Chromosoma (1992) 102:S100-S106
CHROMOSOMA 9 Springer-Verlag 1992
Isolation and characterization of a DNA helicase from cytosolic extracts of calf thymus Suisheng Zhang and Frank Grosse German Primate Center, Department of Virology and Immunology, Kelinerweg 4, W-3400 GSttingen, Federal Republic of Germany
Received September 12, 1992
Abstract. A DNA helicase has been isolated from calf thymus tissue. The enzyme was enriched from crude cytosolic extracts by batchwise chromatography on phosphocellulose, followed by 35% ammonium sulfate precipitation, and subsequent chromatography on phenylSepharose, single-stranded DNA cellulose, and AcA 44 gel filtration. The DNA helicase had a Stokes' radius of about 45 A and a sedimentation coefficient of 4.3 S. The most purified fractions contained three polypeptides with apparent molecular weights of 110, 65, and 34 kDa. UV crosslinking with radioactive dATP stained all three major polypeptides. The helicase catalyzed the unwinding of a DNA primer from a single-stranded DNA template in an ATP- or dATP-dependent manner. DNA unwinding was also observed with CTP or dCTP, but with reduced efficiency. The helicase translocated from 3' to 5' on the single-stranded template it was bound to. Relationships between this DNA helicase and other calf thymus helicases will be discussed.
Introduction DNA unwinding is a prerequisite for many physiological processes, such as DNA replication, DNA repair, DNA recombination and possibly also DNA transcription. In the living cell, DNA unwinding is carried out by enzymes named DNA helicases (Komberg and Baker 1991). Helicases use the energy derived from hydrolysis of the T-phosphate of nucleoside triphosphates to fuel the ener-
getically disfavored unwinding reaction. In the bacterium Escherichia coli and its related bacteriophages up to ten different DNA helicases have been found so far (Matson 1991; Matson and Kaiser-Rogers 1990). Similarly, a variety of different helicases have been detected in higher eukaryotes (Thtmmes et al. 1992; Thtmmes and Hfibscher 1990b). These enzymes have been classified according to their physical properties, their polarity of unwinding and their capability to utilize different nueleoside triphosphate cofactors. Although some of these enzymes partially purify with DNA polymerases (Li et al. 1992; Siegal et al. 1992; ThSmmes and Hfibscher 1990b) their physiological functions have not yet been clarified. Recently, we have isolated and characterized two distinct DNA helicases from calf thymus nuclei (Zhang and Grosse 1991). Since most of the helieases known so far have been isolated from cytosolic extracts of higher eukaryotic cells (like the replicative DNA polymerases 5, and e) we have now attempted to purify DNA helicases from this cellular compartment. Purification of cytosolic DNA helicases turned out to be difficult because contaminating DNases usually destroyed the substrates for the helicase assay. This made it impossible to follow the DNA unwinding reaction during the initial steps of the purification scheme. In spite of this problem, we have developed a simple and efficient procedure for the fast purification of a highly active DNA helicase from calf thymus cytosol. Here we present some properties of this enzyme and discuss possible physiological implications. Materials and methods
Abbreviations: E.coli, Escherichia coli; SV 40, simian virus 40; SSB, single-stranded DNA binding protein; ATP2S , adenosine 5'-O-(3-thiotdphosphate); DTF, dithiothreitol. Correspondence to: F. Grosse
Materials. Unlabeled nucleotides, nucleoside Iriphosphates and deoxynucleoside triphosphates were purchased from Sigma. 3H-labeled and 32p-labeled ribo- and deoxyribonucleoside triphosphates were obtained
S101 from Amersham (Braunschweig, FRG). Phosphocelluloso (P11) was from Whatman (Maidstone, UK). AcA 44 ultrogel was obtained from LKB-IBF-Biotcchnics (Villeneuve-la-Garenne, France). Single-slranded DNA cellulose was prepared by epoxide cross-linking of DNA to cellulose as described (Grosse et al. 1986). Polyethyleneimine-cellulose (PEI-cellulose) thin-layer plates CEL 300 PEL were from MachereyNagel (I)iiren, FRG). M13mp 18 single-stranded DNA was prepared as described (Grosse and Krauss 1984). The DNA 45-mer 5'-ACTCTAGAGGATCCCCGGGTACGT TA'ITGCATGAGCCCGGCTGand the43-mer 5'-AGGTCGACTCTAGAGGATCCCCGGGTACCGAGC TCGAATrCGT were synthesized by the phosphoroamidite method using a 380 B DNA synthesizer (Applied Biosystems). The oligonucleotide 45-mer had a 22 nucleotide long sequence at its 5' end that is complementary to the nucleotides from position 6243 to 6264 of M 13mpl 8 single-stranded DNA, leaving a 23-base-long non-complementary tail at its 3' end when hybridized to its template DNA. The DNA 43-mer was complementary to the sequence from 6228 to 6271 of the M13mpl 8 single-stranded DNA genome. T4 polynucleotide kinase, Klenow fragment ofDNA polymerase I of E.coli, and Sma I were from New England Biolabs (Schwalbach, FRG). The calibration kit for gel filtration was from Pharmaeia (Freiburg, FRG).
