Eur. J. Biochem. 62, 131 - 142 (1976)

A DNA Polymerase from Ustilago maydis 1 . Purification and Properties of the Polymerase Activity Geoffrey K.BANKS, William K. HOLLOMAN, Michael V. KAIRIS, Ad SPANOS, and Geoffrey T. YARRANTON National Institute for Medical Research, London (Received July 23 /September 30, 1975)

A DNA polymerase from Ustilugo rnu-vdis has been purified to apparent homogeneity. The native enzyme possesses a subunit structure consisting of 50000 and 55 000-dalton monomers. The apparent sedimentation coefficient of the polymerase activity in the absence of salt is 8.4 S (M, = 180000-200000),thatinitspresence(0.6MNaClor0.22MKCI)being6.3 S ( M , = 80000100000). Low concentrations of EDTA also converted the 8.4-S to a 6.3-S form, whereas magnesium ions catalysed the reverse association. The enzyme has an absolute requirement for both a DNA or RNA template and a DNA primer. For homopolymer templates the primer requirement was satisfied by a short Complementary oligodeoxynucleotide, but oligoribonucleotides were extremely inefficient primers. With the template-primer poly(dA) * (dT),,, the enzyme added an average of 50 dTMP nucleotides on to each primer molecule, whereas with poly(rA). (dT)12,this figure was 300. The enzyme also possesses an associated deoxyribonuclease activity. No other DNA polymerase activity was detected in cell-free extracts of U. muydis.

DNA polymerase enzymes have now been isolated and characterised from a variety of prokaryotic and eukaryotic organisms [l -291. All such enzymes catalyse the polymerisation of deoxyribonucleoside triphosphates into DNA, directed by a template and initiated at the 3’-terminus of a complementary primer. Because, however, many organisms contain multiple DNA polymerase activities, it has been necessary to employ mutant strains, which contain a defective polymerase, in order to determine its function within the cell [30-331. As yet, such an approach has not been possible with mammalian systems. Purified enzymes and other proteins have also been invaluable in constructing complexes in vitro which mimic replication in i’ivo, inbrder-to probe certain aspects of the mechanisms of DNA replication [34 - 371. __

-

4hhrcwtrtiorzs. 5-Bromodeoxyuridine 5’-triphosphate, dBTP; 5-

broinodeoxyuridine 5’-monophosphate, dBMP. Abbreviations for synthetic polynucleotides follow CBN rules, see Eur. J . Biochem. 15, 203-208 (1970). Enzymes. DNA polymerase or deoxynucleosidetriphosphate : DNA deoxynucleotidyltransferase (EC 2.7.7.7); RNA polymerase or nucleosidetriphosphate : RNA nucleotidyltransferase (EC 2.7. 7.6); terminal transferase or deoxynucleosidetriphosphate: oligodeoxynucleotide deoxynucleotidyltransferase (EC 2.7.7.-) ; deoxyribonuclease (EC 3.1.4.5); ribonuclease H (EC 3.1.4.34); polynucleotide 5’-hydroxyLkinase (EC 2.7.1.78); bacterial alkaline phosphatase (Escherichiu coli) or orthophosphoric-monoester phosphohydrolase (EC 3.1.3.1); exonuclease 111 (E. coli) or nucleate (deoxyribonucleate) 5’-nucleotidohydrolase(EC 3.1.4.9).

The basidiomycete fungus Ustilugo muydis is a simple eukaryote suitable for both biochemical and genetic studies of DNA replication [38].The poll-1 mutant strain is temperature sensitive for nuclear DNA synthesis in vivo, and its DNA polymerase activity detected in crude extracts and after partial purification was found to be thermolabile compared to that from the wild-type strain [39 -411. These observations suggest that the mutant is temperature sensitive for DNA replication in vivo because it contains a temperature-sensitive DNA polymerase, which is, therefore, involved in chromosomal replication. In this paper we present a detail characterisation of the polymerase activity of this enzyme after extensive purification from the wild-type strain, and in the following one, we describe the properties of the associated 3’-+5‘ exonuclease activity [42]. MATERIALS AND METHODS Materials

