Hotta, Y. & Stern, H. (1971a). Develop. BioZ. 26, 87-99. Hotta, Y. & Stern, H. (1971b). Nature New Biol. 234, 83-86. Huberman, J. A., Kornberg, A. & Alberts, B. M. (1971). J. Mol. BioZ. 62, 39-62. Ioannou, P. (1973). Nature New BioZ. 244, 257-260. Jeggo, P. A. (1973). Ph.D. Thesis, C.N.A.A., London. Jeggo, P. A., Unrau, P., Banks, G. R. & Holliday, R. (1973). Nature New BioZ. 242, 14-16. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951). J. Biol. Chem. 193, 265275. Martin, R. G. & Ames, R. N. (1961). J. Biol. Chem. 236, 1372-1379. McGhee, J. D. & von Hippel, P. H. (1974). J. Mol. Biol. 86, 46+490.
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Me&son, M. (1972). J. Mol. Biol. 71, 795-798. Molineux, I. J., Friedman, S. t Gefter, M. L. (1974). J. B&d. Chem. 249, 6090-6098. Oey, J. L. & Knippers, R. (1972). J. Mol. Biol. 68, 125-138. Pratt, D., Laws, P. & Griffith, J. (1974). J. Mol. Bid. 82, 425-439. Reuben, R. C. & Gefter, M. L. (1973). Proc. Nat. Acud. Sci., U.S.A. 70, 184&1850. Reuben, R. C. & Gefter, M. L. (1974). J. Biol. Chem. 249, 3843-3850. Richadson, C. C. (1966). J. Mol. BioZ. 15, 49-61. Richardson, C. C., Lehman, I. R. & Kornberg, A. (1964). J. Biol. Chem. 239, 251-258. Scherzinger, E., Litfin, F. & Jost, E. (1973). Mol. Gen. Genet. 123, 247-262. Sigal, N. & Alberts, B. (1972). J. Mol. BioZ. 71, 789-793. Sigttl, N., Delius, H., Kornberg, T., Gefter, M. L. & Alberts, B. (1972). Proc. Nat. Acad. Sci., U.S.A. 69, 3537-3541. Sinha, N. K. & Snustad, D. P. (1971). J. Mol. BioZ. 62, 267-271. Studier, F. W. (1965). J. Mol. BioZ. 11, 373-390. Tomizawa, J., Anreku, N. & Iwama, Y. (1966). J. Mol. Biol. 21, 247-253. Tsai, R. L. & Green, H. (1973). J. Mol. BioZ. 73, 307-316. Weber, K. & Osborn, M. (1969). J. BioZ. Chem. 244, 4406-4412. Whitehouse, H. L. K. (1963). 2\rature (London), 199, 103P1040. Wright, M., Buttin, 0. & Hurwitz, J. (1971). J. BioZ. Chem. 246, 6543-6555.
The Isolation and Properties of a DNA-unwinding from Ustilago maydis
Protein
G. R. BANKS AND A. SPANOS National Institute for Medical Mill Hill, London NW7 IAA,
Research Engkmd
(Received 17 September 1974) Mitotic cells of the eukaryote U&ago waaydis contain a DNA-unwinding protein. It has been purified to apparent homogeneity and it possesses many of the properties of the proksryotic DNA-unwinding proteins. It has a molecular weight of 20,000, binds tightly and specifically to single-stranded DNA and catalyses its renatumtion at physiological temperatures. It also denatures double-stranded DNA, reducing the melting temperature of poly[d(A-T)] * [d(A-T)] by almost 50 deg. C. One molecule of the protein apparently binds to not more than seven to ten single-stranded DNA nucleotides and there is evidence that the binding may be co-operative. It stimulates the initial rate of DNA synthesis by the Ustilago DNA polymerase when the latter repairs partially single-stranded DNA templates. Conversely, it inhibits the degradation of double-stranded DNA by Eschetichia coli exonuclease III and that of singlestranded DNA by U. naaydb DNAase I. One cell of U. maydis contains 2.5 to 3.0X IO6 molecules of the protein, a number that may suggest 8 structural rather than a catalytic role.
1. Introduction DNA-unwinding proteins have been isolated from several prokaryotic organisms (Albert8 et al., 1972; Oey & Knippers, 1972; Reuben & Gefter, 1973,1974; Scherzinger et al., 1973 ; Sigal et al., 1972). Their properties are similar to those of the product of bacteriophage T4 gene 32, the first of these proteins to be isolated and characterized (Alberts & Frey, 1970; Delius et al., 1972; Huberman et al., 1971). Because of the existence of strains of T4 carrying mutations in this gene, there is direct evidence that the protein is involved in DNA replication and recombination (Epstein et al., 1963; Tomizawa et al., 1966; Berger et al., 1969; Broker & Lehman, 1971; Sinha & Snustad, 1971). This evidence is lacking for the other proteins, but because their properties are so similar, they may also possesssimilar functions. It has been suggested that the proteins may be required at the DNA replication fork (Alberts & Prey, 1970; Alberts, 1970; Huberman et al., 1971; Ioannou, 1973; Pratt e.tal., 1974), and models for recombination invoke unwinding of the DNA strands of homologous chromosomes followed by reannealing to form heteroduplex molecules (Whitehouse, 1963; Holliday, 1964,1968; Meselson, 1972; Sigal & Alberts, 1972). The properties of the binding proteins may suggest a role for them in these processes, because they are able to bind tightly and specifically to single-stranded DNA, unwind double-stranded DNA, stimulate the rate of DNA synthesis by DNA polymerases in vitro, and in some cases catalyse the renaturation of DNA. 63
64
G. R. BANKS
AND
A. SPANOS
Similar proteins have been isolated from eukaryotic sources. Hotta & Stern (1971ab) were able to detect them after partial purification of extracts of meiotic cells, but not of their mitotic counterparts, of Lilium and several mammals. They suggested that such proteins promote pairing and crossing-over of DNA strands of homologous chromosomes during meiosis. The P8 protein of cultured mammalian cells binds to single-stranded DNA, but possesses none of the other properties of the prokaryotic unwinding proteins (Tsai & Green, 1973). Recently, an unwinding protein has been isolated from calf thymus tissue (Herrick & Alberta, 1973). We describe the isolation protein from mitotic cells of the basidioand some properties of a DNA-unwinding mycete fungus Ustilugo may&s.
