29
Biochimica et Biophysica Acta, 561 (1979) 29--41 © Elsevier/North-HollandBiomedicalPress
BBA 9 9 3 5 7
DNA POLYMERASE~ FROM REGENERATING RAT LIVER CATALYTIC PROPERTIES OF THE HIGHLY PURIFIED ENZYME
O D I L E F I C H O T , M A R T I A L P A S C A L *, M A R C E L M E C H A L I and A N N E - M A R I E DE RECONDO
Unitd d 'Enzymologie, Institut de Recherches Scientifiques sur le Cancer, Villejuif (France) ( R e c e i v e d April 19th, 1978)
Key words: DNA polymerase-~; Regeneration; (Rat liver, Properties)
Summary DNA polymerase-a from the cytosol of regenerating rat liver has been highly purified by a procedure which includes affinity chromatography. The purified enzyme sediments at 7.4 S in high ionic strength and at 9--10 S in low ionic strength, i.e. under in vitro polymerization conditions. This enzyme has all the properties of the other mammalian DNA polymerases-a: sensitivity to sulfhydryl-blocking agents, to heparin, and to the level of salt in the assay, neutral pH optimum, use of ribonucleotide-initiated DNA templates, and inability to copy the ribostrand of hybrids. After chromatography on denatured DNA-cellulose, the a-polymerase is completely devoid of exo- and endonuclease activities. Template competition experiments indicate that the binding of the enzyme to the template can be distinguished from the polymerization itself and that the in vitro synthesis catalyzed by this a-polymerase is not distributive in a classical sense. These facts are discussed.
Introduction
The existence of mutiple molecular weight forms of DNA polymerases in eukaryotic cells is well established [1--4]. These DNA polymerases are distinguishable by their physical properties, intracellular localization, template specificity, and requirements for maximal activity, but their biological role in DNA replication and repair is not well understood at this time. However many observations favor the involvement of the DNA polymerase-a in DNA replica* Present address: Unit~ de B i o c h i m i e Cellulaire, Institut de Biologie Mol6culaire, Universit~ de Paris VII, 2, Place Jussieu, 75005 - Paris, France. Abbreviations: d N T P ( s ) , d e o x y n u c l e o s i d e triphosphate(s). T h e abbreviations u s e d for n u c l e o t i d e s and p o l y n u c l e o t l d e s are those of the IUPAC-IUB commission (1970).
30 tion, since it has been shown that the 6 to 8 S activity, but not the 3.4 S enzyme (DNA polymerase-~) is significantly increased under conditions of active cellular proliferation [1,5--10]. Heterogeneity of DNA polymerase-a has been observed in a variety of tissues and cells [1,11,12]. This heterogeneity may be due to aggregation, proteolysis [ 11 ] or possibly to association with other proteins of the replication complex, and these probably account for some of the difficulties encountered during its purification. It has also been suggested that a-polymerase exists in more than one unique form possessing a common catalytic core [13,14]. We have previously described the template specificity of the DNA polymerase-a purified from regenerating rat liver cytosol [15]. We now present a more detailed analysis of the enzymatic properties of this enzyme which we have purified to a higher degree of specific activity by two additional steps (phosphocellulose and affinity chromatography). This procedure allowed us to obtain an enzyme which partially retained the property of elongating the growing chain in vitro in a processive manner; these results will be discussed in comparison to those obtained by Chang with calf thymus DNA polymerase-a and -~ in in vitro systems containing no other components of the replication complex [16].
