Eur. J . Biochem. 62, 143- 150 (1976)
A DNA Polymerase from Ustilago maydis 2. Properties of the Associated Deoxyribonuclease Activity Geoffrey R. BANKS and Geoffrey T. YARRANTON National Institute for Medical Research, London (Received July 23/September 30, 1975)
The polymerase and deoxyribonuclease activities of the purified Ustilago maydis DNA polymerase coeluted from a hydroxyapatite column, cosedimented in sucrose gradients in both the absence and presence of salt, possessed similar thermolabilities and reaction requirements. These observations suggest that both activities are associated with the same enzyme and that the deoxyribonuclease activity is not a contaminant. The initial rate of degradation of native 3'-end-group-labelled DNA was similar to that of a heat-denatured substrate, but the final extent was greater for the former. The enzyme exhibits a high specificity for degradation of DNA in a 3'+5' direction. The degradation of a DNA template was inhibited by the presence of the deoxyribonucleoside triphosphates necessary for simultaneous DNA synthesis, but not that of the newly synthesised DNA. About 50 29 '%; and 13 of the purine, cytosine and thymine deoxyribonucleotide residues incorporated by the enzyme into DNA respectively, were subsequently excised when monitored by the resulting conversion of the triphosphate substrates to free monophosphate. The majority of the purine deoxyribonucleoside monophosphates appear after the synthetic phase of the reaction has ceased. In many respects, therefore, the deoxyribonuclease activity of the U . maydis DNA polymerase is similar to the bacteriophage T4-induced enzyme.
x,
Extensively purified DNA polymerases from prokaryotic organisms appear to possess at least one exonuclease activity, and there is good evidence in many cases that both activities reside on the same polypeptide chain [I - 1I]. Most eukaryotic enzymes, although having polymerase activity with characteristics similar to those of the prokaryotic ones, are devoid of detectable exonuclease activity [12- 171. Nuclease activity may, however, be associated with DNA polymerases purified from bakers' yeast and Tetrahymena pyriformis [18,19]. The DNA polymerase from Ustilago maydis, whose polymerase activity was the subject of the preceding paper [20], also possesses deoxyribonuclease activity. This paper describes its characteristics and provides evidence that it is not a contaminant of the preparation. ~
Ahbrevrarions. Abbreviations for synthetic polynucleotides follow CBN rules, see Eur. J . Biochem. 15, 203 - 208 (1970). Enzymes. D N A polymerase or deoxynucleosidetriphosphate : DNA deoxynucleotidyl-transferase(EC 2.7.7.7) ;deoxyribonuclease (EC 3.1.4.5); polynucleotide 5'-hydroxyl-kinase (EC 2.7.1.78); bacterial alkaline phosphatase (Escherichia coli) or orthophosphoricmonoester phosphohydrolase (EC 3.1.3.1).
MATERIALS AND METHODS
Materials Deoxyribonucleoside mono, di and triphosphates were obtained from PL Biochemicals Inc., and the 'Hlabelled triphosphates from the Radiochcniical Centrc. Both sets were purified by Dowex 1 x 2 chromatography and were a generous gift from Dr S. Linn. Poly(ethy1eneimine)-cellulose thin-layer chromatography sheets (Macherey-Nagel Polygram CEL 300 PEL 20 x 20 cm) were from CamLab.
Nucleic Acids Activated calf-thymus DNA labelled with ["HIdTMP at 3'-termini and with [''Plphosphate groups at 5'-termini was prepared as follows. The 3H-labelled DNA (40 pg) [20] was incubated at 65 "C in 70 mM Tris-HC1, pH 8.0, with 14 pg bacterial alkaline phosphatase. Equal amounts of this enzyme were added after 10 min and 20min. After a total of 30 min, 0.02 ml 1.0 M Tris-HC1, pH 8.0, and 0.01 ml of
144
0.1 M sodium nitriloacetate (pH 8.0) were added, the mixture held at 65 "C for 50 min, cooled and 0.005 ml 1.0 M MgCl,, 0.001 ml 20 mM ATP, 0.2 ml [y-"PIATP (15.6 Ci/nmol, 500 pCi/ml), 0.005 ml 0.1 M 2-mercaptoethanol and 5.4 units of polynucleotide kinase added. Incubation was continued at 37 "C for 20 min and then at 65 "C for 2 min. A further 5.4units of the kinase were added after cooling, with incubation at 37 "C for 20 min and again at 65 "C for 15 min. Unreacted [y-32P]ATPand other low-molecular-weight components were removed by Sephadex G-25 chromatography and the dual-isotope-labelled DNA isolated by alcohol precipitation and centrifugation. It was stored at - 20 "C in 0.01 M Tris-HC1, pH 7.6, 0.02 M NaC1. Unlabelled and [3H]dTMP-labelled poly [d(A-T)] [d(A-T)] (sedimentation coefficient of about 17S) was prepared by the method of Schachman et al. [21].