2. Experimental Procedure (a) Materials Calf thymus DNA, ovalbumin, pancreatic DNAase I and bacterial alkaline phosphatase inhibitor from the Sigma were products of the Worthington Biochemical Corp., trypsin and deoxyribonucleoside triphosphates Chemical Co., lysozyme, poly[d(A-T)].[d(A-T)] from PL Biochemicals Inc., bovine serum albumin from Pentex and Eeclte&chia co& exonuclease III from the Boehringer Corp. Munktell410 cellulose was supplied by Camlab and carboxymethylcellulose (CM32) by Whatman Biochemicals Ltd. Radiochemicals were from the Radiochemical Centre and nitrocellulose filters were type BA86 from Schleicher and Schiill. U. maydia DNAase I was a gift from Dr W. K. Holloman and U. lnuydis DNA polymerase from M. V. K&iris. (b) Method8 (i) Nucleic
a&d-s
T7 DNA, unlabelled and labelled with [3H]thymidine, was extracted from the purified phage particles essentially according to Richardson (1966). It was denatured by heating at 100°C for 10 min and rapidly cooled in ice, or by the alkali treatment of Studier (1965). Native T7 DNA was treated with exonuclease III until 5 to 10% of the total nucleotides were made acid-soluble. (ii) DNA-cellulose Single-stranded calf thymus DNA-cellulose was prepared by the method of Alberts & Herrick (1971). About I.5 mg DNA per g cellulose remained bound after extensive washing. (iii)
Polymylamide
gel electrophwesis
The procedure for native polyacrylamide gel electrophoresis was that of Davis (1964). After electrophoresis, the gels were stained with 0.25% Coomassie blue in 50% methanol, 10% acetic acid, destained and scanned at 640 nm using a Unicam SP1809 scanning densitometer. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate was carried out according to Weber & Osborn (1969), except that the gels contained 5 M-urea. Samples were prepared for electrophoresis by concentration to about 0.5 mg protein/ml by dialysis against 30% polyethylene glycol, followed by dialysis against 0.01 M-sodium phosphate (pH 7+2), 0.1% sodium dodecyl sulphate, O-1 y. 2-mercaptoethanol and 5 M-Urea, and the resulting solution heated at IOO’C for 2 min. (iv) Prot&n determinations Protein concentrations were determined bovine serum albumin as the standard.
by the
method
of Lowry
et al. (1951)
using
(v) Aesczys (1) DNA-binding PO&&. The sssay measures the amount of single-stranded T7 [3H]DNA bound to nitrocellulose filters in the presence of the protein and is essentially that developed by Tad & Green (1973). Denatured T7 [3H]DNA (1 to 5 pg) wss incubated
DNA-UNWINDING
PROTEIN
FROM
U. MAYDIS
66
with the protein (0 to 30 pg) in 20 mu-Tris.HCl (pH 8-O), 1 mu-EDTA, 2 mb4-t-mercaptoethanol, 50 mm-N&l and 5% glycerol (total volume 1.0 ml) at 20°C for 10 min. The solution was then filtered at a flow-rate of 5 ml/min through a nitrocellulose titer prewashed with 10 ml buffer BD (20 ma6-Tris*HCl (pH 8-O), 1 mM-EDTA, 2 mna-2-mercaptoethanol, 50 mM-N&Cl, 5% glycerol and 1% dimethylsulphoxide). The filter was washed 3 times with 3 ml buffer BD, dried and the radioactivity determined in & toluene-bssed scintilla.tion fluid. The nitrocellulose filters were boiled in water and soaked overnight in buffer BD before use. In the absence of the binding protein, about 1% of the DNA in the assay was retained by the filter. Maximum binding activity w&s attained in less than 30 s at 25°C and remained constant for 20 min. It was not possible to assay the protein in crude cell-free extracts by this method because of the presence of endogenous DNA. Even after removal of the latter (fraction II), estimates of binding activity were unreliable, presumably because other proteins interfered, and it was not until fraction III in the purification procedure that the assay became reproducible, and proportionality between the amount of protein and DNA bound WEW achieved (see Fig. 4). The reaction mixtures (0.15 ml) contained 67 mM(2) U. maydis DNA poZymerme. 10 mM-dithiothreitol, 6.7 mM each of Tris.HCl (pH 7.5), 100 m&r-KCl, 6.7 mu-MgCl,, dATP, TTP, dGTP and dCTP, I.25 PCi r3H]TTP, U. rrsaydis DNA polymerase (10 to 15 units) and DNA template as indicated (Banks, Holloman, K&iris, Yarranton & Spanos, unpublished work). After incubation at 37”C, O.l-ml portions of the mixture were assayed by the method of Bollum (1968). (3) Deoxyribonucleases. E. coli exonuclease III was assayed by the method of Richard& Holliday (1973). Fraction son et al. (1964) and U. maydis DNAase I by that of Holloman V of the binding protein was assayed for DNA polymerase activity as described above, exonuclease activity by the method of Holloman & Holliday (1973), endonucleaae activity by that of Wright et al. (197 1) using ColEI [3H]DNA and RNAase H activity by the method of Banks (1974). (vi) Growth of cells U. may&a (wild-type haploid aab, strain) was grown in 300 1 medium containing 3 kg Oxoid yeast extract, 6 kg Oxoid bacteriological Peptone, 6 kg sucrose and 50 ml Silicone RD antifoam at 32°C. The cells (approx. 12 kg) were harvested at late log phase by centrifugation in a Sharples supercentrifuge model 6A at 15,000 revs/min and the packed cells stored at -20°C. (vii)
Purification
All operations
of
the binding protein
were conducted
at 4OC.