Experimentalprocedure Materials UnlabeUed deoxyribonucleotides were purchased from Schwarz-Mann (U.S.A.), and labelled deoxyribonucleotides from the Radiochemical Centre, Amersham (U.K.). Calf thymus DNA was supplied by Choay (France). The double-stranded copolymer poly[d(A-T)" d(T-A)] and the single-stranded homopolymer poly(dT), poly(dC), poly(dA), poly(dG), poly(A) and poly(C) were purchased from Boehringer, Mannheim (G.F.R.) and P.L. Biochemical Inc. (U.S.A.) The oligodeoxyribonucleotides (dT}12-1s, (dA)12-18, (dG)12-1s were obtained from Collaborative Research Inc. (U.S.A.). Double-stranded polydeoxyribonucleotides were prepared by heating at 80°C followed by slow cooling of equimolar quantities of homopolymers (1 mM in mononucleotide concentration) in 10 mM Tris-HC1 (pH 7.5)/100 mM NaC1. The initiator to template ratios were 1 : 4 (in mononucleotide concentration) for the homopolymers containing G and C and 1 : 1 for those containing A and T. Activated calf thymus DNA was prepared by treating native calf thymus DNA with pancreatic DNAase I until 20% of the total nucleotides was acid soluble. Ribonuclease (beef pancreas) was obtained from Worthington, Escherichia coli RNA polymerase and deoxyribonuclease I (grade I, ribonuclease free) from Boehringer (G.F.R.). Enzymes. DNA polymerase or deoxynucleoside triphosphate : DNA nucleotidyltransferase (EC 2.7.7.7.), DNA polymerase-a and DNA polymerase-~, according to the nomenclature proposed by Weissbach et al. [30], DNAase or deoxyribonuclease (EC 3.1.4.5.}, RNAase or ribonuclease (EC 2.7.7.16), alcohol dehydrogenase or alcohol : NAD oxidoreductase (EC 1.1.1.1.), catalase or H:O: : H20: oxidoreductase (EC 1.11.1.6.}.
31
Methods DNA polymerase assay. Unless otherwise stated in the figure legends, the reaction mixture (0.05 ml) contained: 60 mM Tris-HC1 (pH 7.0 for the templates containing A or T and for the DNA templates, and pH 7.6 for the templates containing G or C), 3.4 mM MgC12, 2.4 mM KC1, 50 mM 2-mercaptoethanol, 22 pM synthetic template or 150 gM DNA, 200 pM each dATP, dCTP, dTTP and dGTP, 2.5 gCi 3H-labeUed deoxyribonucleoside triphosphate and DNA polymerase. The concentrations of the synthetic templates are given as mononucleotide concentration(s) of copied strand(s). Incubations were carried out at 38°C, aliquots were withdrawn at various time intervals and the amount of product formed was determined by the glass filter method [15]. 1 enzyme unit is defined as the activity necessary to convert I nmol of total nucleotide to an acid-insoluble product in 1 h at 38°C. Assay of endo- and exonuclease activities. Endo- and exonuclease activities were assayed by the incubation of SV40 [3H]DNA (sonicated to 8--10 S fragments) with the enzyme preparation, under the conditions of the DNA polymerase reaction. Gel filtration on Sephadex G-100 in the presence of SSC (standard saline-citrate solution, 0.0015 M sodium citrate/0.15 M NaC1) was used to monitor the course of the exonucleolytic and endonucleolytic hydrolysis of DNA. 'Nickase' activity was measured by cleavage of supercoiled SV40 [3H]DNA (34 000 cpm/~g) to an open circular form [17]. Protein determination. The protein concentration was determined by the method of Schaffner and Weissman [18]. Sucrose gradient centrifugation. Linear 5--20% (w/v) sucrose gradients were prepared in 50 mM Tris-HC1 (pH 7.5) containing various concentrations of KC1 and 10 mM 2-mercaptoethanol, as described in the legends of the figures. The enzyme solution was layered onto the gradient and the sedimentation was performed for 15 h at 37 000 rev./min and 4°C in a SW50.1 Spinco rotor. At the end of the centrifugation, fractions of equal volume were collected from the bottom of each gradient. Gel electrophoresis. Electrophoresis was carried out in non-denaturing conditions at 4°C, in a Tris-glycine buffer, pH 8.3. Approx. 3--5 gg of protein were layered over a 2.5% stacking gel. The separating gel was 7.5% acrylamide or was a linear gradient of 3--20% polyacrylamide concentration. The gels were stained in 0.0125% Coomassie Blue in 50% methanol/5% acetic acid, then destalned in 20% methanol/5% acetic acid. Sodium dodecyl sulfate polyacrylamide gel electrophoresis was carried out as described by Laemmli [19] with a 20 ml linear gradient of 6--20% polyacrylamide gel concentration used as a separating gel. When necessary, samples were concentrated by lyophylisation or evaporation under nitrogen. Results
