/ . Biochem., 77, 863-866 (1975)
Incorporation of Nucleotides into DNA by Mammalian DNA Polymerase in the Presence of a Single Deoxynucleoside Triphosphate1 Takashi TSURUO and Tyunosin UKITA1 Faculty of Pharmaceutical Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113 Received for publication, September 2, 1974
Rat ascites hepatoma cell DNA polymerases [EC 2.7.7.7], especially low molecular weight polymerase, could incorporate a significant amount of single nucleotide into acid-insoluble products in the absence of the other three deoxynucleoside triphosphates when activated DNA was used as a template. This relaxed requirement for deoxynucleotides was not observed when poIy[d(A—T)-d(T—A)] was used as a template. Nearest-neighbour base analyses of the products formed in the presence of a single deoxynucleoside triphosphate revealed that the reaction is not of a terminal transferase-type but a very limited repair synthesis in which one or a few triphosphates are incorporated at numerous 3'-hydroxyl ends.
Unlike Escherichia coli DNA polymerase [EC 2. 7. 7. 7], mammalian DNA polymerases incorporate significant amounts of deoxynucleotides into acid-insoluble products in the absence of one to three other deoxynucleoside triphosphates (7—5). This relaxed requirement for deoxynucleoside triphosphates has been clearly observed in the reaction catalyzed by the low molecular weight DNA polymerase of mammalian cells ( 1 , 2, 5). Three explanations may account for these observations, (a) The enzyme preparation contains a significant amount of terminal transferase activity, as has been reported in calf thymus (6). (b) The enzyme loses the capacity to carry out the faithful copying of the template base sequences.
This interpretation is possible, because it has been demonstrated that a mutant phage DNA polymerase and a leukemia cell DNA polymerase activity show increased error-frequency during in vitro DNA synthesis (7, 8). (c) The enzyme performs a very limited repair synthesis in which one or a few triphosphates are incorporated at numerous 3'-hydroxyl ends of activated DNA. In this communication, we have investigated the nature of the DNA product synthesized by the DNA polymerase of rat ascites hepatoma cells in the presence of a single deoxyribonucleoside triphosphate, and the reaction was shown to represent a very limited repair synthesis.
1
This work was supported by a grant from the Ministry of Education of Japan. • Deceased on April 25, 1972. Vol. 77, No. 4, 1975
MATERIALS AND METHODS DNA polymerases used in this experiment were £63
T. TSURUO and T. UKITA
864
DNA polymerases C and P-2 which were prepared as described previously (9). Polymerase C belongs to the group of DNA polymerases having higher molecular weight, while polymerase P-2 is a low molecular weight polymerase (Tsuruo, T. et al. unpublished results). [a-82P]dATP (4.2 Ci per mmole) was obtained from International Chemical and Nuclear Corp. [a-"P]dTTP (2.8 Ci per mmole) was prepared enzymatically according to the method of Sugino and Okazaki {10). Nearest-neighbour base analysis was carried out according to the method of Josse and Swartz ( / / ) . The assay of polymerase, and other materials and methods have been described previously (9). RESULTS AND DISCUSSION Fidelity of Replication—For maximal activity, ascites cell DNA polymerases required the presence of all four deoxyribonucleoside triphosphates when activated DNA was used as a template (Table I). In the presence of only a single deoxyribonucleoside triphosphate, the rate of incorporation of the nucleotide by polymerase C was reduced by 92%. In the presence of two and three deoxynucleoside triphosphates, 12 and 20% of maximum incorporation was observed, respectively. With the P-2 enzyme, incorporation occurred at pH 7.3
in the absence of one deoxynucleoside triphosphate up to 88% of the value with four triphosphates, and even in the absence of three triphosphates, synthesis occurred up to 60% of the control level. Similar results were obtained when the single deoxynucleoside triphosphate used was either [8H]dATP or ['H]dTTP. As calf thymus terminal transferase incorporates a single deoxyribonucleoside triphosphate at a maximal rate in the absence of the other three triphosphates and the reaction velocity with terminal transferase is faster with dATP than with dTTP (12), this observation suggests that these polymerases do not contain terminal transferase-type activity of calf thymus. 8 The results described above suggest that the DNA polymerase, especially P-2, might not carry out faithful copying of the template base sequences. The fidelity of replication by polymerase P-2 was examined with activated poly[d(A-T)-d(T-A)] as a template. As shown in Table II, incorporation of both dATP and dTTP by P-2 enzyme was observed in the presence of both triphosphates, but little in3
The absence of terminal transferase activity in the enzyme preparations was confirmed because neither preparation could incorporate a single deoxynucleoside triphosphate (dATP or dTTP) into (pT)B.