Sucrose Gradient Sedimentation The details are given in the preceding paper [20]. Fractions were collected and assayed immediately for polymerase and nuclease activity, using activated calf-thymus DNA without and with ['HH]dTMPlabelled 3'-termini respectively. Molecular weights were estimated by the method of Martin and Ames [22] using ovalbumin (3.6 S) as an internal standard. Its sedimentation position in the gradient was determined by assaying fractions for protein [20].
Deoxyribonuclease Activity of a
(I.muydis
D N A Polymerase
phosphate was made by published procedures [23,24]. A standard polymerase assay mixture (0.15 ml) [20] was incubated at 37 "C and at recorded intervals of time a 0.005-ml or 0.01-ml sample was applied to the origin of a prewashed poly(ethy1eneimine)-cellulose thin-layer chromatography sheet, which had been divided into several lanes. EDTA, deoxyribonucleoside mono and triphosphate markers had previously been dried on to each origin. The sheets were eluted with 1.2 M LiCl (for [3H]dATP or [3H]dGTP) or 1.0 M LiCl (for ['HIdTTP or [3H]dCTP), which cleanly separated the DNA product, the deoxyribonucleosidemono, di- and triphosphates.The nucleotide markers were located by inspection of the dried chromatogram over a low-intensity ultraviolet light and each lane cut into l-cm strips, which were counted directly in a toluene-based scintillation fluid. To determine the specific activity of the label, the total number of counts/min in each lane was divided by the amount (pmol) of the labelled deoxynucleotide in the 0.005-ml or 0.01-ml sample of the reaction mixture at zero time. The amount of label incorporated into DNA and its subsequent conversion to the monophosphate could be determined from the counts/min on the appropriate region of the chromatogram. Other materials and procedurcs were described in the preceding paper [20].
RESULTS
Enzyme Assays
Evidence for an Association of Polymerase and Nuclease Activities
DNA polymerase activity was assayed as described previously [20]. The assay for the degradation of prelabelled DNA measures the amount of radioactivity made acidsoluble by the enzyme. A standard polymerase assay (0.15 ml), but without deoxynucleoside triphosphates, contained 2.5 pg activated calf-thymus DNA labelled at 3'-termini with t3H]dTMP (prepared as described [20] in a reaction mixture containing ['HIdTTP at 9.5 x lo3 counts min-' pmol-') or 5 nmol [3H]poly[d(A-T)] . [d(A-T)] (20 counts min-' pmol [3H]dTMP-'), and an aliquot of DNA polymerase. After incubation at 37 "Cfor 10 min, the tubes were cooled in ice and 0.02 ml calf-thymus DNA solution (2 mg/ml) followed by 0.2 ml of 10% trichloroacetic acid containing 1 tetrasodium pyrophosphate added. They were left in ice for 30 min, centrifuged at 7000 rev./min for 10 min and the radioactivity in a 0.25-ml sample of the supernatant fraction determined in a dioxan-based scintillation fluid. The simultaneous measurement of 3H-labelled deoxynucleoside triphosphate incorporation into DNA and its subsequent hydrolysis to release free mono-