Purification of DNA polymerase-a DNA polymerase-a was prepared from regenerating rat liver using a slight modification of the procedure described previously [15]. Two-third partial hepatectomies were performed on 30--50 male WAG rats (200--300 g) by removal of the left lateral and median lobes. 40 h after the hepatectomy the
32 rats were killed by decapitation, and regenerating livers rapidly removed and placed on ice. All subsequent fractionation steps were performed at 4°C. After preparation of the postmicrosomal supernatant by centrifugation at 105 000 × g (Step I), precipitation by (NH4)2SO4 (30--40%, Step II), chromatography on DEAE cellulose (elution at 0.15 M NaC1, Step III), and chromatography on hydroxyapatite in the presence of 0.5 M NaCI (elution between 0.12 and 0.15 M potassium phosphate, Step IV), the enzyme was further purified by chromatography on phosphocellulose and affinity chromatography on denatured-DNA cellulose. After dialysis against a solution containing Tris-HC1 (pH 7.5)/1 mM MgC12/6 mM KC1/1 mM 2-mercaptoethanol and 30% glycerol (Buffer A), Fraction IV (17 mg protein) was loaded onto a phosphocellulose column (P 11 Whatman 0.8 × 16 cm) which was freshly regenerated and previously equilibrated with the same buffer. The column was washed overnight with Buffer A containing 0.15 M NaC1. Then the absorbed proteins were eluted with 60 ml of a linear NaC1 gradient (0.2 to 0.6 M) in Buffer A. DNA polymerase activity was eluted between 0.25 and 0.35 M NaC1. Fractions containing polymerase activity were pooled, dialyzed against Buffer A containing 2 mM EDTA, and loaded onto a denatured-DNA cellulose column (0.5 × 8 cm), prepared according to Alberts and Herrick [20] and equilibrated with Buffer A containing 2 mM EDTA (Buffer B). The unadsorbed proteins were eluted with the same buffer. The DNA polymerase-a activity was eluted with Buffer B containing 0.15 M NaCl. The enzyme fractions were dialyzed against Buffer A containing 50% glycerol and stored at --80°C. Under these conditions, the activity remained relatively stable for many weeks. A summary of the purification is shown in Table I. Homogeneity of the enzyme and estimation of its molecular weight. Electrophoresis of the purified enzyme preparation in 7.5% polyacrylamide gels indicated that one major protein band was present in addition to some aggregates which were not able to migrate in this gel even after treating the preparation with DNAase I (not shown). Electrophoresis was also carried out by using a 3--20% polyacrylamide gel concentration in order to obtain a better resolution of total proteins than in a homogeneous gel. In these conditions a single protein band was reproducibly observed (Fig. 1). DNA polymerase activity was tested
TABLE I PURIFICATION OF DNA POLYMERASE-a
FROM REGENERATING
RAT LIVER
The reaction conditions were as described in Materials and Methods. 1 u n i t = 1 n m o l of total nucleotide i n c o r p o r a t e d p e r h in i n i t i a l v e l o c i t y c o n d i t i o n s . Fraction
I lI IIl IV V VI
Supernatant ( N H 4 ) 2 SO 4 D E A E cellulose Hydroxyapatite PhosphoceUulose D N A cellulose
Total protein (rag)
poly(dC) • ( d G ) 12-I 8
Activated DNA
(units)
(units/rag)
(units)
(units/mg)
4 500 514 30 4.75 0.69 0.030
31 84 14 11 2
7 165 450 2 420 3 165 16 560
92 95 24 9 1
20 185 805 2 074 2 520 16 146
350 000 050 490 185 557
300 250 250 850 910 545
33
!
