JOURNAL OF BACTERIOLOGY, Mar. 1975, P. 1214-1215 Copyright i 1975 American Society for Microbiology
Vol. 121, No. 3 Printed in U.S.A.
Role of Ribonucleic Acid Synthesis in Replication of Deoxyribonucleic Acid MARTIN L. PATO
National Jewish Hospital and Research Center, Denver, Colorado 80206* and University of Colorado School of Medicine, Denver, Colorado 80220
Received for publication 12 December 1974
An experiment previously interpreted to show a ribonucleic acid requirement for propagation of deoxyribonucleic replication is reexamined and the earlier interpretation is shown to be incorrect.
It is known that initiation of deoxyribonucleic acid (DNA) replication requires prior synthesis of ribonucleic acid (RNA) (2, 4, 5). The observation that Okazaki Pieces are synthesized with RNA primers (6) strongly suggests that RNA synthesis is required also for propagation of DNA replication. An early report of Doudney (1) is of interest in this context as it concluded that RNA synthesis in fact is required for propagation of DNA replication. The interpretation of these data is reexamined in this note. In the original experiments of Doudney (1), E. coli 15 TAU bar was starved for thymine and the required amino acids arg, pro, trp, and met. Readdition of thymine immediately after its removal allowed an increase of about 60% in the amount of DNA, reflecting completion of one round of replication in the absence of required amino acids; addition of thymine at subsequent times resulted in decreasing amounts of residual DNA replication. After 1 h of starvation for thymine and amino acids, no residual DNA synthesis was observed upon addition of thymine; however, the simultaneous addition of chloramphenicol and thymine at this time yielded the expected amount of residual synthesis. Doudney suggested that chloramphenicol reversed the normal stringent control of RNA synthesis, and that the regained ability to complete a round of replication was due to the synthesis of required RNA molecules. The observation of Doudney is reproduced in Figure la. When E. coli 15 TAU bar is starved for thymine and the amino acids arg, pro, trp, and met, no residual DNA replication occurs upon addition of thymine after 90 min of starvation; addition of chloramphenicol with thymine does allow approximately 40% residual replication. However (Fig. lb), if methionine is not removed when the cells are starved for thymine and the other required amino acids, residual DNA synthesis is no longer dependent upon the
presence of chloramphenicol. Furthermore, rifampicin, which inhibits all measurable RNA synthesis, does not prevent the DNA synthesis seen in the presence of chloramphenicol. The finding that residual DNA replication is prevented only when methionine is absent suggested that this phenomenon might involve the E. coli 15 restriction system (3) which recognizes and degrades unmethylated DNA. Therefore, the experiment was repeated with a mutant of E. coli 15 deficient in this restriction system. Figure 2 shows the residual replication observed upon readdition of thymine after 90 min of starvation in the wild-type and mutant strains, in the presence and absence of methionine. In the absence of chloramphenicol, the B
7
E
Il -60
0
60
120
l 180
l -60
0
60
120
180
FIG. 1. Residual DNA replication in E. coli 15 TAU bar. Cultures were grown in minimal medium plus glucose and requirements, and labeled for 5 to 10 generations with [3H]thymidine (2 Ag/ml; 10 ,uCi/ml) in the presence of 200 Mg of deoxyadenosine per ml and 200 uig of uridine per ml in order to fully label chromosomes. Thymidine and required amino acids were removed by filtration, and cultures were incubated at 37 C minus (A) and plus (B) 50 ,ug of methionine per ml. After 90 min [3H]thymidine (2 Mg/ml; 10 MCi/ml) was added back alone (0, *), or in the presence of 200 Mg of chloramphenicol per ml (A, A), or chloramphenicol plus 200 Mg of rifampicin per liter (0, U). Samples of 50X were removed into 5% trichloroacetic acid, filtered and washed with trichloroacetic acid and then with hot water, and counted in a toluene-based scintillation mixture.
1214
The details of the mechanism by which methionine starvation leads to a loss of potential for residual DNA replication in the cells with a functional restriction system remain unclear, as does the involvement of RNA synthesis in propagation of DNA replication.
3.0 1
B
A
25-
20~~~~~~~~~~~~ E
I05101 -60
0
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NOTES
VOL. 121, 1975
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FIG. 2. Residual DNA replication in a restriction deficient mutant. E. coli 15 (555-7), wild type (A) and restriction deficient (B), were treated as in Fig. 1. Closed symbols, minus 50 pg/ml methionine; open symbols, plus methionine; (0, *) plus [3H]thymidine at t 0; (A, A) plus thymidine and chloramphenicol.