Ribonucleoside and deoxyribonucleoside triphosphates, polydeoxyribonucleotides and oligodeoxyribonucleotides were from PL Biochemicals Inc., polyribonucleotides from the Boehringer Corporation and (rU)& from Calbiochem Ltd. Radiochemicals were obtained from the Radiochemical Centre, calf-thymus

132

DNA, pancreatic DNase I and Escherichia coli alkaline phosphatase from the Worthington Biochemical Corporation. Phosphocellulose (P-1 1) was from Whatman Biochemicals Ltd., Sephadex G-25 and G-150 from Pharmacia Fine Chemicals and hydroxyapatite (Bio-Gel HT) from Bio-Rad Laboratories. E. coli DNA polymerase I was purified according to Richardson et al. [43], and polynucleotide kinase was a generous gift of D r I . Molineux. Bromodeoxyuridine triphosphate was synthesised by bromination of dUTP.

Nucleic Acids Calf-thymus DNA was dissolved in 20 mM TrisHCl, pH 7.5, 20 mM NaCl at 2.0 mg/ml. It was denatured by heating in boiling water for 10 min, followed by rapid cooling in ice-water. Activated calfthymus DNA was prepared by the method ofAposhian and Kornberg [44] until 15-20% of the DNA had been made acid soluble by pancreatic DNase I. Activated DNA labelled at 3'-termini with [3H]dTMP was prepared as follows. Activated calf-thymus DNA (400 pg) was incubated in a 1.0-ml reaction mixture containing 0.2 M glycine, pH 9.0, 100 pCi [3H]dTTP (25Ci/mmol), 3 mM 2-mercaptoethanol, 20 mM MgCI, and 12 units of E. coli DNA polymerase I for 60 min at 37 'C. Protein was removed by extraction with chloroform/isoamyl alcohol (24/1) and the aqueous phase chromatographed on a column (1.1 x 45 cm) of Sephadex G-25, which was eluted with 1 0 m M Tris-HC1, pH 7.6, 20 mM NaC1. The radioactive solution eluting at the void volume of the column was made 0.1 M in NaCl and three volumes of cold ethanol added. After holding overnight in ice, the precipitated DNA was collected by centrifugation and dissolved in 1.0 ml of 10 mM Tris-HCI, p H 7.6, 20 mM NaCI. T7 [3H]DNAwas extracted from the purified phage particles according to Richardson [45]. Double-stranded homopolynucleotides and homopolymer * oligomer complexes were constructed by mixing appropriate amounts of the component single strands dissolved in 10 mM Tris-HCI, pH 7.8, 0.1 M NaCl, followed by incubation at 37 "C (for adenine, thymine or uracil combinations) or at 60 "C (for guanine and cytosine combinations) for 30 min. Depurinated poly(dC) was prepared by acid hydrolysis as described by Harwood and Wells [46]. Nucleotide concentrations of the homopolymers were estimated spectrophotometrically using the data of Riley et al. [47] and of Chamberlin and Patterson [48]. [5'-32P](dT),, was prepared as follows. A reaction mixture (0.135 ml), containing 250 mM Tris-HCI, p H 8.0, 130 nmol (dT)12 and 14 yg bacterial alkaline phosphatase, was incubated at 70 "C for 60 min, a further 14 pg of the phosphatase being added after 30 min.

Polymcrase Activity of a U . muydis DNA Polymerase

The mixture was adjusted to pH 13.5 by addition of 1 N NaOH, incubated at 100 "C for 30 min, cooled and readjusted to p H 8.0 by addition of 1 N HCI. After addition of 0.05 ml 0.01 M MgCl,, 0.001 ml 20 mM ATP and 50 pCi [ Y ~ ~ P I A T (15.6 P Ci/mmol, 500 pCi/ml), the solution was again incubated at 100 "C for 2 min, cooled and 0.05 ml 0.1 M 2-mercaptoethanol, 5.4 units of polynucleotide kinase and distilled water were added to a final volume of 0.5 ml. This reaction mixture was incubated at 37 "C for 60 min, a further 5.4 units of the kinase being added after 20 min. The reaction was terminated by incubation at 100 "C for 2 min. Unreacted [ Y - ~ ~ P I Aand T P other low-molecular-weight components were removed by chromatography of the mixture through Sephadex G-50, the [5-32P](dT),, being concentrated by lyophilisation. Po lyacrylamide Gel Electrophoresis