The last stage of the purification procedure involved hydroxyapatite chromatography to give fraction V [20]. Both DNA polymerase and deoxyribonuclease activities coeluted from the column at about a 0.25 M KH,P04 concentration and the ratios of their activities across the peak were approximately constant (Fig. 1). Glycerol gradient sedimentation demonstrated that salt induced a conversion of the polymerase activity from an 8.4-S to 6.3-S form [20]. This conversion was also observed by sucrose gradient sedimentation, and in both the presence and absence of 0.12 M KC1 the nuclease activity cosedimented with that of the polymerase (Fig. 2). Thus the nuclease also underwent the same sdlt-induced change in sedimentation coefficient. The inactivation of fraction V at 50 "C was determined. Both activities possessed a similar temperature sensitivity (Fig. 3). Table 1 compares the reaction requirements for the polymerase and deoxyribonuclease activities. Both required a sulphydryl reagent and a divalent metal cation for the maximum rate of activity, but in
145
G . R. Banks and G . T. Yarrmton
100 00
.e 40 .Ee
10 0
1
5
Fraction number
Fig. I . D N A polytnerase arid tleosj'rihonuclease activity profiles during Iijxiroxyapatite chroniatogruphy. The chromatography conditions were described prcviously [20]. 0.01-ml samples of 3-ml fractions were assayed for polymerase and deoxyribonuclease activities in standard assay mixtures containing activated calf-thymus DNA, without and with 3'-["H]dTMP label respectively. Reactions were deoxyribonuclease at 37 "C for 30 min. (0)Polymerase, (0)
I
I
I
10 15 20 Time at 50°C (min)
I
1
25
30
Fig. 3. Heat inactivation oj the p l j r w r u s ~ ' arid rIc.o.~~rihoiiuclea~e uctivities. Fraction V (380 units/ml) of the enzyme was incubatcd at 50 "C and 0.01-ml samples assayed immediately for polymerasc and deoxyribonuclease (M activities ) as described in the legend to Fig. 1
(M)
Table 1. Reaction requirements ,for the polymerase and deoxyrihonuclease activiiies Assay mixtures with 6 units of fraction V of the enzyme were us described in the legend to Fig. 1, with incubation at 37 "C for 10 min. Additions and subtractions to the standard mixture are shown Assay mixture
Activity polymerase dcoxyribonuclease
x ~
Complete - KCI - Dithiothreitol - Dithiothreitol + 20 mM N-eth ylmaleimide - MgClz MgCI, + 5 mM EDTA ~
100
100
15
110
8
10
A DNA Polymerase from Ustilago maydis 2. Properties of the Associated Deoxyribonuclease Activity Geoffrey R. BANKS and Geoffrey T. YARRANTON National Institute for Medical Research, London (Received July 23/September 30, 1975)
The polymerase and deoxyribonuclease activities of the purified Ustilago maydis DNA polymerase coeluted from a hydroxyapatite column, cosedimented in sucrose gradients in both the absence and presence of salt, possessed similar thermolabilities and reaction requirements. These observations suggest that both activities are associated with the same enzyme and that the deoxyribonuclease activity is not a contaminant. The initial rate of degradation of native 3'-end-group-labelled DNA was similar to that of a heat-denatured substrate, but the final extent was greater for the former. The enzyme exhibits a high specificity for degradation of DNA in a 3'+5' direction. The degradation of a DNA template was inhibited by the presence of the deoxyribonucleoside triphosphates necessary for simultaneous DNA synthesis, but not that of the newly synthesised DNA. About 50 29 '%; and 13 of the purine, cytosine and thymine deoxyribonucleotide residues incorporated by the enzyme into DNA respectively, were subsequently excised when monitored by the resulting conversion of the triphosphate substrates to free monophosphate. The majority of the purine deoxyribonucleoside monophosphates appear after the synthetic phase of the reaction has ceased. In many respects, therefore, the deoxyribonuclease activity of the U . maydis DNA polymerase is similar to the bacteriophage T4-induced enzyme.