(1) Preparation of crude eztruct. Frozen cells (40 g) were suspended and washed twice in buffer A (20 m&f-Tris.HCl (pH &+O), 1 mM-EDTA, 1 mrvr-2-mercaptoethanol, 50 mu-N&l and 10% glycerol). The cells were then suspended in 80 ml buffer A containing 2.0 M-N&~ by sonic&ion (30 s at full power with a Dawe Soniprobe fitted with a microtip) and crushed by passage through a French pressure cell at 16,000 lb/in2. Cell debris w&s removed by centrifugation at 12,000 revs/min for 30 min to give fraction I. (2) Removal of nucleic a&da. Nucleic acids were removed by precipitation with polyethylene glycol (Carbowax 6000) (Alberts & Herrick, 1971). Polyethylene glycol (30% in 2.0 x-N&l) was added slowly with stirring to fraction I to a flnel concentration of lo%, and after stirring for a further 20 min, the suspension was centrifuged at 18,000 revs/min for 20 min to remove the nucleic acid precipitate. The resulting clear supernatant fraction W&S dialysod against two 5-1 changes in buffer A for 20 h. The precipitate formed during dialysis was removed by centrifugation to give fraction II. (3) DNA c*olumn washed 6
cellulose chromatography. Fraction II (95 ml) was loaded at 10 ml/h on a (3 cm x 12 cm) of single-stranded DNA cellulose that. had been extensively with buf%r A. The column w&8 washed with buffer A containing O-1 M-NaCl
G. R. BANKS
66
AND
A. SPANOS
(300 ml) and then eluted with a gradient of O-16 M to 2.0 ~-Nacl in buffer A (total 300 ml). The resulting fraotions were each dialysed overnight against buffer A and for DNA binding activity. The peak fractions were combined to give fraction III. preparations, the O-1 M-NaCI wash was followed by 0.6 u-NaCl (200 ml), and the activity eluted by 2-O M-NaCl (all in buffer A).
volume assayed In later binding
Fraction III (50 ml) was loaded on a column of CM(4) CM-ceZZ&se c!~onza~gqn~y. cellulose (2 cm x 20 cm), which was then washed with buffer A (100 ml) and then eluted with a 0.1 to 0.6 M-NaCl linear gradient in the same buffer system (total volume 300 ml). The single-stranded DNA binding activity was found in the fractions eluted during loading and washing. The peak fractions were concentrated by dialysis against 30% glycerol in buffer A or, when necessary, 6rst by dialysis against 30% polyethylene glycol in buffer A to give fraction IV. It wss stored at - 15°C at 0.1 to 0.15 mg/ml and was stable for 3 to 4
1
(a) I.6 -
I.2 -
0.6 -
0 Origin
I
2
3
4
5
6
7
Gel length (cm)
Fra. 1. Polyacrylamide gel eleotrophoresis of Ustilqo binding protein fraotions. After eleotrophorcsis, the bands on the gels were visualized as desoribed in Experimental Procedure. The traoes are of fraotion III (a) or IV (b) in the presence of sodium dodlecyl sulphate and uwa (20 ,qq protein for each). Migration was from left to right.
DNA-UNWINDINU
PROTEIN
FROM
U. MAYDIS
67
months under these conditions. The recovery of the protein cannot be calculated precisely, because it was not possible to essay binding activity in crude extracts (seeabove). Fraction IV was purified I-fold over fraction III with a yield of 86%.
3. Results When a single-stranded DNA cellulose column was loaded with fraction II and then &ted with a 0.15 to 2.0 M-N&I gradient, binding activity appeared at 1.6 u-salt. Sometimes 5 to 10% of the total binding activity was not retained by the column, but was retained when applied to B second column. When about half the usual amount of protein was ohromatographed on a DNA-cellulose column of the same dimensions as those given above, the binding activity wa,s less strongly bound, being eluted at 04 to 1.0 M-N&L On analysis of fraction III by polyacrylamide gel electrophoresis either in the presence (Fig. l(a)) or absence of sodium dodecyl sulphete and urea, four protein bands were revealed by staining with Coomassie blue. Identical analysis of fraction IV revealed a single protein band (Fig. l(b)). The molecular weight of this protein, determined by comparison of its relative mobility during gel electrophoresis in the presence of sodium dodeoyl sulphate with those of proteins of known molecular weights, was about 20,000 (Fig. 2).