The procedure for native polyacrylamide gel electrophoresis was essentially that of Davis [49], and for electrophoresis in the presence of sodium dodecylsulphate that of Weber and Osborn [50], except that the gels contained 5 M urea. The gels were stained in 0.25% Coomassie blue in 50% methanol/lO% acetic acid, then destained in lo"/, methanol/7% acetic acid. The gels were scanned at 640nm using a Unicam SP 1809 scanning densitometer. For the determination of DNA polymerase activity from native gels, the gel was sliced into 0.25-cm segments and each assayed, without prior elution, in a standard assay mixture for 90 min.

Protein Determination Protein was determined by the method of Lowry et al. [51], using bovine serum albumin as the standard.

Grudient Sedimentation Glycerol Gradients. A sample of fraction IV was dialysed overnight against 50 mM Tris-HCI, pH 7.5, 10 mM 2-mercaptoethanol and 10% glycerol (with or without 0.6 M NaCl or 0.12 M KCI) at 4 "C. It was then dialysed for several hours against this buffer containing 30% poly(ethyleneglyco1)(Carbowax 6000) because of an increase in volume of the sample. Alternatively, fraction IV was dialysed for 4 h against the first of the above buffers in a tight dialysis bag, which prevented the volume increase, and used directly. The latter procedure resulted in a smaller loss in enzyme activity. The prepared sample (0.2 ml) was layered on to a preformed 20 - 40% glycerol gradient in 50 mM Tris-HCI, pH 7.5, and 10 mM 2-mercaptoethanol, with or without 0.6 M NaCl or 0.12 M KC1, in a polyallomer tube of a Beckman SW56 rotor. Centri-

G. R. Banks. W. K. Holloman, M. V. Kairis, A. Spanos, and G. T. Yarranton

fugation was at 45000 rev./min at 2-4 "C for 21 h in a Beckman L2 65B ultracentrifuge. Fractions were collected in polyallomer tubes through a needle insertcd into the base of the centrifuge tube, and an aliquot of each assayed for DNA polymerase activity. Sucrose Gradients. Fraction IV of the enzyme (or fraction V for one experiment; see Results section) was dialysed in a tight dialysis bag against 10 mM KPO,, pH 7.5, 10 mM 2-mercaptoethanol for 4 h, with or without 0.12 M KC1. 0.2 ml was then layered on to preformed 5 - 20% sucrose gradient, which was Centrifuged for 15 h under conditions identical to the glycerol gradients. Molecular weights were determined by the method of Martin and Ames [52] using E. coli alkaline phosphatase (6.3 S) a s an internal marker. It was assayed according to Garen and Levinthal [53]. D N A Polymerase Assay

The assay measures the incorporation of [3H]dTTP into acid-insoluble DNA. A 0.15-ml reaction contained 67 rnM Tris-HCI, pH 7.5, 6.7 mM MgCl,, 100 mM KCl, 17 mM dithiothreitol, 8 pg activated calf-thymus DNA, 5 pg bovine serum albumin, 13.3 pM each of dATP, dTTP, dGTP and dCTP, 2.5 pCi [3H]dTTP (25 Ci/mmol) and aliquots of DNA polymerase. After incubation at 37 "C for 30 min, the reaction was terminated in ice, 0.02 ml calf-thymus DNA (2 mg/ml) added, followed by 0.1 ml of 0.1 M tetrasodium pyrophosphate and 2.0 ml cold 5 %, trichloroacetic a c i d / l x tetrasodium pyrophosphate. The precipitate was collected by filtration on Whatman GFjC glass-fibre filter discs, which were extensively washed with cold 5 ?4 tricliloroacetic acid/l % tetrasodium pyrophosphate and finally ethanol. The discs were dried and radioactivity determined in a toluene-based scintillation fluid. When column chromatography fractions were assayed, 0.1 ml of the reaction mixture was absorbed on Whatman 3 MM filter squares, which were assayed as described by Bollum [54]. One unit of DNA polymerase activity is the amount of enzyme catalysing the incorporation of 10 pmol dTMP into acid-insoluble DNA in 30 min at 37 "C. E n q w e Purifkation