Methods. Preparation ofDNA helicase substrates, the DNA unwinding assay and the DNA dependent ATPase assaywere performed exactly as described elsewhere (Zhang and Grosse 1991). UV photocrosslinking was conducted as described (Nasheuer and Grosse 1988). SDS-polyacrylamide gel electrophoresis was performed according to Laemmll (Laemmli 1970).
pH 7.8, 0.5 M (NH4)2SO4,1 mM EDTA. The column was washed with 90 ml of this buffer and proteins were eluted batchwise with 1 L of a buffer containing 20 mM potassium phosphate, pH 7.8, 1 mM EDTA. The protein containing fractions that eluted at low salt concentrations were combined and directly loaded onto 30 ml single-stranded DNA cellulose (3x40 era), equilibrated with 20 mM potassitnn phosphate, pH 7.8, 50 mM NaC1, 1 mM EDTA. The column was washed with 90 ml of this buffer and then eluted with a 300 ml gradient of 50 mM to 500 mM NaCI in equilibration buffer. Fractions of 3 ml were collected and assayed for DNA helicase activity. From there on, a DNA heliease activity was identified unambiguously and quantified directly by using the strand-displacement assay (Fig. 1A). DNA helicase activity eluted at a conductivity equivalent to about 150 mM NaCI. These fractions were combined and concentrated to a final volume of 4 ml by using an Amicon protein concentrator. The concentrated fraction was loaded onto an AeA 44 column (1.5x80 era) equilibrated with a buffer of 30 mM potassium phosphate, pH 7.8, 100 mM KCI, 200 mM NaCI, 1 mM EDTA. The flow rate was 20 ml/h and fractions of 3 ml were collected. Fructions 22-25 contained both DNA-dependent ATPase and DNA unwinding activities (Fig. 1B). The heliease-containing fractions were combined, dialyzed against a buffer of 30 mM potassium phosphate, pH 7.8,1 mM EDTA, 50% (v/v) glycerol, and stored at-20~ The enzymatic activity was stable for at least 6 months. A summary of the purification procedure is shown in Table 1.
Table 1. Purification ofa eytosolic DNA helicase from calf thymus
Helicase Activity Fraction
Protein nag
Total units
Specific unitshng
I. Crude exlraet II. Phosphccellulose
36,000 1,000
N.D. N.D.
N.D. N.D.
N.D. N.D.
III. 35% (NH4)2SO4 IV. Phenyl-Sepharose V. DNA-ceUulose VI. AcA44
450 150 5.8 0.8
N.D. 120,000 18,000 16,000
N.D. 800 3,000 20,000
N.D. 100 15 13
Purification of a DNA helicasefrom calf thymuscytosol. All purification steps were carried out at 4~ and all buffers were prepared containing 10 mM Na2S205, 7 mM 2-mercaptoethanol, 1% (v/v) Trasylol (Bayer, Germany), and 1 mM phenylmethanesulfonyl fluoride. One kg calf thymus was thawed overnight at 4 ~ and homogenized in 3 L buffer containing 50 mM Tris/HC1, pH 7.8, 25 mM KC1, 5 mM MgC12, and 250 mM sucrose for 3 min at the lowest setting of a Waring Blendor. The homogenate was centrifuged for 10 rain at 10,000 g. The supematant was filtered through two layers of Miracloth (Calbiochem, Bad-Soden, FRG). The crude extract was adsorbed to 500 ml phosphoeellulose P11, equilibrated with 30 mM potassium phosphate, pH 7.0, by stirring for 30 rain at 4~ The slurry was filtered through a sintered glass funnel by applying water aspirator vacuum to remove non-adsorbed proteins. The phosphocellulose was washed on the funnel by applying water aspirator vacuum with 5 L of 70 mM potassium phosphate, pH 7.0, 1 mM EDTA. The proteins were eluted in a batchwise manner with 1.8 L of 0.25 M potassium