Pm. 2. Migration of the Ustilago binding protein during polyaorylamide gel eleotrophoresis. Fraction IV was run in gels containing sodium dodeoyl sulpbate and urea along with the marker proteins lysozyme (L), trypsin inhibitor (TI), panoreatio DNAI (D), ovalbumin (0), bovine serum elbumin (BSA) and its dimer (BSAs). Molecular weights were taken from the Handbook of Biochmniatry (ed. by H. A. Sober, the Chemical Rubber Co., Cleveland, 1970).
A sample of fraction IV was sedimented through a glycerol gradient and fractions from the gradient assayed for binding activity (Fig. 3). A single peak of activity was found with a sedimentation coefficient of 26 S, which agrees well with a molecular weight of about 20,000, assuming an essentially spherical molecule and a partial specific volume similar to that of the marker protein (Martin & Ames, 1901). Fraction IV possessed no detectable exo- or endodeoxyribonuclease, DNA poly merase or RNAase H activity when assayed m described in Experimental Procedure.
88
G. R. BANKS
AND
A. SPANOS
Fraction no
Pm. 3. Glycerol gradient sedimentation of the U&ago binding protein. Fraction IV (60 pg) was dialysed into 60 mnn-Tris.HCl (pH ‘7.6), 10 m&r-2-mercaptoethanol, 10% glycerol and layered on a preformed 20% to 40% glycerol gradient in the same buffer. Centrifugation was for 21 h at 46,000 revs/mm and 4°C in a SW66 rotor of a Beckman L266B ultracentrifuge. Fractions were collected after puncturing the bottom of the tube and a portion of each assayed in the standard binding assay. Bacterial alkaline phosphatase (6.3 S) was included as a marker and was assayed by the method of Garen & Levinthal (1960).
Although fraction IV, eluted from the CM-cellulose column before the salt gradient, was unable to bind to double-stranded T7 DNA (see Fig. 4), two proteins that could bind to this DNA were eluted by the subsequent gradient. Both bound less efficiently to single-stranded T7 DNA, in that an amount of each su&ient to bind 5 pg doublestranded DNA to nitrocellulose filters, bound only 0.6 pg single-stranded DNA in the standard assay. The properties of these proteins are now under investigation. (b) Binding of the protein to DNA The Ustikqo protein bound to single, but not to double-stranded T7 [3H]DNA (with and without 5 x 10v4 J/mm2 U.V. irradiation), when assayed by the standard binding reaction (Fig. 4). Single-stranded E. coli [3H]DNA was also equally e0icient in the binding assay. When the binding activity with single-stranded DNA was plotted as a function of binding protein concentration, a pronounced sigmoidal curve was generated, which was not altered by decreasing the degree of washing of the nitrocellulose Glters (Fig. 4), in contrast to the binding found for E. coli RNA polymerase to DNA (Hinkle & Chamberlin, 1972). To determine if the protein itself was able to bind to the filters, 50 pg was incubated in a standard assay mixture but lacking DNA. The solution was filtered through a nitrocellulose fdter and the filtrate collected. To this was then added single-stranded T7 [3H]DNA and, after further incubation, filtered through a second alter. No DNA (less than 1% of the input) was bound to the latter, suggesting that the binding protein itself had bound to the first filter in the absence of DNA. It has been observed that other proteins will also bind to nitrocellulose filters (Hinkle t
DNA-UNWINDING
PROTEIN
FROM
U. M,4 YDZS
69
,
0 Binding protein (rg)
Fro. 4. DNA binding aativity of the U&&go protein as a function of its oonoentration. The atanderd binding assay included inareclsing amounts of protein using 6 erg denatured T7 [3H]DNA u&radiated or irradiated with 6 x 10 - l J/mm” (-a-e-) or native T7 [3H]DNA (-O-O-), U.V. irradiation. The in& shows the initial portion of the ourve. Duplicate aaasys were run and after filtration of the reaction mixtures, one membrane was wsahed es usual (--a-@-) and the other was dried and oounted without washing (-+-+-). Control ~sseys contained no binding protein, and the radioaotivity thet was ret.sined wea subtracted from the experimental V8lU08.