I +
T
Dye
I
Fig. 1. N o n - d e n a t u r i n g p o l y a c w l a m i d e gel e l e c t r o p h o r e s i s of r e g e n e r a t i n g r a t liver D N A polymerase-C~. F r a c t i o n VI D N A p o l y m e r a s e - ~ ( 2 . 5 pg) was l o a d e d o n t o a l i n e a r 3 - - 2 0 % p o l y a c r y l a m i d e g r a d i e n t gel o v e r l a y e r e d b y a 2.5% p o l y a c r y l a m i d e s t a c k i n g gel. E l e e t r o p h o r e s i s was p e r f o r m e d in 10 m M T r i s / 7 5 m M glycine, p H 8.3, at 9.7 m A / c m 2 gel surface. S t a i n i n g a n d d e s t a i n i n g w e r e as d e s c r i b e d in Materials a n d M e t h o d s . T h e s t a i n e d gel was s c a n n e d w i t h a m i c r o d e n s i t o m e t e r a n d t h e t r a c i n g o b t a i n e d is aligned in register b e l o w t h e gel.
after extraction of proteins from the gel. These experiments suggested that activity corresponds to the stained band b u t the percentage of activity recovered was less than 2% and so far we could not ascertain that any activity should be found in other parts of the gel. In sodium dodecyl sulfate acrylamide gels, all the preparations (Fraction VI) studied exhibited t w o major bands in the molecular weight range of 6 to 7 • 104 (68%) and 1.6- l 0 s (25%); occasionally a discrete band in the molecular weight range of 9 . 1 0 4 appeared (
Biochimica et Biophysica Acta, 561 (1979) 29--41 © Elsevier/North-HollandBiomedicalPress
BBA 9 9 3 5 7
DNA POLYMERASE~ FROM REGENERATING RAT LIVER CATALYTIC PROPERTIES OF THE HIGHLY PURIFIED ENZYME
O D I L E F I C H O T , M A R T I A L P A S C A L *, M A R C E L M E C H A L I and A N N E - M A R I E DE RECONDO
Unitd d 'Enzymologie, Institut de Recherches Scientifiques sur le Cancer, Villejuif (France) ( R e c e i v e d April 19th, 1978)
Key words: DNA polymerase-~; Regeneration; (Rat liver, Properties)
Summary DNA polymerase-a from the cytosol of regenerating rat liver has been highly purified by a procedure which includes affinity chromatography. The purified enzyme sediments at 7.4 S in high ionic strength and at 9--10 S in low ionic strength, i.e. under in vitro polymerization conditions. This enzyme has all the properties of the other mammalian DNA polymerases-a: sensitivity to sulfhydryl-blocking agents, to heparin, and to the level of salt in the assay, neutral pH optimum, use of ribonucleotide-initiated DNA templates, and inability to copy the ribostrand of hybrids. After chromatography on denatured DNA-cellulose, the a-polymerase is completely devoid of exo- and endonuclease activities. Template competition experiments indicate that the binding of the enzyme to the template can be distinguished from the polymerization itself and that the in vitro synthesis catalyzed by this a-polymerase is not distributive in a classical sense. These facts are discussed.