TABLE I. Requirement for deoxynucleoside triphosphates of the DNA polymerase reaction. The standard assay mixture (9) was used except for changes in deoxyribonucleoside triphosphates as indicated. With polymerase P-2, assay was also carried out at optimal pH (40 mM glycine-KOH, pH 9.5). The specific activity of both [3H]dATP and ['H]dTTP was 75 mCi per mmole. Incorporation of labeled nucleotide (pmoles) Deoxyribonucleoside triphosphate added [3H]dATP, dGTP, dCTP, dTTP [3H]dATP, dGTP, dCTP [>H]dATP, dGTP [•H]dATP 3
[ H]dTTP, dGTP, dCTP, dATP [3H]dTTP, dGTP, dCTP [3H]dTTP, dGTP [>H]dTTP
Polymerase C
P-2
pH 7.3
pH 7.3
1,460
272
756
338
231
425
170
208
352
104
153
272
1,320
. 288
748
266
223
442
169
210
362
116
191
312
pH 9.5
/ . Biochem.
FIDELITY OF REPLICATION BY MAMMALIAN DNA POLYMERASE
865
corporation occurred when either of these tri- were not incorporated into poly[d(A—T)phosphates was omitted from the reaction mix- d(T-A)]. These results strongly suggest that ture. dGTP and dCTP were not incorporated the P-2 enzyme faithfully replicates poly[d(A— when they were added alone. Even in the T)-d(T-A)]. presence of dATP and dTTP, dGTP and dCTP Nature of the Product Synthesized in the Presence of a Single Triphosphate—The nature of the DNA product synthesized by polymerase TABLE II. Incorporation of deoxyribonucleotides P-2 in the presence of a single triphosphate into poly[d(A-T)-d(T-A)] by P-2 polymerase. Assay was performed by the standard method (9) was examined. Reactions were carried out except that 40 mM glycine-KOH, pH 9.5, and 600 ftM with P-2 enzyme in the presence of [a- n P]of activated poly[d(A—T)-d(T—A)] were used, and dATP or [a-"P]dTTP using activated polydeoxyribonucleoside triphosphates were added as [d(A-T)-d(T-A)] and activated calf thymus indicated in the table. The specific activity of DNA as templates, and subjected to nearestlabeled nucleoside triphosphates was 100 mCi per neighbour base sequence analysis (Table III). mmole. The values were corrected for background In the reaction where activated poly[d(A—T)and the numerals in parentheses indicate relative d(T—A)] was used as a template in the presactivities (%). ence of both dATP and dTTP, the " P was Deoxyribonucleoside Incorporation of labeled transferred almost completely from 5'-deoxynucleotide (pmoles) triphosphate added adenylate to 3'-deoxythymidylate. This result also strongly indicates that P-2 enzyme faith[3H]dTTP, dATP 530 (100) fully copies the nucleotide sequences of the 3 [ H]dATP, dTTP 536 (101) template. [3H]dTTP 10.1 (1.9) In the presence of a single "P-labsled [5H]dATP 14.7 (2.8) deoxynucleoside triphosphate, the extent of [3H]dGTP, dATP, dTTP
Incorporation of Nucleotides into DNA by Mammalian DNA Polymerase in the Presence of a Single Deoxynucleoside Triphosphate1 Takashi TSURUO and Tyunosin UKITA1 Faculty of Pharmaceutical Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113 Received for publication, September 2, 1974
Rat ascites hepatoma cell DNA polymerases [EC 2.7.7.7], especially low molecular weight polymerase, could incorporate a significant amount of single nucleotide into acid-insoluble products in the absence of the other three deoxynucleoside triphosphates when activated DNA was used as a template. This relaxed requirement for deoxynucleotides was not observed when poIy[d(A—T)-d(T—A)] was used as a template. Nearest-neighbour base analyses of the products formed in the presence of a single deoxynucleoside triphosphate revealed that the reaction is not of a terminal transferase-type but a very limited repair synthesis in which one or a few triphosphates are incorporated at numerous 3'-hydroxyl ends.