x,
Extensively purified DNA polymerases from prokaryotic organisms appear to possess at least one exonuclease activity, and there is good evidence in many cases that both activities reside on the same polypeptide chain [I - 1I]. Most eukaryotic enzymes, although having polymerase activity with characteristics similar to those of the prokaryotic ones, are devoid of detectable exonuclease activity [12- 171. Nuclease activity may, however, be associated with DNA polymerases purified from bakers' yeast and Tetrahymena pyriformis [18,19]. The DNA polymerase from Ustilago maydis, whose polymerase activity was the subject of the preceding paper [20], also possesses deoxyribonuclease activity. This paper describes its characteristics and provides evidence that it is not a contaminant of the preparation. ~
Ahbrevrarions. Abbreviations for synthetic polynucleotides follow CBN rules, see Eur. J . Biochem. 15, 203 - 208 (1970). Enzymes. D N A polymerase or deoxynucleosidetriphosphate : DNA deoxynucleotidyl-transferase(EC 2.7.7.7) ;deoxyribonuclease (EC 3.1.4.5); polynucleotide 5'-hydroxyl-kinase (EC 2.7.1.78); bacterial alkaline phosphatase (Escherichia coli) or orthophosphoricmonoester phosphohydrolase (EC 3.1.3.1).
MATERIALS AND METHODS
Materials Deoxyribonucleoside mono, di and triphosphates were obtained from PL Biochemicals Inc., and the 'Hlabelled triphosphates from the Radiochcniical Centrc. Both sets were purified by Dowex 1 x 2 chromatography and were a generous gift from Dr S. Linn. Poly(ethy1eneimine)-cellulose thin-layer chromatography sheets (Macherey-Nagel Polygram CEL 300 PEL 20 x 20 cm) were from CamLab.
Nucleic Acids Activated calf-thymus DNA labelled with ["HIdTMP at 3'-termini and with [''Plphosphate groups at 5'-termini was prepared as follows. The 3H-labelled DNA (40 pg) [20] was incubated at 65 "C in 70 mM Tris-HC1, pH 8.0, with 14 pg bacterial alkaline phosphatase. Equal amounts of this enzyme were added after 10 min and 20min. After a total of 30 min, 0.02 ml 1.0 M Tris-HC1, pH 8.0, and 0.01 ml of
144
0.1 M sodium nitriloacetate (pH 8.0) were added, the mixture held at 65 "C for 50 min, cooled and 0.005 ml 1.0 M MgCl,, 0.001 ml 20 mM ATP, 0.2 ml [y-"PIATP (15.6 Ci/nmol, 500 pCi/ml), 0.005 ml 0.1 M 2-mercaptoethanol and 5.4 units of polynucleotide kinase added. Incubation was continued at 37 "C for 20 min and then at 65 "C for 2 min. A further 5.4units of the kinase were added after cooling, with incubation at 37 "C for 20 min and again at 65 "C for 15 min. Unreacted [y-32P]ATPand other low-molecular-weight components were removed by Sephadex G-25 chromatography and the dual-isotope-labelled DNA isolated by alcohol precipitation and centrifugation. It was stored at - 20 "C in 0.01 M Tris-HC1, pH 7.6, 0.02 M NaC1. Unlabelled and [3H]dTMP-labelled poly [d(A-T)] [d(A-T)] (sedimentation coefficient of about 17S) was prepared by the method of Schachman et al. [21].
Sucrose Gradient Sedimentation The details are given in the preceding paper [20]. Fractions were collected and assayed immediately for polymerase and nuclease activity, using activated calf-thymus DNA without and with ['HH]dTMPlabelled 3'-termini respectively. Molecular weights were estimated by the method of Martin and Ames [22] using ovalbumin (3.6 S) as an internal standard. Its sedimentation position in the gradient was determined by assaying fractions for protein [20].
Deoxyribonuclease Activity of a
(I.muydis
D N A Polymerase
phosphate was made by published procedures [23,24]. A standard polymerase assay mixture (0.15 ml) [20] was incubated at 37 "C and at recorded intervals of time a 0.005-ml or 0.01-ml sample was applied to the origin of a prewashed poly(ethy1eneimine)-cellulose thin-layer chromatography sheet, which had been divided into several lanes. EDTA, deoxyribonucleoside mono and triphosphate markers had previously been dried on to each origin. The sheets were eluted with 1.2 M LiCl (for [3H]dATP or [3H]dGTP) or 1.0 M LiCl (for ['HIdTTP or [3H]dCTP), which cleanly separated the DNA product, the deoxyribonucleosidemono, di- and triphosphates.The nucleotide markers were located by inspection of the dried chromatogram over a low-intensity ultraviolet light and each lane cut into l-cm strips, which were counted directly in a toluene-based scintillation fluid. To determine the specific activity of the label, the total number of counts/min in each lane was divided by the amount (pmol) of the labelled deoxynucleotide in the 0.005-ml or 0.01-ml sample of the reaction mixture at zero time. The amount of label incorporated into DNA and its subsequent conversion to the monophosphate could be determined from the counts/min on the appropriate region of the chromatogram. Other materials and procedurcs were described in the preceding paper [20].