phosphate, pH 7.8, 1 mM EDTA, also under water aspirator vacuum. Fractions of 300 ml were collected. Since DNA helicases cannot be detected at this step because o fcontaminating DNase activities, we followed the protein absorption peak at 280 nm. The fractions with the highest UV-absorption were combined (900 ml). Pulverized (NH4)2SO4 was added slowly to the phosphoeellulose eluate (0.2 g/ml) under stirring over a period of at least 30-45 rain. Stirring was continued for another 30 min at 4~ During the addition of (NH4)2SO4, the pH of the solution was kept at 7.8 by adding an appropriate amount of KOH. The precipitated proteins were collected by centrifugation at 15,000 g for 20 min. The pellet was suspended in about 30 ml of 20 mM potassium phosphate, pH 7.8, 0.5 M (NH4)2SO4,1mM EDTA. The ammonium sulfate fraction was directly loaded onto a 30 ml phenyl-Sepharose column (3.0x40 era; Pharmacia, Freiburg, FRG) equilibrated with 20 mM potassium phosphate,
Yield %
DNA helicase activity could not be quantified (N.D.) during steps I to III. Accordingto the enrichmerlt ofDNA polymerase o~andthe removal of unrelated proteins, it was estimated that phosphocollulose gave an about 50-fold purification and the ammonium sulfate and phenylSepharose steps an approximate 10-fold purification.
Results Isolation o f a DNA helicase from calf thymus cytosol D N A helicases c a n n o t be detected in crude extracts o f c a l f t h y m u s and at early purification steps because contaminating D N a s e activities rapidly degrade the D N A substrate used for a s s a y i n g helicase activity (see e.g. Thfimmes et al. 1992). Thus, w e a s s u m e d that helicases m i g h t bind to p h o s p h o c e l l u l o s e and substantially be enriched b y this step, similar as it has b e e n s h o w n for m a n y other e n z y m e s i n v o l v e d in D N A replication. After phosphocellulose c h r o m a t o g r a p h y w e used a m m o n i u m
S102 A
B
Single-Stranded DNA Cellulose
AcA 44 Gel Filtration 1
10
tt
0.3
2
3
4
t
tit
5
6
7
8
9
t
t
t3o A m
14.0~ Am o
-2.5 O 12.0
E
1=
0.2
7.5 84
CHROMOSOMA 9 Springer-Verlag 1992
Isolation and characterization of a DNA helicase from cytosolic extracts of calf thymus Suisheng Zhang and Frank Grosse German Primate Center, Department of Virology and Immunology, Kelinerweg 4, W-3400 GSttingen, Federal Republic of Germany
Received September 12, 1992
Abstract. A DNA helicase has been isolated from calf thymus tissue. The enzyme was enriched from crude cytosolic extracts by batchwise chromatography on phosphocellulose, followed by 35% ammonium sulfate precipitation, and subsequent chromatography on phenylSepharose, single-stranded DNA cellulose, and AcA 44 gel filtration. The DNA helicase had a Stokes' radius of about 45 A and a sedimentation coefficient of 4.3 S. The most purified fractions contained three polypeptides with apparent molecular weights of 110, 65, and 34 kDa. UV crosslinking with radioactive dATP stained all three major polypeptides. The helicase catalyzed the unwinding of a DNA primer from a single-stranded DNA template in an ATP- or dATP-dependent manner. DNA unwinding was also observed with CTP or dCTP, but with reduced efficiency. The helicase translocated from 3' to 5' on the single-stranded template it was bound to. Relationships between this DNA helicase and other calf thymus helicases will be discussed.