Unlike Escherichia coli DNA polymerase [EC 2. 7. 7. 7], mammalian DNA polymerases incorporate significant amounts of deoxynucleotides into acid-insoluble products in the absence of one to three other deoxynucleoside triphosphates (7—5). This relaxed requirement for deoxynucleoside triphosphates has been clearly observed in the reaction catalyzed by the low molecular weight DNA polymerase of mammalian cells ( 1 , 2, 5). Three explanations may account for these observations, (a) The enzyme preparation contains a significant amount of terminal transferase activity, as has been reported in calf thymus (6). (b) The enzyme loses the capacity to carry out the faithful copying of the template base sequences.
This interpretation is possible, because it has been demonstrated that a mutant phage DNA polymerase and a leukemia cell DNA polymerase activity show increased error-frequency during in vitro DNA synthesis (7, 8). (c) The enzyme performs a very limited repair synthesis in which one or a few triphosphates are incorporated at numerous 3'-hydroxyl ends of activated DNA. In this communication, we have investigated the nature of the DNA product synthesized by the DNA polymerase of rat ascites hepatoma cells in the presence of a single deoxyribonucleoside triphosphate, and the reaction was shown to represent a very limited repair synthesis.
1
This work was supported by a grant from the Ministry of Education of Japan. • Deceased on April 25, 1972. Vol. 77, No. 4, 1975
MATERIALS AND METHODS DNA polymerases used in this experiment were £63
T. TSURUO and T. UKITA
864
DNA polymerases C and P-2 which were prepared as described previously (9). Polymerase C belongs to the group of DNA polymerases having higher molecular weight, while polymerase P-2 is a low molecular weight polymerase (Tsuruo, T. et al. unpublished results). [a-82P]dATP (4.2 Ci per mmole) was obtained from International Chemical and Nuclear Corp. [a-"P]dTTP (2.8 Ci per mmole) was prepared enzymatically according to the method of Sugino and Okazaki {10). Nearest-neighbour base analysis was carried out according to the method of Josse and Swartz ( / / ) . The assay of polymerase, and other materials and methods have been described previously (9). RESULTS AND DISCUSSION Fidelity of Replication—For maximal activity, ascites cell DNA polymerases required the presence of all four deoxyribonucleoside triphosphates when activated DNA was used as a template (Table I). In the presence of only a single deoxyribonucleoside triphosphate, the rate of incorporation of the nucleotide by polymerase C was reduced by 92%. In the presence of two and three deoxynucleoside triphosphates, 12 and 20% of maximum incorporation was observed, respectively. With the P-2 enzyme, incorporation occurred at pH 7.3
in the absence of one deoxynucleoside triphosphate up to 88% of the value with four triphosphates, and even in the absence of three triphosphates, synthesis occurred up to 60% of the control level. Similar results were obtained when the single deoxynucleoside triphosphate used was either [8H]dATP or ['H]dTTP. As calf thymus terminal transferase incorporates a single deoxyribonucleoside triphosphate at a maximal rate in the absence of the other three triphosphates and the reaction velocity with terminal transferase is faster with dATP than with dTTP (12), this observation suggests that these polymerases do not contain terminal transferase-type activity of calf thymus. 8 The results described above suggest that the DNA polymerase, especially P-2, might not carry out faithful copying of the template base sequences. The fidelity of replication by polymerase P-2 was examined with activated poly[d(A-T)-d(T-A)] as a template. As shown in Table II, incorporation of both dATP and dTTP by P-2 enzyme was observed in the presence of both triphosphates, but little in3
The absence of terminal transferase activity in the enzyme preparations was confirmed because neither preparation could incorporate a single deoxynucleoside triphosphate (dATP or dTTP) into (pT)B.
