Eur. J. Biochem. 84, 123- 131 (1978)

Studies on the Purification and Properties of a 6.8-S DNA Polymerase Activity Found in Calf-Thymus DNA Polymerase-a Fraction Ian P. HESSLEWOOD, Andrew M. HOLMES, William F. WAKELING, and Irving R. JOHNSTON Department of Biochemistry, University College London (Received September IS, 1977)

The heterogeneity of calf thymus DNA polymerase-a has been further investigated. In particular, an enzyme (enzyme D) which exhibits higher activity on poly(dA) . (dT)lo (A :T = 20 : 1) compared with that on activated DNA, has been further purified and its properties compared with two other activities of the DNA polymerase-a fraction (enzymes A1 and C) which do not show a preference for poly(dA) . (dT)lo over activated DNA. As with A1 and C, enzyme D was shown to have many of the characteristic properties of DNA polymerase-a in that it is an acidic protein as judged by its binding to DEAE-cellulose, has a molecular weight of about 140000, does not use a poly(A) . (dT)Io template-initiator complex and is inhibited by N-ethylmaleimide. It exhibits anomalous gel filtration behaviour on Sepharose 6B and it binds relatively weakly to DNA-cellulose compared with DNA polymerase-p. The extreme sensitivity of enzyme D to inhibition by N-ethylmaleimide distinguishes it from A1 and C, as does its elution position from a DEAE-cellulose column. On the other hand enzymes C and D are readily inactivated by heating at 45 "C unlike enzyme Al. The possible interrelationships of the multiple activities of calf thymus DNA polymerase-a are discussed.

Several different types of DNA polymerase activity have been described in mammalian cells [1,2]. Of these DNA polymerase-a is of particular interest since its activity is highest in situations where DNA synthesis is occurring [3]. A further point of interest concerns the heterogeneity of DNA polymerase-a reported in several instances, for example in calf thymus [4- 71 rat liver and spleen [5], Chinese hamster cells [8], mouse myeloma [9- 111 and baby hamster kidney (BHK) cells [12]. When preparations of DNA polymerase-a from calf thymus, freed of DNA polymerase-p and terminal transferase, are fractionated on DEAE-cellulose, several peaks of polymerase-a activity can be detected using activated DNA in assays carried out at pH 7.8 [5,13]. From tissue frozen shortly after removal from the animal these are enzymes A1, A2 and C. A further enzyme B appears occasionally and is probably derived by proteolysis from enzyme C [6] (see also [14]). However, assay of the same DEAEcellulose profile with the synthetic template-initiator complex poly(dA) . (dT)lo at pH 7.0 reveals a further Enzjlmes. Catalase (EC 1.11.1.6); lactate dehydrogenase (EC 1.1.1.27); /l-galactosidase (EC 3.2.1.23); D N A polymerase (EC 2.7. 7.7); deoxyribonuclease (EC 3.1.4.5).

peak of activity differing in position from AI, A2, B and C [6,13]. This activity, designated enzyme D [6,13], has been further purified and some of its properties compared with similarly purified samples of enzymes A1 and C. The relationships of the various DNA polymerases of the a-polymerase fraction is discussed. MATERIALS AND METHODS Matevials

All materials were as described [ 5 ] except as mentioned below. (dA)4, (dT)S and (dT)lo were purchased from P. L. Biochemicals as were some batches of poly(dA) ( M , z 300000). (dC)5 was a gift from F. J. Bollum. Most of the poly(dA) used in this work was prepared using terminal transferase and a (dA), initiator as described by Bollum [15]. The product had an average chain length of 750 residues ( M , M 250000). Poly(dC) and poly(dT) were similarly prepared using (dC)s and (dT)S, respectively. [3H]Poly(dA) and [3H]poly(dT) were prepared by the same reaction. DNAcellulose was prepared as described by Litman [16].