The method is based on that developed by Jeggo [41]. U . nwydis (wild-type haploid a2bl strain) was grown as described previously [55] and the packed cells stored at - 20 "C. All operations were at 4 'C. Preparation of Crude Extract. Frozen cells (500 g) were thawed, washed twice in buffer A (50 mM TrisHC1, pH 7.5, 1 mM EDTA, 10 mM 2-mercaptoethano1 and 10% glycerol) containing 1.7 M NaCl and suspended by sonication in 1 1 of this buffer. The cells were broken by passage of the suspension through a French pressure-cell at 20000 lb/in2 (140 MPa) pres-

133

sure. Cell debris was removed by centrifugation at 20000 rev./min in an SS-34 rotor of a Sorvall centrifuge for 60 min, and the supernatant fraction passed through a layer of cheese-cloth to remove floating lipid-like material to give fraction I. Removal of Nucleic Acids. To fraction I (960 ml) was added 480 ml 30 poly(ethyleneglyco1) (Carbowax 6000) in 2 M NaCl over a period of 30 min with constant stirring. After a further 15 min the precipitate was removed by centrifugation at 9000 rev./min for 30 min. The clear supernatant fraction was dialysed for 48 h against two 10-1 volumes of buffer A to give fraction 11. D N A Celltilose Chronzatogvapliy. Fraction I1 (2020 ml) was centrifuged to remove a precipitate which formed during dialysis and then loaded on to a column of single-stranded calf-thymus DNA cellulose (4 x 7 cm), which had been extensively washed with buffer A. The column was washed with 1 1 of the same buffer and eluted with a linear gradient (2 I) of 0.05 - 0.5 M NaCl in buffer A. DNA polymerase activity eluted at a 0.25 M NaCl concentration. Active fractions were pooled and dialysed against two 2-1 volumes of buffer B (50 niM KPO, pH 7.5, 10 m M 2-mercaproethanol and 1Oo/, glycerol) overnight to give fraction 111. Phosphocellulose Chromatography. Fraction 111 ( 150 ml) was loaded on to a column of phosphocellulose (2.5 x 14 cm) equilibrated with buffer B. It was washed with 500 ml of this buffer and then eluted with a linear gradient (500 ml) of 0.05-0.5 M KH2P04in buffer B. DNA polymerase activity was eluted at 0.3 M KH,PO, concentration. The active fractions were pooled and dialysed against 2 1 buffer to give fraction IV. HjJdroxyapatite Chromatography. Fraction IV (27 ml) was loaded on to a column of hydroxyapatite (1.5 x 10 cm) equilibrated with buffer B. The column was washed with 100 ml of this buffer and eluted with a linear gradient (200 ml) of 0.05 - 0.5 M KH2P04 in buffer B. Fractions were collected in polyallomer tubes because polymerase activity was lost with glass tubes, presumably by non-specific adsorption of protein on to the walls. DNA polymerase activity eluted at 0.25 M KH,PO, concentration. The peak fractions were pooled and dialysed against buffer B containing 50% glycerol to give fraction V. It was stored at -20 C and was then stable for several months. RESULTS Enzyme Puriti. and PoI!.cic.i.!.ltiiiii~~e Gel Electrophoresis

The purification procedure described above resulted in over a 11000-fold purification (Table 1). No other DNA polymerase activity was detected during any of