This investigation was supported by Public Health Service grant AI11075 from the National Institute of Allergy and Infectious Diseases. I am indebted to Michael Gallagher for his expert technical assistance and to Mogens T. Hansen for many helpful discussions.
LITERATURE CITED
=
mutant organism shows residual DNA synthesis
in either the presence or absence of methionine, whereas the wild type shows residual synthesis only in the presence of methionine. These results show that the lack of residual DNA synthesis originally observed by Doudney (1) is a consequence of the effect of the E. coli 15 restriction system in cells starved for methionine. Therefore, this observation does not provide evidence for a requirement for RNA synthesis during propagation of DNA replication. The effect of chloramphenicol in allowing residual replication during methionine starvation may be due, at least in part, to the prevention of reutilization by protein synthesis of methionine liberated by protein turnover, and utilization of that methionine for the methylation of DNA.
1. Doudney, C. 0. 1966. Requirement for ribonucleic acid synthesis for deoxyribonucleic acid replication in bacteria. Nature (London) 211:39-41. 2. Lark, K. G. 1972. Evidence for the direct involvement of RNA in the initiation of DNA replication in Escherichia coli 15 T-. Methods Mol. Biol. 64:47-60. 3. Lark, C., and W. Arber. 1970. Host specificity of DNA produced by Escherichia coli. XIm. Breakdown of cellular DNA upon growth in ethionine of strains with rl5+, rPl + or rN3+ restriction phenotypes. J. Mol. Biol.
52:337-348. 4. Laurent, S. J. 1973. Initiation of deoxyribonucleic acid replication in a temperature-sensitive mutant of B. subtilis: evidence for a transcriptional step. J. Bacteriol.
116:141-145. 5. Messer, W. 1972. Initiation of deoxyribonucleic acid replication of Escherichia coli B/r: chronology of events and transcriptional control of initiation. J. Bacteriol. 112:7-12. 6. Sugino, K., S. Hirose, and R. Okazaki. 1972. RNA-linked DNA fragments in Escherichia coli. Proc. Nat. Acad. Sci. U.S.A. 69:1863-1867.
Vol. 121, No. 3 Printed in U.S.A.
Role of Ribonucleic Acid Synthesis in Replication of Deoxyribonucleic Acid MARTIN L. PATO
National Jewish Hospital and Research Center, Denver, Colorado 80206* and University of Colorado School of Medicine, Denver, Colorado 80220
Received for publication 12 December 1974
An experiment previously interpreted to show a ribonucleic acid requirement for propagation of deoxyribonucleic replication is reexamined and the earlier interpretation is shown to be incorrect.
It is known that initiation of deoxyribonucleic acid (DNA) replication requires prior synthesis of ribonucleic acid (RNA) (2, 4, 5). The observation that Okazaki Pieces are synthesized with RNA primers (6) strongly suggests that RNA synthesis is required also for propagation of DNA replication. An early report of Doudney (1) is of interest in this context as it concluded that RNA synthesis in fact is required for propagation of DNA replication. The interpretation of these data is reexamined in this note. In the original experiments of Doudney (1), E. coli 15 TAU bar was starved for thymine and the required amino acids arg, pro, trp, and met. Readdition of thymine immediately after its removal allowed an increase of about 60% in the amount of DNA, reflecting completion of one round of replication in the absence of required amino acids; addition of thymine at subsequent times resulted in decreasing amounts of residual DNA replication. After 1 h of starvation for thymine and amino acids, no residual DNA synthesis was observed upon addition of thymine; however, the simultaneous addition of chloramphenicol and thymine at this time yielded the expected amount of residual synthesis. Doudney suggested that chloramphenicol reversed the normal stringent control of RNA synthesis, and that the regained ability to complete a round of replication was due to the synthesis of required RNA molecules. The observation of Doudney is reproduced in Figure la. When E. coli 15 TAU bar is starved for thymine and the amino acids arg, pro, trp, and met, no residual DNA replication occurs upon addition of thymine after 90 min of starvation; addition of chloramphenicol with thymine does allow approximately 40% residual replication. However (Fig. lb), if methionine is not removed when the cells are starved for thymine and the other required amino acids, residual DNA synthesis is no longer dependent upon the