Chamberlin, 1972). The sigmoidal nature of the binding activity versus protein concentration curve may indicate co-operative binding, or that more than one protein molecule per DNA strand is required to bind the latter to the filter (see Discul3sion). (c) Denuturatima alad rencduration of poly[d(A-T)] in the presence of the protein
*[d(A-T)]
When an excess of the U&ago protein (see Discussion for the method of calculation) was incubated with poly[d(A-T)].[d(A-T)] at 25°C for 30 minutes, a l-3-fold increase in the optical density of the solution at 260 nm was observed. This hyperchromic shift is characteristic of the helix-coil transition of DNA, and could be eliminated by increasing the NaCl concentration to 0.6 Y or decreasing the temperature to 4°C. The temperature at which this transition occurred was determined by the experiments shown in Figure 5. The optical density at 260 nm of a mixture of an exceaa of the protein and poly[d(A-T)]-[d(A-T)] was determined at increasing temperatures from 5 to 16°C. The results of these experimenta show that, in the absence of magnesium ions, the transition occurred at 10*7”C!. This, however, is not an equilibrium value, because when the optical densities were determined after the temperature cycle wag reversed, the lower melting temperature of 9*5”C! was found. In the presence of
70
C4. R. BANKS
I 7
AND
I 3
A. SPANOS
I II
Temperature
I
13
AL.-..I!5
I
(“C)
FIG. 6. The absorbance versus temperature profile of poly[d(A-T)]+[d(A-T)] in the presenoe of the U&lag0 binding protein. The reaotion mixtures contained the alternating copolymer (6.3 pg) and binding protein (60 pg) in 10 maa-potassium phosphate (pH 76), 33 m&r-KC& 0.3 maa-EDTA, 1 mn-2meroeptoethanol, 6% glyoerol with and without 10 mna-MgCls at 6% (total volume 1-O ml). The blank oontained all oomponents except the alternating oopolymer. The mixtures were incubated in 1 am p&h-length ouvettes in 8 thermostatted auvette holder of a Zeiss PMQll speotrophotometer. The temperature, aontrolled by a water bath containing a cooling ooil, was increased by about 1 deg. C, and 16 mm later the temperature in B seoond blank ouvette determined by a Mettler TM16 temperature probe and the absorbanoe at 260 nm of the reaction mixtures determined. When the temperature reached 16°C (maximum hyperohromiaity), the temperature cycle was reversed and readings taken as described above. Reaction mixtures without (-a--e-, -O-O-) or with (-A-A-, --A-A-) magnesium ions, increasing (-a--e--, -A-A-) or deoreesing (-O---O-, -A-A-) the temperature. The absorbance values are uucorreoted for volume ohanges.
magnesium ions the transition on increasing the temperature occurred at 126°C and there was no hysteresis on decreasing the temperature again. The melting temperetures for poly[d(A-T)]+[d(A-T)] under identical conditions but in the absence of the protein, were 54°C and 62°C in the absence and presence of magnesium ions, respeotively. Thus the melting temperature of this alternating copolymer appears to be decreased by some 50 deg. C by the Ustilczgo protein. The transition is sharp, occurring over a range of only 2 to 3 deg. C ; and magnesium ions increase the rate of renaturation induced by the protein. No turbidity of the above reaction mixtures was observed, but when attempts were made to measure the melting temperature of T7 DNA in the presence of the protein, the solutions rapidly became turbid at the higher temperatures required. (d) Renaturation of single-stranded T7 DNA cutalysed by the protein To demonstrate the renaturation of single strands of T7 DNA by the Ustilago protein, use was made of the different buoyant densities of the single and doublestranded DNAs. The protein was incubated in the presence of magnesium ions with alkali-denatured T7 DNA as described in the legend to Figure 6. After treatment with
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1.723
U. MAYDIS
71
D
FIG. 6. Analytiaal ultreoentrifugation in E oaesium ohloride gradient of renatured T7 DNA. The reaction mixtures (total volume 0.56 ml) oontained alkali-denatured DNA (7.6 pg) with (a) no, (b) 11.6 clg (O-28 x saturation) or (0) 40 ~lg (1.12 x saturation) binding protein in 2 rnMTria.HCl (pH 8*0), 1 m-2.meroeptoethenol, 0.1 ma6-EDTA, 120 -.-Kc1 and 10 mxu-MgCl,. After incubation at 37°C for 30 min, 100 pg sodium dodeayl eulphate was added to disrupt proteinDNA oomplexes, and a sample of eaoh diluted and C&l added to 1.26 g/ams. Yeast mitoohondrial DNA waa added aa a marker (ref., p = 1.683 g/am3). The solutions were centrifuged to equilibrium in a Be&man model E analytical ultracentrifuge at 44,000 reva/min for 20 h at 20°C. The photographed gradients were scanned by a Joyae-Loebl densitometer. Both native (arrow N) and alkali-denatured (arrow D) were also run with the marker DNA.
sodium dodecyl sulphate to destroy protein-DNA complexes, the buoyant density of the DNA was determined by analytical ultracentrifugation in a caesium chloride gradient. After incubation with subsaturating levels of protein (Fig. 6(b)), a relatively broad band of DNA was observed, possessing a mean density between those for double and single-stranded T7 DNAs. However, when saturating levels of the protein were used (Fig. 6(c)), almost all of the DNA was in a sharp band with a density of that for
U. R. BANKS
72
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native T7 DNA. The U&ago protein is therefore able to catalyse the conversion of single-stranded DNA molecules to their fully double-stranded state. Little, if any, renaturation was observed in the absence of the protein (Fig. 6(a)). (e) Effect of the binding protein on in vitro DNA q/rztbis by the Ustilago DNA polymerase Only one DNA polymerase has been detected so far in cell-free extracts of U. maya% (Jeggo, 1973), and there is some evidence that it is responsible for DNA replication in viva (Jeggo et al., 1973). The purified enzyme preferentially uses native DNA templates that have been extensively nicked with pancreatic DNAase I or partially degraded with E. coli exonuclease III, is also active to a limited extent with heatdenatured calfthymus DNA, but almost inactive on denatured T7 DNA (Jeggo, 1973; Banks et al., unpublished work). When the polymerase activity using an exonuclease III-treated native DNA template was determined in the presence of increasing amounts of the Ustdugo binding protein, a twofold stimulation was found (Fig. 7(a)). If the amount of binding protein present in the assay was increased above that necessary for this stimulation, the latter was gradually inhibited. DNA synthesis by the Ustilugo polymerase with exonuclease III-treated templates appears to be of the repair type, because enough deoxyribonucleotides are incorporated into acid-insoluble DNA to make the terminal single-stranded regions, generated by the exonuclease III, double-stranded. No more nucleotides are then incorporated. Other evidence using homopolydeoxyribonucleotide templates with complementary homooligodeoxyribonucleotide primers also lead to this conclusion (Banks et al., unpublished work). Addition of the binding protein does not influence the final extent of synthesis, but does increase the initial rate (Fig. 7(b)). Very little, if any, stimulation occurred if DNAase I-nicked calf thymus DNA templates were used, but a twofold 200
100*
Binding protein (pg/ossoy) (a)
lncubotion
time IminI ib)
FIG. 7. Stimulation of DNA synthesis by the U.&&go binding protein. Assays with the U.&ago DNA polymer&se were oar&d out as described in Experimental Proooedure and oontained 2.26 nmol nuoleotide equivalent of native T7 DNA, of whioh 170 pmol (7.6%) had been mede single&ended by treatment with exonuolease III. In (a) assays were for 6 min and inoluded increasing @mounts of the binding protein, end in (b) 0.2 pg binding protein was present (--O-O-) or ebeent (-e-a--) and the reaction mixtures inoubated for the times indioeted.