Polymerase Activity of a U.may& DNA Polymerase

134 Table 1. Summary Of’puriJkution 01U. maydis DNA polymerase Protein concentrations for fraction V were too low to be determined accurately Fraction

I. Crude extriict 11. N ucleic-acid-free 111. DNA cellulose

IV. Phosphocellulose V. Hydroxyapati te

Protein

Activity

Specific activity

Yield

n1g

units

unitsjmg protein

:/,

25728 4 444

39 50 I 103026 63 557 35315 17000

33 2.2

-

I .j4 23.2 1926

16050 -

I00 260 160 89 43

Fig. 1. Sodiuin f ~ i ~ r ~ ~ ~ p l s z i l p l i ~ i t ~ i ~ ~gel f ~ felectro~~p~l~~i~r~/a~iiid~ phor~sic-of D N A p d y / w m w frciclions. Technical details are given

in the Materials and Methods section. (A) Fraction IV; (B) fraction V ; (C) fraction V after sucrose gradient sedimentation. Molecular weights increase from left to right. The small arrows point to discrete bands observed by visual inspection of the Coornassie blue stained gels and the larger arrow the position of the broinophenol blue tracker dye. Thc gel for fraction IV (A) was longer than for the other two (B,C),and a left-hand portion has been oinittcd so that the 50000 and 55000-dalton bands in all three gels could be lined up

t h e fractionation procedures or by DEAE-cellulose chromatography (results not shown). Fractions from DNA-ceIlulose and phosphoce]]ulose have also been assayed using reaction conditions differentfrom those described above in their pH, KC1, MgClz and deoxyribonucleolide triphosphate concentrations. In every case only one activity was detected.

Fig. 2. S O d i U / ? l d f ~ r k l ~ ~ ’ ~ , S l 4 ~ ~ J h t / / ~ ’ ; l f ~gC.~1 ’ c’/t.l’f/’Ot / ~ ~ ~ ~ phoresb. (A) Fraction V after sucrose gradient sedimcntation; ( 8 ) protein eluted froln the excised band which was associLitcd w i t h polynierase activity I’rom 2) gel run under non-denaturing protein conditions olfrxction IV(see text for dctails). Dircction ofmigration is from the top to the bottom of the gels

~ ~ ~ U ~ ’

G. R. Banks. W. K. Holloman, M. V. Kairis, A. Spanos. and G. T. Yarranton

Fraction V of the enzyme possessed no detectable RNase H activity when assayed with calf-thymus [3H]RNA .DNA [56] or DNA-directed RNA polymerase activity using the standard assay conditions (Table2). Native T7 [3H]DNA (2.5 pg) was incubated for 30 min with 36 units of fraction V in a standard assay mixture which lacked the deoxyribonucleoside triphosphates. After being made alkaline, the reaction mixture was sedimented through an alkaline 5 - 20x sucrose gradient. There was no change in the sedimentation profile of the DNA compared with that of an untreated sample (results not shown). Thus fraction V of the enzyme possessed no detectable endonuclease activity. It did, however, possess exonuclease activity, which is the subject of the following paper. Sodium dodecylsulphate/urea/polyacrylamide gel electrophoresis of fraction V, prepared from 100 g of cells, revealed two protein bands of approximately equal staining intensities. Their molecular weights were about 50 000 and 55 000 when determined by a comparison of their relative mobilities with those of marker proteins run in parallel gels. After a similar analysis of fraction V prepared from 500g of cells, a third major protein band of molecular weight about 45 000 was also seen, its staining intensity varying over the peak of activity from the hydroxyapatite chromatography column. When this fraction was sedimented in a 5 -20% sucrose gradient in the absence of salt, the three fractions of peak DNA polymerase activity (sedimentation coefficient 8.4 S , see below) combined, dialysed, concentrated and analysed by sodium dode-