Introduction
The existence of mutiple molecular weight forms of DNA polymerases in eukaryotic cells is well established [1--4]. These DNA polymerases are distinguishable by their physical properties, intracellular localization, template specificity, and requirements for maximal activity, but their biological role in DNA replication and repair is not well understood at this time. However many observations favor the involvement of the DNA polymerase-a in DNA replica* Present address: Unit~ de B i o c h i m i e Cellulaire, Institut de Biologie Mol6culaire, Universit~ de Paris VII, 2, Place Jussieu, 75005 - Paris, France. Abbreviations: d N T P ( s ) , d e o x y n u c l e o s i d e triphosphate(s). T h e abbreviations u s e d for n u c l e o t i d e s and p o l y n u c l e o t l d e s are those of the IUPAC-IUB commission (1970).
30 tion, since it has been shown that the 6 to 8 S activity, but not the 3.4 S enzyme (DNA polymerase-~) is significantly increased under conditions of active cellular proliferation [1,5--10]. Heterogeneity of DNA polymerase-a has been observed in a variety of tissues and cells [1,11,12]. This heterogeneity may be due to aggregation, proteolysis [ 11 ] or possibly to association with other proteins of the replication complex, and these probably account for some of the difficulties encountered during its purification. It has also been suggested that a-polymerase exists in more than one unique form possessing a common catalytic core [13,14]. We have previously described the template specificity of the DNA polymerase-a purified from regenerating rat liver cytosol [15]. We now present a more detailed analysis of the enzymatic properties of this enzyme which we have purified to a higher degree of specific activity by two additional steps (phosphocellulose and affinity chromatography). This procedure allowed us to obtain an enzyme which partially retained the property of elongating the growing chain in vitro in a processive manner; these results will be discussed in comparison to those obtained by Chang with calf thymus DNA polymerase-a and -~ in in vitro systems containing no other components of the replication complex [16].
Experimentalprocedure Materials UnlabeUed deoxyribonucleotides were purchased from Schwarz-Mann (U.S.A.), and labelled deoxyribonucleotides from the Radiochemical Centre, Amersham (U.K.). Calf thymus DNA was supplied by Choay (France). The double-stranded copolymer poly[d(A-T)" d(T-A)] and the single-stranded homopolymer poly(dT), poly(dC), poly(dA), poly(dG), poly(A) and poly(C) were purchased from Boehringer, Mannheim (G.F.R.) and P.L. Biochemical Inc. (U.S.A.) The oligodeoxyribonucleotides (dT}12-1s, (dA)12-18, (dG)12-1s were obtained from Collaborative Research Inc. (U.S.A.). Double-stranded polydeoxyribonucleotides were prepared by heating at 80°C followed by slow cooling of equimolar quantities of homopolymers (1 mM in mononucleotide concentration) in 10 mM Tris-HC1 (pH 7.5)/100 mM NaC1. The initiator to template ratios were 1 : 4 (in mononucleotide concentration) for the homopolymers containing G and C and 1 : 1 for those containing A and T. Activated calf thymus DNA was prepared by treating native calf thymus DNA with pancreatic DNAase I until 20% of the total nucleotides was acid soluble. Ribonuclease (beef pancreas) was obtained from Worthington, Escherichia coli RNA polymerase and deoxyribonuclease I (grade I, ribonuclease free) from Boehringer (G.F.R.). Enzymes. DNA polymerase or deoxynucleoside triphosphate : DNA nucleotidyltransferase (EC 2.7.7.7.), DNA polymerase-a and DNA polymerase-~, according to the nomenclature proposed by Weissbach et al. [30], DNAase or deoxyribonuclease (EC 3.1.4.5.}, RNAase or ribonuclease (EC 2.7.7.16), alcohol dehydrogenase or alcohol : NAD oxidoreductase (EC 1.1.1.1.), catalase or H:O: : H20: oxidoreductase (EC 1.11.1.6.}.