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stimulation was also obtained with heat-denatured calf thymus DNA (results not shown). The protein did not stimulate the rate of DNA synthesis by either E. coli DNA polymerase I or the Micrococcus luteus polymerase when repairing partially single-stranded templates. (f) Effect of the binding protein on deoxyribonuclease activities (i) E. coli exonucleme III The degradation of native T7 [3H]DNA by exonuclease III was assayed in the presence of increasing amounts of the binding protein. A complete inhibition of activity could be obtained, 5076 inhibition occurring when enough protein was added to saturate the DNA present (Fig. 8). This inhibition presumably reflects the ability of the protein to denature the double-stranded DNA, the specific substrate for exonuclease III. The fd gene 5 and T7 unwinding proteins also inhibit this exonuclease (Oey & Knippers, 1972; Scherzinger et al., 1973). (ii) U. maydis DNAase I Figure 8 also shows that the binding protein could inhibit completely the degradation of single-stranded T7 [3H]DNA by Us&go DNAase I. This enzyme exhibits a high specificity for single-stranded substrates (Rolloman t Holliday, 1973 ; Holloman, 1973), but it can nick double-stranded DNA containing mismatched bases (Holloman & Ahmad, personal communication) and is implicated in recombination (Holloman & Holliday, 1973). Its inhibition may result from the protein binding to the singlestranded DNA, which is then inaccessible to the nucleolytic action of the enzyme. About a threefold excess of protein necessary to saturate the DNA was required to generate 50% inhibition of the DNAase I, however.
FIO. 8. Inhibition of deoxyribonuclease activity by the u8tdago binding protein. Assays were oarried out aa desoribed in Experimental Prooedure and contained in 0.16 ml, native T7 [sH]DNA (0.18 H) and exonuolease III (0.19 units), incubated for 30 min at 37°C (-•-a-), or heatdenatured T7 [sH]DNA (0.2 pg) and Uatila~o DNAaae T (0.7 units), incubated for 16 min at 37°C (-o-o-).
74
Cl. R. BANKS
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4. Discussion A DNA-binding protein has been purified to apparent homogeneity from mitotic cells of the simple eukaryote U. may&s. It is not known if it is nuclear and/or cytoplasmic in origin. Its properties are very similar to those of the unwinding proteins isolated from several prokaryotic sources (Alberts & Frey, 1970; Huberman et al., 1971; Alberts et al., 1972; Oey & Knippers, 1972; Reuben & Gefter, 1973,1974; Sigal et al., 1972; Scherzinger et al., 1973). The single protein band has a molecular weight of about 20,000 when determined by polyacrylamide gel electrophoresis under protein denaturing conditions. This value was also found for the binding activity from glycerol gradient sedimentation, which suggests that self-association, found with the gene 32 and fd gene 5 products, and the E. mli protein (Carroll et al., 1972 ; Oey & Knippers, 1972; Molineux et al., 1974) does not occur to a significant extent under these conditions. The T4 protein forms stable dimers under a variety of conditions, although higher aggregates of the monomer may predominate under others (Carroll et al., 1972), while the E. coli protein exists as a stable tetramer (Molineux et al., 1974). The degree of self-association of the former depends on salt and protein concentrations, pH and the age of the preparation. In particular, it has been shown to be unstable under the low ionic strength used for the glycerol gradient sedimentation of the Ustilago protein. Further molecular weight determinations under a variety of conditions should determine if this protein is also capable of self-association. The binding activity is specific for single-stranded DNA and a sigmoidal curve is found when binding activity versus protein concentration is plotted. The nature of this curve was not perturbed by the degree of washing of the nitrocellulose filters, and because the protein was apparently bound to the latter in the absence of any DNA, it may indicate co-operative binding by the protein to single-stranded DNA or a requirement for more than one protein molecule per single strand to bind the latter to the filter. McGhee & von Hippel (1974) have recently analysed in detail the problem in interpreting binding curves for the non-specific (with respect to the base sequence in the DNA) class of DNA-binding proteins. These arise because potential binding sites overlap one another and so, at any degree of binding saturation, the remaining number of sites depends on the distribution of bound protein molecules as well as on their number. The fact that the strength of binding of the Ustilago protein to singlestranded DNA-cellulose is related to its concentration is also circumstantial evidence for binding involving protein-protein