135

cylsulphate/urea gel electrophoresis, this third band was no longer detectable (Fig. 1 C and 2A). Only the M, 50000 and 55000 protein bands were observed, although not of equal staining intensities, as wdS found for the smaller-scale fraction V preparation. We conclude that the protein is a contaminant of the largerscale purification procedure, or possibly a protease degradation product. The traces of the stained bands of fraction IV (phosphocellulose step), V (hydroxyapatite step) and the sucrose gradient peak fraction (Fig. 1) strongly suggest that the two proteins copurify with the polymerase activity. Gel electrophoresis of the first of the above fraction V preparations under non-denaturing protein conditions (7'x acrylamide) gave a single proteinstaining band of mobility 0.16 relative to the bromophenol blue tracker dye. Attempts to recover DNA polymerase activity from slices of these gels did not meet with success, but did with fraction IV (with a yield of about 5%). Two gels were run in parallel, one being stained as usual when several bands were observed (Fig.1A and 3). The second gel was sliced into two longitudinal halves and one of these then cut into segments, which were assayed for polymerase activity (see Materials and Methods section). The activity possessed a mobility of about 0.16 relative to the tracker dye and coincided with a protein band stained in the first of these gels (Fig.3; although the faint band was clearly visible in the original stained gel, it is not very distinct in the resulting photograph). The segment corresponding to the activity was excised

136

Polymerase Activity of +: U.nzaydis DNA Polymerase Table 2. Reaction requirements of' rhe IJ. maydis D N A po1j:mera.w The standard assay mixture was used with the additions or subtractions shown below Assay mixture

Complete - KCI MgCI, - Dithiothreitol DNA - dATP dGTP dATP, - dGTP - dATP, - dGTP, - dCTP 4dNTPs, + 4rNTPs (13.3 pM) - 4dNTPs. - MgCI, + rNTPs (13.3 FM), + MnCI, (100 pM) - dATP. + rATP (13.3 pM) - MgC12, MnCI, (100 pM)

Activity

:4

100 18 7 12

~

~

Fraction number

Fig. 4. G!i'cwol gratlient .sedinieti/u/ion (4' D N A poljtnerase octivities. Fraction 1V of thc cnzyme was sedimented in a glyccrol or (B) 0.6 M NaCl gradient containing (A) no salt (O---) (w) 8 s described in Materials and Methods. The profile in the presence of 0.12 M KCI was identical to that shown for (B). Thc yield of activity was 40- 50'x in each case. The 6 . 3 3 marker protein was B. coli alkaline phosphatase

from the remaining half of the second gel and protein eluted from it with a buffer containing sodium dodecylsulphate and urea. When this solution was analysed by gel electrophoresis under protein-denaturing conditions, two proteins of molecular weights 50000 and 55000 were stained, but again, not of equal intensities (Fig. 2B). Glycerol Gradient Sedimentation ?f the Enzymt)

Attempts to determine the molecular weight of the Ustilago DNA polymerase activity of fraction V by gel filtration or glycerol and sucrose gradient sedimentation were unsatisfactory because of losses of activity. Bovine serum albumin added as carrier protein before or after these procedures failed to stabilise the activity significantly. The determinations were, therefore, made with fraction IV. An aliquot of fraction IV was chromatographed on a calibrated column (1.2 x 45 cm) of Sephadex G-150, equilibrated and eluted with 50mM KPO,, pH 7.5, 10 mM 2-mercaptoethanol and 10% glycerol. A single sharp peak of activity emerged 10 ml after the void volume of the column,which corresponds to an apparent molecular weight of about 180000 (results not shown). Fraction IV was also sedimented through glycerol gradients in the presence and absence of salt, as described in the Materials and Methods section. The results are presented in Fig.4, from which it can be seen that the apparent sedimentation coefficients of the activity were about 8.4 S and 6.3 S in the absence and presence of salt respectively, E. coli alkaline phosphatase being the internal marker protein. These determinations have also been made using sucrose gradient sedimentation with ovalbumin as the protein marker with identical results (see following paper).

A DNA polymerase from Ustilago maydis. 1. Purification and properties of the polymerase activity.

A DNA polymerase from Ustilago maydis has been purified to apparent homogeneity. The native enzyme possesses a subunit structure consisting of 50000 a...
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