Mammalian DNA Polymerase-a Fraction

124

N-Ethylnialeiniide was obtained from British Drug Houses and Biogel HT was obtained from BioRad Laboratories. Except where indicated, all buffers contained 20% glycerol and 1 mM dithiothreitol. Linear standard phosphate gradients were run between 0.03 M and 0.25 M potassium phosphate, pH 7.8. Gradient salt concentrations were measured at 22 "C using a Radiometer conductivity meter, type CDN 2d. Partially purified DNA polymerase-a from rat liver and spleen were prepared as described previously [5] as was rat liver DNA polymerase$ [17]. D N A Polymerase Assays Assays Using Activated DNA. DNA polymerase-a was assayed as before [5] except that a pH of 7.8 was used. DNA polymerase-P was assayed at pH 8.5 [17]. Assays Using poly(dA) . ( d T j l o ( A :T = 2 0 : l ) . Assays of column fractions using poly(dA) . (dT)lo were incubated at 30 "C in 0.125 ml containing 20 mM sodium/potassium phosphate buffer, pH 7.0 (final ionic strength, 0.032 M), 10 mM MgC12, 1 mM dithiothreitol, 62.5 pg bovine serum albumin, 0.1 mM r3H]dTTP (25 counts x min-' x pmol-I), 1 pg poly(dA) . (dT)lo (A:T = 20: 1) and enzyme protein up to 4 units. Poly(dA) . (dT)lo was prepared as previously [5]. Assays were processed as previously [5]. The pH optimum of enzyme D on poly(dA). (dT)lo changed from 7.2 at step V to 6.8 at step IX; enzymes A1 and C had optima of pH 6.4 on this synthetic complex. These optima were used in the characterisation of the enzymes. Nuclease Assays

These were performed in 0.1 ml of solution containing 25 mM Tris-HC1 pH 8.5, 8 mM MgC12, 3 mM CaC12, 100 pg bovine serum albumin, 1 pg 3H-labelled polynucleotide and enzyme fraction at ten times the amount used in polymerase assays. The reaction was started with enzyme protein and incubated at 30 "C for 2 h. 0.05 ml of a solution containing bovine serum albumin (1 mg ml-') and calf thymus DNA (1 mg m1-I) in 50 mM EDTA was added followed by 0.15 ml ice-cold 10% trichloracetic acid. After 20 min in ice, samples were centrifuged and 0.15 ml of the supernatant was added directly to 10 ml of scintillation fluid and counted. Controls contained bovine serum albumin instead of enzyme fractions. Other Methods

Molecular weights were calculated as before [5] by combining estimates of S Z O , ~ , determined on standard glycerol gradients and of Dzo, w, determined

on a standard Sepharose 6B column, in the Svedberg equation to obtain the molar mass. Polyacrylamide gel electrophoresis in sodium dodecyl sulphate was performed by a standard method [18] and in non-denaturing conditions as previously described [19]. RESULTS Enzyme D Preparation

The enzyme in the supernatant (20 1) from 6 kg of calf thymus, homogenised in 0.05 M potassium phosphate, pH 7.0 (without glycerol) and centrifuged as previously [5] (step l), was adsorbed batchwise on to 4.2 I phosphocellulose. After washing with 20 1 of 0.1 M potassium phosphate, pH 7.0, DNA polymerase activity was eluted with 0.5 M potassium phosphate, pH 7.0 (400- 500-ml fractions) from the resin packed into a 25 x 16-cm column (step 11). The resulting eluate (about 1 1) was fractionated with ammonium sulphate (30- 55 % cut, step 111) and then chromatographed on Sepharose 6B (Fig. 1). Fractions were pooled to avoid inclusion of terminal transferase (step IV) and then chromatographed on DEAE-cellulose as previously [6,13]. Enzyme A1 eluted from the DEAE-cellulose column (50 x 1.6 cm) between 0.055 M and 0.075 M, A2 between 0.075 M and 0.095 M, D between 0.1 M and 0.125 M and C between 0.125 M and 0.160 M potassium phosphate, pH 7.8. Fractions containing enzyme D were pooled and concentrated by ammonium sulphate precipitation at 55 saturation (step V). After dialysis against 30 mM potassium phosphate, pH 7.8, step V enzyme was re-chromatographed on DEAE-cellulose (9 x 1 cm) to remove contaminating A2 and C. Following concentration of appropriate fractions by vacuum dialysis to 0.5 M NaC1, 0.05 M Tris-HC1, pH 8.5. (step VI), the enzyme was chromatographed on Sepharose 6 B (93 x 1.5 cm) in the same buffer where it eluted at 1.90 times the void volume (step VII). After concentration and dialysis against 0.03 M potassium phosphate, pH 7.8, the enzyme was chromatographed on phosphocellulose (8 x 1 cm) with a 150-ml standard phosphate gradient, collecting 3-ml fractions, when it eluted between 0.12 M and 0.16 M phosphate (step VIII). Finally, active fractions from phosphocellulose were pooled, concentrated by vacuum dialysis against 0.03 M potassium phosphate, pH 7.8 and loaded onto hydroxyapatite (3 x 1 cm) in the same buffer. The column was developed with a 60-ml linear gradient up to 0.5 M phosphate, pH 7.8 collecting 1.6-ml fractions. Enzyme D eluted at 0.1 M phosphate (step IX). Using the activated DNAdependent assay a second peak of activity eluted at 0.15 M phosphate; this was not apparent using the poly(dA) . (dT)lo-dependent assay (Fig. 2). Like en-