interactions (see Results, section (a)). Sucrose gradient sedimentation of [3H]protein-DNA complexes with varying protein-DNA ratios, as done for other DNA-binding proteins, should resolve this question. Experiments are in progress to examine protein-protein interactions using [“Hlleucinelabelled material and to visualize the protein-DNA complexes in the electron microscope. If the linear part of the binding curve (Fig. 4) reflects the stoichiometry between DNA and the protein bound to it, a simple calculation reveals that 1 pg single-stranded DNA binds 6 to 7 pg protein. This gives a figure of one protein molecule per 7 to 10 single-stranded nucleotides. The calculation rests on the assumption that at each point on the curve, the DNA bound to the filter is saturated with protein. Thus when the minimum amount of protein that binds all the DNA in the assay to the filter is present, no more protein is able to bind to that DNA. Further binding would not be detected because the assay measures only DNA bound to the filter. The above figure for the number of nucleotides bound by a single protein
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molecule is, therefore, only a maximum. If the DNA bound is only half saturated with protein, then one molecule would bind to 3 to 6 nucleotides. The proteins investigated by other workers bind to 3 to 11 nucleotides, depending on the size of the protein monomer. A calculation of the number of protein molecules per cell is difhcult, because it was found not to be possible to assay accurately the binding protein in crude extracts, but assuming 100% recovery of activity from the DNA-cellulose chromatography, 1 g of cells yields 25 to 30 pugprotein. This gives a figure of 2.5 to 3.0~ IO6 molecules of binding protein per cell. The Ustilago binding protein stimulates the initial rate of in vitro DNA synthesis by the Ustilago DNA polymerase when it repairs partially single-stranded DNA templates, but does not influence the final amount of synthesis. The maximum stimulation is observed when about enough protein is present to saturate the singlestranded regions (calculated from the above figures) end is independent of the amount of polymerase. This suggests that its action is mediated principally via interaction with the template rather than the enzyme. The stimulation of in vitro DNA synthesis by binding proteins from other organisms does show specificity, however, because it is found only when both the polymerase and binding protein originate from the same organism, although the E. coli and T7-induced binding proteins both stimulate synthesis by the T7 polymerase (Huberman et al., 1971; Herrick t Alberts, 1973; Reuben & Gefter, 1973,1974; Scherzinger et al., 1973). We have not investigated the specificity of the Ustihzgo binding protein fully, but no stimulation of DNA synthesis by the E. coli polymerase I and M. lute%9 polymerase was detected using partially single-stranded DNA templates. Reuben & Gefter (1974) have concluded that unwinding of the DNA template is not a sufficient explanation for the stimulation of DNA synthesis, but suggest that in addition, each polymerase may require the template to be aligned in a very specific configuration, which is induced only by the unwinding protein from the same organism as the polymerase. Our results support this suggestion, but it is perhaps surprising if the template for replication were aligned in a different way for each organism. We have attempted to detect an association of the binding protein with the Ustilago DNA polymerase by sedimentation of a mixture of the two through a glycerol gradient as described for the binding protein alone. No shift in the peaks of the binding (2.6 S) or polymerase (8.3 S) activities from those of the one in the absence of the other was detected, but it is possible that the method would not detect a weak association. It is clear from our investigations that, in contrast to the conclusions of Hotta & Stern (1971a,b), DNA-unwinding proteins can occur in mitotic cells of a eukaryotic organism. These results must weaken their evidence for the involvement of these proteins in meiotic recombination only. We now hope to determine if the same or a different protein is present in meiotio cells of U. may&s. We have assessed the levels of the binding protein in three mutant strains. Two of these, 1~011-1and tsd432, are temperature sensitive for DNA replication. There is evidence that the former possesses a temperature sensitive replicative DNA polymerase (Jeggo et al., 1973) and tsd432 is also defective in mitotic allelic recombination (Hollidey et al., 1975). Both were grown at the permissive temperature (22°C) and after switching to the restrictive temperature (32°C) for five hours, the binding protein was purified to fraction III. Wild-type levels were found in poll-l, but tsd432 contained two to three times the binding activity of the wild-type strain. Both