31
Methods DNA polymerase assay. Unless otherwise stated in the figure legends, the reaction mixture (0.05 ml) contained: 60 mM Tris-HC1 (pH 7.0 for the templates containing A or T and for the DNA templates, and pH 7.6 for the templates containing G or C), 3.4 mM MgC12, 2.4 mM KC1, 50 mM 2-mercaptoethanol, 22 pM synthetic template or 150 gM DNA, 200 pM each dATP, dCTP, dTTP and dGTP, 2.5 gCi 3H-labeUed deoxyribonucleoside triphosphate and DNA polymerase. The concentrations of the synthetic templates are given as mononucleotide concentration(s) of copied strand(s). Incubations were carried out at 38°C, aliquots were withdrawn at various time intervals and the amount of product formed was determined by the glass filter method [15]. 1 enzyme unit is defined as the activity necessary to convert I nmol of total nucleotide to an acid-insoluble product in 1 h at 38°C. Assay of endo- and exonuclease activities. Endo- and exonuclease activities were assayed by the incubation of SV40 [3H]DNA (sonicated to 8--10 S fragments) with the enzyme preparation, under the conditions of the DNA polymerase reaction. Gel filtration on Sephadex G-100 in the presence of SSC (standard saline-citrate solution, 0.0015 M sodium citrate/0.15 M NaC1) was used to monitor the course of the exonucleolytic and endonucleolytic hydrolysis of DNA. 'Nickase' activity was measured by cleavage of supercoiled SV40 [3H]DNA (34 000 cpm/~g) to an open circular form [17]. Protein determination. The protein concentration was determined by the method of Schaffner and Weissman [18]. Sucrose gradient centrifugation. Linear 5--20% (w/v) sucrose gradients were prepared in 50 mM Tris-HC1 (pH 7.5) containing various concentrations of KC1 and 10 mM 2-mercaptoethanol, as described in the legends of the figures. The enzyme solution was layered onto the gradient and the sedimentation was performed for 15 h at 37 000 rev./min and 4°C in a SW50.1 Spinco rotor. At the end of the centrifugation, fractions of equal volume were collected from the bottom of each gradient. Gel electrophoresis. Electrophoresis was carried out in non-denaturing conditions at 4°C, in a Tris-glycine buffer, pH 8.3. Approx. 3--5 gg of protein were layered over a 2.5% stacking gel. The separating gel was 7.5% acrylamide or was a linear gradient of 3--20% polyacrylamide concentration. The gels were stained in 0.0125% Coomassie Blue in 50% methanol/5% acetic acid, then destalned in 20% methanol/5% acetic acid. Sodium dodecyl sulfate polyacrylamide gel electrophoresis was carried out as described by Laemmli [19] with a 20 ml linear gradient of 6--20% polyacrylamide gel concentration used as a separating gel. When necessary, samples were concentrated by lyophylisation or evaporation under nitrogen. Results
Purification of DNA polymerase-a DNA polymerase-a was prepared from regenerating rat liver using a slight modification of the procedure described previously [15]. Two-third partial hepatectomies were performed on 30--50 male WAG rats (200--300 g) by removal of the left lateral and median lobes. 40 h after the hepatectomy the
32 rats were killed by decapitation, and regenerating livers rapidly removed and placed on ice. All subsequent fractionation steps were performed at 4°C. After preparation of the postmicrosomal supernatant by centrifugation at 105 000 × g (Step I), precipitation by (NH4)2SO4 (30--40%, Step II), chromatography on DEAE cellulose (elution at 0.15 M NaC1, Step III), and chromatography on hydroxyapatite in the presence of 0.5 M NaCI (elution between 0.12 and 0.15 M potassium phosphate, Step IV), the enzyme was further purified by chromatography on phosphocellulose and affinity chromatography on denatured-DNA cellulose. After dialysis against a solution containing Tris-HC1 (pH 7.5)/1 mM MgC12/6 mM KC1/1 mM 2-mercaptoethanol and 30% glycerol (Buffer A), Fraction IV (17 mg protein) was loaded onto a phosphocellulose column (P 11 Whatman 0.8 × 16 cm) which was freshly regenerated and previously equilibrated with the same buffer. The column was washed overnight with Buffer A containing 0.15 M NaC1. Then the absorbed proteins were eluted with 60 ml of a linear NaC1 gradient (0.2 to 0.6 M) in Buffer A. DNA polymerase activity was eluted between 0.25 and 0.35 M NaC1. Fractions containing polymerase activity were pooled, dialyzed against Buffer A containing 2 mM EDTA, and loaded onto a denatured-DNA cellulose column (0.5 × 8 cm), prepared according to Alberts and Herrick [20] and equilibrated with Buffer A containing 2 mM EDTA (Buffer B). The unadsorbed proteins were eluted with the same buffer. The DNA polymerase-a activity was eluted with Buffer B containing 0.15 M NaCl. The enzyme fractions were dialyzed against Buffer A containing 50% glycerol and stored at --80°C. Under these conditions, the activity remained relatively stable for many weeks. A summary of the purification is shown in Table I. Homogeneity of the enzyme and estimation of its molecular weight. Electrophoresis of the purified enzyme preparation in 7.5% polyacrylamide gels indicated that one major protein band was present in addition to some aggregates which were not able to migrate in this gel even after treating the preparation with DNAase I (not shown). Electrophoresis was also carried out by using a 3--20% polyacrylamide gel concentration in order to obtain a better resolution of total proteins than in a homogeneous gel. In these conditions a single protein band was reproducibly observed (Fig. 1). DNA polymerase activity was tested
TABLE I PURIFICATION OF DNA POLYMERASE-a
FROM REGENERATING
RAT LIVER
The reaction conditions were as described in Materials and Methods. 1 u n i t = 1 n m o l of total nucleotide i n c o r p o r a t e d p e r h in i n i t i a l v e l o c i t y c o n d i t i o n s . Fraction
I lI IIl IV V VI
Supernatant ( N H 4 ) 2 SO 4 D E A E cellulose Hydroxyapatite PhosphoceUulose D N A cellulose
Total protein (rag)
poly(dC) • ( d G ) 12-I 8
Activated DNA
(units)
(units/rag)
(units)
(units/mg)
4 500 514 30 4.75 0.69 0.030
31 84 14 11 2
7 165 450 2 420 3 165 16 560
92 95 24 9 1
20 185 805 2 074 2 520 16 146
350 000 050 490 185 557
300 250 250 850 910 545
33
!
I +
T
Dye
I
Fig. 1. N o n - d e n a t u r i n g p o l y a c w l a m i d e gel e l e c t r o p h o r e s i s of r e g e n e r a t i n g r a t liver D N A polymerase-C~. F r a c t i o n VI D N A p o l y m e r a s e - ~ ( 2 . 5 pg) was l o a d e d o n t o a l i n e a r 3 - - 2 0 % p o l y a c r y l a m i d e g r a d i e n t gel o v e r l a y e r e d b y a 2.5% p o l y a c r y l a m i d e s t a c k i n g gel. E l e e t r o p h o r e s i s was p e r f o r m e d in 10 m M T r i s / 7 5 m M glycine, p H 8.3, at 9.7 m A / c m 2 gel surface. S t a i n i n g a n d d e s t a i n i n g w e r e as d e s c r i b e d in Materials a n d M e t h o d s . T h e s t a i n e d gel was s c a n n e d w i t h a m i c r o d e n s i t o m e t e r a n d t h e t r a c i n g o b t a i n e d is aligned in register b e l o w t h e gel.
after extraction of proteins from the gel. These experiments suggested that activity corresponds to the stained band b u t the percentage of activity recovered was less than 2% and so far we could not ascertain that any activity should be found in other parts of the gel. In sodium dodecyl sulfate acrylamide gels, all the preparations (Fraction VI) studied exhibited t w o major bands in the molecular weight range of 6 to 7 • 104 (68%) and 1.6- l 0 s (25%); occasionally a discrete band in the molecular weight range of 9 . 1 0 4 appeared (