Introduction DNA unwinding is a prerequisite for many physiological processes, such as DNA replication, DNA repair, DNA recombination and possibly also DNA transcription. In the living cell, DNA unwinding is carried out by enzymes named DNA helicases (Komberg and Baker 1991). Helicases use the energy derived from hydrolysis of the T-phosphate of nucleoside triphosphates to fuel the ener-
getically disfavored unwinding reaction. In the bacterium Escherichia coli and its related bacteriophages up to ten different DNA helicases have been found so far (Matson 1991; Matson and Kaiser-Rogers 1990). Similarly, a variety of different helicases have been detected in higher eukaryotes (Thtmmes et al. 1992; Thtmmes and Hfibscher 1990b). These enzymes have been classified according to their physical properties, their polarity of unwinding and their capability to utilize different nueleoside triphosphate cofactors. Although some of these enzymes partially purify with DNA polymerases (Li et al. 1992; Siegal et al. 1992; ThSmmes and Hfibscher 1990b) their physiological functions have not yet been clarified. Recently, we have isolated and characterized two distinct DNA helicases from calf thymus nuclei (Zhang and Grosse 1991). Since most of the helieases known so far have been isolated from cytosolic extracts of higher eukaryotic cells (like the replicative DNA polymerases 5, and e) we have now attempted to purify DNA helicases from this cellular compartment. Purification of cytosolic DNA helicases turned out to be difficult because contaminating DNases usually destroyed the substrates for the helicase assay. This made it impossible to follow the DNA unwinding reaction during the initial steps of the purification scheme. In spite of this problem, we have developed a simple and efficient procedure for the fast purification of a highly active DNA helicase from calf thymus cytosol. Here we present some properties of this enzyme and discuss possible physiological implications. Materials and methods
Abbreviations: E.coli, Escherichia coli; SV 40, simian virus 40; SSB, single-stranded DNA binding protein; ATP2S , adenosine 5'-O-(3-thiotdphosphate); DTF, dithiothreitol. Correspondence to: F. Grosse
Materials. Unlabeled nucleotides, nucleoside Iriphosphates and deoxynucleoside triphosphates were purchased from Sigma. 3H-labeled and 32p-labeled ribo- and deoxyribonucleoside triphosphates were obtained
S101 from Amersham (Braunschweig, FRG). Phosphocelluloso (P11) was from Whatman (Maidstone, UK). AcA 44 ultrogel was obtained from LKB-IBF-Biotcchnics (Villeneuve-la-Garenne, France). Single-slranded DNA cellulose was prepared by epoxide cross-linking of DNA to cellulose as described (Grosse et al. 1986). Polyethyleneimine-cellulose (PEI-cellulose) thin-layer plates CEL 300 PEL were from MachereyNagel (I)iiren, FRG). M13mp 18 single-stranded DNA was prepared as described (Grosse and Krauss 1984). The DNA 45-mer 5'-ACTCTAGAGGATCCCCGGGTACGT TA'ITGCATGAGCCCGGCTGand the43-mer 5'-AGGTCGACTCTAGAGGATCCCCGGGTACCGAGC TCGAATrCGT were synthesized by the phosphoroamidite method using a 380 B DNA synthesizer (Applied Biosystems). The oligonucleotide 45-mer had a 22 nucleotide long sequence at its 5' end that is complementary to the nucleotides from position 6243 to 6264 of M 13mpl 8 single-stranded DNA, leaving a 23-base-long non-complementary tail at its 3' end when hybridized to its template DNA. The DNA 43-mer was complementary to the sequence from 6228 to 6271 of the M13mpl 8 single-stranded DNA genome. T4 polynucleotide kinase, Klenow fragment ofDNA polymerase I of E.coli, and Sma I were from New England Biolabs (Schwalbach, FRG). The calibration kit for gel filtration was from Pharmaeia (Freiburg, FRG).
Methods. Preparation ofDNA helicase substrates, the DNA unwinding assay and the DNA dependent ATPase assaywere performed exactly as described elsewhere (Zhang and Grosse 1991). UV photocrosslinking was conducted as described (Nasheuer and Grosse 1988). SDS-polyacrylamide gel electrophoresis was performed according to Laemmll (Laemmli 1970).
pH 7.8, 0.5 M (NH4)2SO4,1 mM EDTA. The column was washed with 90 ml of this buffer and proteins were eluted batchwise with 1 L of a buffer containing 20 mM potassium phosphate, pH 7.8, 1 mM EDTA. The protein containing fractions that eluted at low salt concentrations were combined and directly loaded onto 30 ml single-stranded DNA cellulose (3x40 era), equilibrated with 20 mM potassitnn phosphate, pH 7.8, 50 mM NaC1, 1 mM EDTA. The column was washed with 90 ml of this buffer and then eluted with a 300 ml gradient of 50 mM to 500 mM NaCI in equilibration buffer. Fractions of 3 ml were collected and assayed for DNA helicase activity. From there on, a DNA heliease activity was identified unambiguously and quantified directly by using the strand-displacement assay (Fig. 1A). DNA helicase activity eluted at a conductivity equivalent to about 150 mM NaCI. These fractions were combined and concentrated to a final volume of 4 ml by using an Amicon protein concentrator. The concentrated fraction was loaded onto an AeA 44 column (1.5x80 era) equilibrated with a buffer of 30 mM potassium phosphate, pH 7.8, 100 mM KCI, 200 mM NaCI, 1 mM EDTA. The flow rate was 20 ml/h and fractions of 3 ml were collected. Fructions 22-25 contained both DNA-dependent ATPase and DNA unwinding activities (Fig. 1B). The heliease-containing fractions were combined, dialyzed against a buffer of 30 mM potassium phosphate, pH 7.8,1 mM EDTA, 50% (v/v) glycerol, and stored at-20~ The enzymatic activity was stable for at least 6 months. A summary of the purification procedure is shown in Table 1.