I. P. Hesslewood, A. M. Holmes, W. F. Wakeling, and 1. R . Johnston

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Fig. 1. Prrpurative Srphurose 6 B c/?romu/ogruphyof cavthynzus D N A poljmcvusr-cc. Step 111 enzyme (1 I70000 units, 2700 mg protein) was loaded onto a 68 x 4.5-cm Sepharose 6 B column equilibrated in 0.5 M NaCI, 0.05 M Tris-HC1 p H 8.5. Approximately 10-ml fractions were collected and, after dilution of samples 26-fold, 10-pl aliquots were assayed for 5 min on activated DNA -.( -0)and on poly(dA) . (dT)," (A:T = 20: 1) (0-~0). 5-kI aliquots were assayed undiluted for 60 min for terminal transferase activity (A-A) using [3H]dATP and (dA)a [IS]. (--- --) Absorbance at 280 nm. Active fractions were pooled as indicated by the bar to exclude termInal transferase activity

Frattion number

Fig.2. Hydroxyupatite chromatography ofenz-vmr D.Step V l l l enzyme (13000 units, 1.7 mg protein) was loaded onto a 3 x 1-cm hydroxyapatite column in 0.03 M potassium phosphate pH 7.8. The column was developed with a 60-ml linear gradient between 0.03 M and 0.5 M potassium phosphate p H 7.8. 1.6-ml fractions were collected, a sample of each fraction diluted 10-fold in 0.05 M Tris-HCI pH 7.5, 1 mg/ml -.() Activated DNA or (O---O) poly(dA). (dT),{, bovine serum albumin, and 10 pl was assayed for 15 min as described in the text. @ ( A : T = 20: 1) were used as templates. (-----) Phosphate gradient. Active fractions were pooled as indicated by the bar

zyme D this species sedimented at about 6.8-6.9 S but it was not further studied. For comparison, samples of A1 and C were also further purified from the first DEAE-cellulose column as described for enzyme D [6,19] omitting however, the second run on Sepharose 6B. The final specific activities of A1 and C were 3000 and 6000 units/mg, respectively, when assayed on activated D N A ; for enzyme D assayed with poly(dA) . (dT)lo it was about 9000 units/ mg protein (Table 1). In all three cases specific activities fell by up to 20% at the final hydroxyapatite step which nevertheless removed contaminating protein. These partially purified preparations of Al, C and D were used in the comparison of some of their properties. Nuclease Activity Preparations of A1 and C solubilised less than 2 % of the acid-precipitable 3H in [3H]poly(dA), ["HI-

poly(dT), [3H]poly(dA) . poly(dT) or [3H]poly(dT) . poly(dA). For the preparation of enzyme D, the corresponding values were 14, 19, 12 and 3 %. Therefore enzyme D shows a low level of nuclease activity but the nature of this or of its association with the polymerase has not been further studied.