presence of chloramphenicol. Furthermore, rifampicin, which inhibits all measurable RNA synthesis, does not prevent the DNA synthesis seen in the presence of chloramphenicol. The finding that residual DNA replication is prevented only when methionine is absent suggested that this phenomenon might involve the E. coli 15 restriction system (3) which recognizes and degrades unmethylated DNA. Therefore, the experiment was repeated with a mutant of E. coli 15 deficient in this restriction system. Figure 2 shows the residual replication observed upon readdition of thymine after 90 min of starvation in the wild-type and mutant strains, in the presence and absence of methionine. In the absence of chloramphenicol, the B
7
E
Il -60
0
60
120
l 180
l -60
0
60
120
180
FIG. 1. Residual DNA replication in E. coli 15 TAU bar. Cultures were grown in minimal medium plus glucose and requirements, and labeled for 5 to 10 generations with [3H]thymidine (2 Ag/ml; 10 ,uCi/ml) in the presence of 200 Mg of deoxyadenosine per ml and 200 uig of uridine per ml in order to fully label chromosomes. Thymidine and required amino acids were removed by filtration, and cultures were incubated at 37 C minus (A) and plus (B) 50 ,ug of methionine per ml. After 90 min [3H]thymidine (2 Mg/ml; 10 MCi/ml) was added back alone (0, *), or in the presence of 200 Mg of chloramphenicol per ml (A, A), or chloramphenicol plus 200 Mg of rifampicin per liter (0, U). Samples of 50X were removed into 5% trichloroacetic acid, filtered and washed with trichloroacetic acid and then with hot water, and counted in a toluene-based scintillation mixture.
1214
The details of the mechanism by which methionine starvation leads to a loss of potential for residual DNA replication in the cells with a functional restriction system remain unclear, as does the involvement of RNA synthesis in propagation of DNA replication.
3.0 1
B
A
25-
20~~~~~~~~~~~~ E
I05101 -60
0
1215
NOTES
VOL. 121, 1975
60
I
120
I~ 180
I
-60
I
0
I
60
i
120
180
minutes
FIG. 2. Residual DNA replication in a restriction deficient mutant. E. coli 15 (555-7), wild type (A) and restriction deficient (B), were treated as in Fig. 1. Closed symbols, minus 50 pg/ml methionine; open symbols, plus methionine; (0, *) plus [3H]thymidine at t 0; (A, A) plus thymidine and chloramphenicol.
This investigation was supported by Public Health Service grant AI11075 from the National Institute of Allergy and Infectious Diseases. I am indebted to Michael Gallagher for his expert technical assistance and to Mogens T. Hansen for many helpful discussions.
LITERATURE CITED
=
mutant organism shows residual DNA synthesis
in either the presence or absence of methionine, whereas the wild type shows residual synthesis only in the presence of methionine. These results show that the lack of residual DNA synthesis originally observed by Doudney (1) is a consequence of the effect of the E. coli 15 restriction system in cells starved for methionine. Therefore, this observation does not provide evidence for a requirement for RNA synthesis during propagation of DNA replication. The effect of chloramphenicol in allowing residual replication during methionine starvation may be due, at least in part, to the prevention of reutilization by protein synthesis of methionine liberated by protein turnover, and utilization of that methionine for the methylation of DNA.
1. Doudney, C. 0. 1966. Requirement for ribonucleic acid synthesis for deoxyribonucleic acid replication in bacteria. Nature (London) 211:39-41. 2. Lark, K. G. 1972. Evidence for the direct involvement of RNA in the initiation of DNA replication in Escherichia coli 15 T-. Methods Mol. Biol. 64:47-60. 3. Lark, C., and W. Arber. 1970. Host specificity of DNA produced by Escherichia coli. XIm. Breakdown of cellular DNA upon growth in ethionine of strains with rl5+, rPl + or rN3+ restriction phenotypes. J. Mol. Biol.
52:337-348. 4. Laurent, S. J. 1973. Initiation of deoxyribonucleic acid replication in a temperature-sensitive mutant of B. subtilis: evidence for a transcriptional step. J. Bacteriol.
116:141-145. 5. Messer, W. 1972. Initiation of deoxyribonucleic acid replication of Escherichia coli B/r: chronology of events and transcriptional control of initiation. J. Bacteriol. 112:7-12. 6. Sugino, K., S. Hirose, and R. Okazaki. 1972. RNA-linked DNA fragments in Escherichia coli. Proc. Nat. Acad. Sci. U.S.A. 69:1863-1867.