TABLE I. Requirement for deoxynucleoside triphosphates of the DNA polymerase reaction. The standard assay mixture (9) was used except for changes in deoxyribonucleoside triphosphates as indicated. With polymerase P-2, assay was also carried out at optimal pH (40 mM glycine-KOH, pH 9.5). The specific activity of both [3H]dATP and ['H]dTTP was 75 mCi per mmole. Incorporation of labeled nucleotide (pmoles) Deoxyribonucleoside triphosphate added [3H]dATP, dGTP, dCTP, dTTP [3H]dATP, dGTP, dCTP [>H]dATP, dGTP [•H]dATP 3
[ H]dTTP, dGTP, dCTP, dATP [3H]dTTP, dGTP, dCTP [3H]dTTP, dGTP [>H]dTTP
Polymerase C
P-2
pH 7.3
pH 7.3
1,460
272
756
338
231
425
170
208
352
104
153
272
1,320
. 288
748
266
223
442
169
210
362
116
191
312
pH 9.5
/ . Biochem.
FIDELITY OF REPLICATION BY MAMMALIAN DNA POLYMERASE
865
corporation occurred when either of these tri- were not incorporated into poly[d(A—T)phosphates was omitted from the reaction mix- d(T-A)]. These results strongly suggest that ture. dGTP and dCTP were not incorporated the P-2 enzyme faithfully replicates poly[d(A— when they were added alone. Even in the T)-d(T-A)]. presence of dATP and dTTP, dGTP and dCTP Nature of the Product Synthesized in the Presence of a Single Triphosphate—The nature of the DNA product synthesized by polymerase TABLE II. Incorporation of deoxyribonucleotides P-2 in the presence of a single triphosphate into poly[d(A-T)-d(T-A)] by P-2 polymerase. Assay was performed by the standard method (9) was examined. Reactions were carried out except that 40 mM glycine-KOH, pH 9.5, and 600 ftM with P-2 enzyme in the presence of [a- n P]of activated poly[d(A—T)-d(T—A)] were used, and dATP or [a-"P]dTTP using activated polydeoxyribonucleoside triphosphates were added as [d(A-T)-d(T-A)] and activated calf thymus indicated in the table. The specific activity of DNA as templates, and subjected to nearestlabeled nucleoside triphosphates was 100 mCi per neighbour base sequence analysis (Table III). mmole. The values were corrected for background In the reaction where activated poly[d(A—T)and the numerals in parentheses indicate relative d(T—A)] was used as a template in the presactivities (%). ence of both dATP and dTTP, the " P was Deoxyribonucleoside Incorporation of labeled transferred almost completely from 5'-deoxynucleotide (pmoles) triphosphate added adenylate to 3'-deoxythymidylate. This result also strongly indicates that P-2 enzyme faith[3H]dTTP, dATP 530 (100) fully copies the nucleotide sequences of the 3 [ H]dATP, dTTP 536 (101) template. [3H]dTTP 10.1 (1.9) In the presence of a single "P-labsled [5H]dATP 14.7 (2.8) deoxynucleoside triphosphate, the extent of [3H]dGTP, dATP, dTTP