Physical Properties Enzyme D sedimented at 6.8-6.9 & 0.2 S (10 determinations) when centrifuged on standard 1030 % glycerol gradients made in 0.5 M NaCl[5]. When chromatographed on Sepharose 6B under the same conditions of ionic strength and pH as used in sedimentation studies, in the presence of 20 %, glycerol in the buffer, it eluted at 1.90 times the void volume. This corresponded to an apparent molecular weight of approximately 250000 and to a diffusion coefficient D ~ oof, 42 ~ pm2 sC1 [5]. When ~ 2 0 and . ~ D ~ Owere , ~ combined in the Svedberg equation (using va of 0.725 ml/g) the calculated molar mass was approxi-

126

Mammalian D N A Polymerase-a Fraction

Table 1. Purification of enzyme D One unit of enzyme is that amount which catalyses the incorporation of 1 nmol of [’HIdTMP per hour into an acid-insoluble form in the assay using poly(dA) (dT)lo (A:T = 20: 1) Fraction

I. 11. 111. IV. V. V1. VII. VIII. IX. a

Supernatant Phosphocellulose Ammonium sulphate 30- 55 ”/, 1st Sepharose 6B 1st DEAE-cellulose 2nd DEAE-cellulose 2nd Sepharose 6B Phosphocellulose Hydroxyapatite

Total activity

Specific activity

Purification

units

units/mg

-fold

1200000” 836700 750000 701 000 181209h 98 000 51 000 18 942 2816

3.2 153 279 692 2 960 4 206 5 992 I0 582 8 800

48 87 216 925 1314 1872 3307 2750

1

Absorbance ratio A280/AZ60

0.73 1.28 1.30 1.67 1.75 1.8 1.8 1.8 1.8

Fraction I activity was not always linear with respect t o time or protein. Only enzyme D peak was taken from the 1st DEAE-cellulose column, representing approximately one-third total poly(dA) . (dT)lOdependent activity.

mately 143000 g/mol. Under the same conditions AI and C sedimented at 8.4 S and 7.3 S and had estimated Dzo,,, values of 36 pm2 s p l and 39 pm2 s-’, respectively, giving calculated molar masses of 225 000 and 160000 g/mol. When a sample of enzyme D (step IX, 50pg protein) was subjected to electrophoresis under nondenaturing conditions [19] it moved as a single peak of activity with a relative mobility of 0.25 (compared with 0.1 1,0.14 and 0.26 for AI, AZand C respectively). The peaks of enzyme activity extracted from five similar non-denaturing gels were pooled to give 10 units of activity (approx. 2 yg protein) and were then electrophoresed on a 5 % dodecyl sulphate/polyacrylamide gel. A main band at a position corresponding to a molecular weight of 135000 was found together with a minor one at M , 68000. Since non-denaturing electrophoresis can result in removal of considerable amounts of protein impurity [19], the band at M , 135000 may represent enzyme protein (compare M , from calculated molar mass). Finally, preincubation o f enzyme D with 2.8 M urea for urea for 30 min did not alter either its sedimentation coefficient or its elution position on DEAEcellulose [13]. General Properties of Enzyme D Compared with A1 and C pH optima for the three enzymes were dependent on the template used. Using optimal Mg2+ concentration in the assay (10 mM) and constant ionic strength buffers (final ionic strength in assay due to buffer was 0.04 M) reasonably sharp optima for A, and C, on activated DNA, were seen at pH 7.8; for D, however, a broader optimum occurs between pH 7.3 and