Table 1. Purification ofa eytosolic DNA helicase from calf thymus
Helicase Activity Fraction
Protein nag
Total units
Specific unitshng
I. Crude exlraet II. Phosphccellulose
36,000 1,000
N.D. N.D.
N.D. N.D.
N.D. N.D.
III. 35% (NH4)2SO4 IV. Phenyl-Sepharose V. DNA-ceUulose VI. AcA44
450 150 5.8 0.8
N.D. 120,000 18,000 16,000
N.D. 800 3,000 20,000
N.D. 100 15 13
Purification of a DNA helicasefrom calf thymuscytosol. All purification steps were carried out at 4~ and all buffers were prepared containing 10 mM Na2S205, 7 mM 2-mercaptoethanol, 1% (v/v) Trasylol (Bayer, Germany), and 1 mM phenylmethanesulfonyl fluoride. One kg calf thymus was thawed overnight at 4 ~ and homogenized in 3 L buffer containing 50 mM Tris/HC1, pH 7.8, 25 mM KC1, 5 mM MgC12, and 250 mM sucrose for 3 min at the lowest setting of a Waring Blendor. The homogenate was centrifuged for 10 rain at 10,000 g. The supematant was filtered through two layers of Miracloth (Calbiochem, Bad-Soden, FRG). The crude extract was adsorbed to 500 ml phosphoeellulose P11, equilibrated with 30 mM potassium phosphate, pH 7.0, by stirring for 30 rain at 4~ The slurry was filtered through a sintered glass funnel by applying water aspirator vacuum to remove non-adsorbed proteins. The phosphocellulose was washed on the funnel by applying water aspirator vacuum with 5 L of 70 mM potassium phosphate, pH 7.0, 1 mM EDTA. The proteins were eluted in a batchwise manner with 1.8 L of 0.25 M potassium phosphate, pH 7.8, 1 mM EDTA, also under water aspirator vacuum. Fractions of 300 ml were collected. Since DNA helicases cannot be detected at this step because o fcontaminating DNase activities, we followed the protein absorption peak at 280 nm. The fractions with the highest UV-absorption were combined (900 ml). Pulverized (NH4)2SO4 was added slowly to the phosphoeellulose eluate (0.2 g/ml) under stirring over a period of at least 30-45 rain. Stirring was continued for another 30 min at 4~ During the addition of (NH4)2SO4, the pH of the solution was kept at 7.8 by adding an appropriate amount of KOH. The precipitated proteins were collected by centrifugation at 15,000 g for 20 min. The pellet was suspended in about 30 ml of 20 mM potassium phosphate, pH 7.8, 0.5 M (NH4)2SO4,1mM EDTA. The ammonium sulfate fraction was directly loaded onto a 30 ml phenyl-Sepharose column (3.0x40 era; Pharmacia, Freiburg, FRG) equilibrated with 20 mM potassium phosphate,
Yield %
DNA helicase activity could not be quantified (N.D.) during steps I to III. Accordingto the enrichmerlt ofDNA polymerase o~andthe removal of unrelated proteins, it was estimated that phosphocollulose gave an about 50-fold purification and the ammonium sulfate and phenylSepharose steps an approximate 10-fold purification.
Results Isolation o f a DNA helicase from calf thymus cytosol D N A helicases c a n n o t be detected in crude extracts o f c a l f t h y m u s and at early purification steps because contaminating D N a s e activities rapidly degrade the D N A substrate used for a s s a y i n g helicase activity (see e.g. Thfimmes et al. 1992). Thus, w e a s s u m e d that helicases m i g h t bind to p h o s p h o c e l l u l o s e and substantially be enriched b y this step, similar as it has b e e n s h o w n for m a n y other e n z y m e s i n v o l v e d in D N A replication. After phosphocellulose c h r o m a t o g r a p h y w e used a m m o n i u m
S102 A
B
Single-Stranded DNA Cellulose
AcA 44 Gel Filtration 1
10
tt
0.3
2
3
4
t
tit
5
6
7
8
9
t
t
t3o A m
14.0~ Am o
-2.5 O 12.0
E
1=
0.2
7.5 84