RESULTS
Enzyme Assays
Evidence for an Association of Polymerase and Nuclease Activities
DNA polymerase activity was assayed as described previously [20]. The assay for the degradation of prelabelled DNA measures the amount of radioactivity made acidsoluble by the enzyme. A standard polymerase assay (0.15 ml), but without deoxynucleoside triphosphates, contained 2.5 pg activated calf-thymus DNA labelled at 3'-termini with t3H]dTMP (prepared as described [20] in a reaction mixture containing ['HIdTTP at 9.5 x lo3 counts min-' pmol-') or 5 nmol [3H]poly[d(A-T)] . [d(A-T)] (20 counts min-' pmol [3H]dTMP-'), and an aliquot of DNA polymerase. After incubation at 37 "Cfor 10 min, the tubes were cooled in ice and 0.02 ml calf-thymus DNA solution (2 mg/ml) followed by 0.2 ml of 10% trichloroacetic acid containing 1 tetrasodium pyrophosphate added. They were left in ice for 30 min, centrifuged at 7000 rev./min for 10 min and the radioactivity in a 0.25-ml sample of the supernatant fraction determined in a dioxan-based scintillation fluid. The simultaneous measurement of 3H-labelled deoxynucleoside triphosphate incorporation into DNA and its subsequent hydrolysis to release free mono-
The last stage of the purification procedure involved hydroxyapatite chromatography to give fraction V [20]. Both DNA polymerase and deoxyribonuclease activities coeluted from the column at about a 0.25 M KH,P04 concentration and the ratios of their activities across the peak were approximately constant (Fig. 1). Glycerol gradient sedimentation demonstrated that salt induced a conversion of the polymerase activity from an 8.4-S to 6.3-S form [20]. This conversion was also observed by sucrose gradient sedimentation, and in both the presence and absence of 0.12 M KC1 the nuclease activity cosedimented with that of the polymerase (Fig. 2). Thus the nuclease also underwent the same sdlt-induced change in sedimentation coefficient. The inactivation of fraction V at 50 "C was determined. Both activities possessed a similar temperature sensitivity (Fig. 3). Table 1 compares the reaction requirements for the polymerase and deoxyribonuclease activities. Both required a sulphydryl reagent and a divalent metal cation for the maximum rate of activity, but in
145
G . R. Banks and G . T. Yarrmton
100 00
.e 40 .Ee
10 0
1
5
Fraction number
Fig. I . D N A polytnerase arid tleosj'rihonuclease activity profiles during Iijxiroxyapatite chroniatogruphy. The chromatography conditions were described prcviously [20]. 0.01-ml samples of 3-ml fractions were assayed for polymerase and deoxyribonuclease activities in standard assay mixtures containing activated calf-thymus DNA, without and with 3'-["H]dTMP label respectively. Reactions were deoxyribonuclease at 37 "C for 30 min. (0)Polymerase, (0)
I
I
I
10 15 20 Time at 50°C (min)
I
1
25
30
Fig. 3. Heat inactivation oj the p l j r w r u s ~ ' arid rIc.o.~~rihoiiuclea~e uctivities. Fraction V (380 units/ml) of the enzyme was incubatcd at 50 "C and 0.01-ml samples assayed immediately for polymerasc and deoxyribonuclease (M activities ) as described in the legend to Fig. 1
(M)
Table 1. Reaction requirements ,for the polymerase and deoxyrihonuclease activiiies Assay mixtures with 6 units of fraction V of the enzyme were us described in the legend to Fig. 1, with incubation at 37 "C for 10 min. Additions and subtractions to the standard mixture are shown Assay mixture
Activity polymerase dcoxyribonuclease
x ~
Complete - KCI - Dithiothreitol - Dithiothreitol + 20 mM N-eth ylmaleimide - MgClz MgCI, + 5 mM EDTA ~
100
100
15
110
8
10