pH 7.8. On poly(dA). (dT),, (A:T = 20:l) both A1 and C had optima at pH 6.4 while for D this was at pH 6.8 (with step V enzyme it was pH 7.2). On poly(dT) optimal activity was observed at pH 7.8 for all three enzymes. With optimal pH, maximal activity on the above templates was attained with 10 mM MgC12. At the same pH values, replacement of 10 mM MgClz by 1 m M MnCIz (optimal for all three enzymes) reduced activity on poly(dA) . (dT)lo to about 20% of the value with MgC12 for all three enzymes (see Table 2). Assays of A1, C and D are inhibited by increasing ionic strength; at 0.1 M NaCl A1 and C were inhibited by 75 % and D by 100 % on activated DNA. On poly(dA) . (dT)lo all three enzymes were completely inhibited by 75 mM NaCl in the assay. DNA binding properties were tested on a DNAcellulose column. A sample of enzyme D pooled from the first DEAE-cellulose column was chroniatographed on a DNA-cellulose column (Fig. 3). The activated DNA-dependent activity, consisting mainly o f enzymes C and AZ as well as D, eluted at 0.24 M NaCl while activity using poly(dA) . (dT)lo assayed at pH 7.0 eluted at 0.26 M NaCl. On the same column calf thymus DNA polymerase$ eluted at 0.93 M NaCl. Enzyme D, therefore, has the relatively weak DNA binding properties characteristic of DNA polymeraseCI [20,21]. For K, determinations of dTTP and dATP, 1 pg template with addition of the appropriate amount of initiator was used at the optimal pH and MgC12 concentration. For dTTP, the K, on poly(dA) . (dT),, (A:T = 20:l) was 1 0 p M with enzymes Al and D but was 20 pM for enzyme C. For incorporation of dATP into poly(dT) . (A)lo ( T :A = 1 : 1) the K , was 5 pM with all three enzymes. These K , values are

127

I. P. Hesslewood, A. M. Holmes, W. F. Wakeling, and I. R. Johnston

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Fig. 3. DNA-wllulose chromatography of cvzyne D.Step V enzyme (4500 units, 2.3 mg protein) was loaded onto a 15 x 2-cm DNA-cellulose column equilibrated in 0.05 M potassium phosphate, pH 6.5, 0,001 M EDTA. The column was developed with a 200-ml linear gradient u p to 1 M NaCl in the same buffer. 3.3-ml fractions were collected and 10 p1 were assayed for 60 min. ( 0 4 )Activated D N A ; ( O - - O ) poly(dA) . (dT),, (A :T = 20: 1 ) ; (-----) NaCl gradient. The arrow indicates the elution position of calf thymus DNA polymerase-p in a separate experiment

Table 2. Template utilifation Values are given relative to incorporation of .'H-labelled deoxyribonucleotide at pH 7.8 on activated DNA as 100%. The full complement of nucleotides was included for assays using activated DNA. For incorporations of [3H]dAMP and [3H]dGMP only values at optimal pH are given. Between 0.3- 1.8 units enzyme were used. Where indicated enzyme Az, which resembles A1 in every respect except charge (see [6]), was used. The divalent cation concentration was either 10 mM MgClz or 1 mM MnC12. Phosphate buffers were used for pH 6.4 or pH 6.8 and Tris-HCI at pH 7.8. The final ionic strength of buffers in the assays was 0.04 M. All incubations were for 10 min at 30 "C except that those with activated DNA were done at 37 'C. 1 pg of synthetic template or SO pg activated calf thymus DNA was used. When base ratios of 1 : 1 are indicated 1 pg of the appropriate initiator was used. Assays with homopolynucleotides, e. g. poly(dA), poly(dT), poly(dC), poly(A), (dT),,, (A)loor (dG)," were done hut in all cases incorporation was less than 1 %, of that with complete template-initiator complex Template

Divalent cation

'H-labelled dNTP

Activity of enzyme ~

C at pH

A1 at pH .

Activated DNA Poly(dA). (dT)lo ( A T = 20: 3 ) Poly(dA) . (dT),, ( A : T = 20:l) Poly(A) . (dT)," ( A : T = 1 :1) Activated DNA Poly(dT) . (A)lo (T:A = 1 : 1) Activated DNA Poly(dC) . (dG)lo (C:G = 1 : 1)

MgZ+ MgZf MnZi M g ' or Mn2+ ME'+ Mg" Mg2' Mg'+ Mn2 Mg2+ or Mn' +

Poly(C) . (dG)," ( C : G = 1 : 1)

rH]dTTP [3H]dTTP ['HIdTTP [3H]dTTP [3H]dATP [3H]dATP [3H]dGTP [3H]dGTP [3H]dGTP ['HIdGTP

D at pH ~

~~~~-

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~

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6.4

6.8

7.8

6.4

6.8

7.8

6.4

6.8

7.8

20 50 11