76
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proteins
were able to denature poly[d(A-T)]*[d(A-T)] and stimulate the wild-type DNA polymerase. The third mutant, reel-1, is sensitive to ultraviolet and ionising radiation and defective in spontaneous and radiation-induced mitotic allelic recombination (Holliday, 1967 ; Holliday et al., 1975). This also possessed a wild-type binding protein level. Thus the function of the binding protein in U. may&s is not yet known, but since its properties are so similar to those of the T4 gene 32 product, its role in DNA replication and recombination may also be similar. Furthermore, its ability to inhibit deoxyribonucleases might also be important in protecting single-stranded DNA within the cell from these enzymes, which might otherwise be lethal. U&ago
We thank Dr D. H. Williamson for carrying out the analytical ultraoentrifugation runs, Dr P. Unrau for providing the strains of U. maydis and Drs R. Ho&day and W. K. Holloman for many valuable discussions. REFERENCES Alberts, B. M. (1970). Fed. Proc. Fed. Amer. Sot. Eqn. Biol. 29, 11541163. Alberts, B. M. & Frey, L. (1970). Nature (London), 227, 1313-1318. Alberts, B. & Herrick, G. (1971). Methods in Enzymology (Grossman, L. & Moldave, K., eds), vol. 21D, pp. 198217, Academic Press, New York. Alberts, B., Frey, L. & Delius, H. (1972). J. ilfol. Biol. 68, 139-152. Banks, G. R. (1974). Eur. J. Biochem. 47, 499-507. Berger, H., Warren, A. J. & Fry, K. E. (1969). J. ViroZ. 3, 171-175. Bollum, F. J. (1968). Methods in Enzymology (Grossman, L. & Moldave, K., eds), vol. 21B, pp. 591-611, Academic Press, New York. Broker, T. R. & Lehman, I. R. (1971). J. MoZ. BioZ. 60, 131-149. Carroll, R. B., Neet, K. E. & Goldthwait, D. A. (1972). Proc. Nat. Acad. Sci., U.S.A. 69, 2741-2744. Davis, B. J. (1964). Ann. N. Y. Acad. Sci. 121, 404-427. Delius, H., Mantell, N. J. & Alberta, B. (1972). J. MoZ. BioZ. 67, 341-360. Epstein, R. H., Bolle, A., Steinberg, C. M., Kellenberger, E., Boy de la Tour, E., Chevalley, R., Edgar, R. S., Susman, M., Denhardt, G. H. & Lielausis, H. (1963). CoZo!Spring Harbor Symp. Quant. BioZ. 28, 375-392. Garen, A. & Levinthal, C. (1960). Biochim. Biophys. Acta, 38, 470-483. Herrick, G. & Alberts, B. (1973). Fed. Proc. Fed. Amer. Sot. Exp. BioZ. 32, 497. Hinkle, D. C. & Chamberlin, M. J. (1972). J. Mol. Biol. 70, 157-185. Holliday, R. (1964). Genet. Re.s. 5, 282-304. Holliday, R. (1967). Mutat. Rea. 4, 275-288. Holliday, R. (1968). In RepZicution and Recombination of Genetic Materiul (Peacock, W. J. & Brock, R. D., eds), pp. 157-174, Australian Academy of Science, Canberra. Holliday, R., Holloman, W. K., Banks, G. R., Unrau, P. & Pugh, J. E. (1975). Oak Ridge Symposium on Mechaniems in Recombination. Holloman, W. K. (1973). J. BioZ. Chem. 248, 8114-8119. Holloman W. K. & Holliday, R. (1973). J. BioZ. Chem. 248, 8107-8113. Hotta, Y. & Stern, H. (1971a). Develop. BioZ. 26, 87-99. Hotta, Y. & Stern, H. (1971b). Nature New Biol. 234, 83-86. Huberman, J. A., Kornberg, A. & Alberts, B. M. (1971). J. Mol. BioZ. 62, 39-62. Ioannou, P. (1973). Nature New BioZ. 244, 257-260. Jeggo, P. A. (1973). Ph.D. Thesis, C.N.A.A., London. Jeggo, P. A., Unrau, P., Banks, G. R. & Holliday, R. (1973). Nature New BioZ. 242, 14-16. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951). J. Biol. Chem. 193, 265275. Martin, R. G. & Ames, R. N. (1961). J. Biol. Chem. 236, 1372-1379. McGhee, J. D. & von Hippel, P. H. (1974). J. Mol. Biol. 86, 46+490.
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Me&son, M. (1972). J. Mol. Biol. 71, 795-798. Molineux, I. J., Friedman, S. t Gefter, M. L. (1974). J. B&d. Chem. 249, 6090-6098. Oey, J. L. & Knippers, R. (1972). J. Mol. Biol. 68, 125-138. Pratt, D., Laws, P. & Griffith, J. (1974). J. Mol. Bid. 82, 425-439. Reuben, R. C. & Gefter, M. L. (1973). Proc. Nat. Acud. Sci., U.S.A. 70, 184&1850. Reuben, R. C. & Gefter, M. L. (1974). J. Biol. Chem. 249, 3843-3850. Richadson, C. C. (1966). J. Mol. BioZ. 15, 49-61. Richardson, C. C., Lehman, I. R. & Kornberg, A. (1964). J. Biol. Chem. 239, 251-258. Scherzinger, E., Litfin, F. & Jost, E. (1973). Mol. Gen. Genet. 123, 247-262. Sigal, N. & Alberts, B. (1972). J. Mol. BioZ. 71, 789-793. Sigttl, N., Delius, H., Kornberg, T., Gefter, M. L. & Alberts, B. (1972). Proc. Nat. Acad. Sci., U.S.A. 69, 3537-3541. Sinha, N. K. & Snustad, D. P. (1971). J. Mol. BioZ. 62, 267-271. Studier, F. W. (1965). J. Mol. BioZ. 11, 373-390. Tomizawa, J., Anreku, N. & Iwama, Y. (1966). J. Mol. Biol. 21, 247-253. Tsai, R. L. & Green, H. (1973). J. Mol. BioZ. 73, 307-316. Weber, K. & Osborn, M. (1969). J. BioZ. Chem. 244, 4406-4412. Whitehouse, H. L. K. (1963). 2\rature (London), 199, 103P1040. Wright, M., Buttin, 0. & Hurwitz, J. (1971). J. BioZ. Chem. 246, 6543-6555.
