Summary
Mutagenesis by 5-bromodeoxyuridine (BrdUrd) can result from base-pairing errors either during replication of a BrdUrd-containing template or at the nucleotide incorporation step. Replication errors give rise predominantly to AT-to-GC transitions, while incorporation errors, in which 5-bromo-dUTP competes with dCTP at a template guanine site, should give rise to GC-to-AT transitions. The latter pathway should be sensitive to deoxyribonucleoside triphosphate (dNTP) pool fluctuations. Since dNTP pools are regulated through allosteric control of ribonucleotide reductase, the control of this enzyme should be a determinant of BrdUrd mutagenesis — if mutagenesis results largely from incorporation errors. Since T4 phage-encoded ribonucleotide reductase is insensitive to feedback inhibition, we established conditions under which phage DNA replication is dependent upon ribonucleotide reductase of the host, Escherichia coli. We examined BrdUrd mutagenesis of rII mutants known to revert to wild type either by AT-to-GC or GC-to-AT transition pathways. While both reversion pathways were stimulated under all conditions analyzed, the AT-to-GC pathway was stimulated more when the E. coli reductase was functioning, while the GC-to-AT pathway was more specifically enhanced when the T4 reductase was active. These results confirm that ribonucleotide reductase is a determinant of BrdUrd mutagenesis, but our observations, plus experiments showing that BrdUrd has relatively small effects upon dNTP pool sizes, indicate that the relationship between deoxyribonucleotide metabolism and BrdUrd mutagenesis is more complex than anticipated.
Similar content being viewed by others
References
Ashman CR, Davidson RL (1981) Bromodeoxyuridine mutagenesis in mammalian cells is related to deoxyribonucleotide pool imbalance. Mol Cell Biol 1:254–260
Berglund Ö (1972) Ribonucleoside diphosphate reductase induced by bacteriophage T4. J Biol Chem 247:7270–7281
Bick MD, Davidson R (1974) Total substitution of bromodeoxyuridine for thymidine in the DNA of a bromodeoxyuridine-dependent cell line. Proc Natl Acad Sci USA 71:2082–2086
Davidson RL, Broeker P, Ashman CR (1988) DNA base sequence changes and sequence specificity of bromodeoxyuridine-induced mutations in mammalian cells. Proc Natl Acad Sci USA 85:4406–4410
Drake JW (1970) The Molecular Basis of Mutation. Holden-Day, New York
Hopkins RL, Goodman MF (1980) Deoxyribonucleotide pools, base pairing, and sequence configuration affecting bromodeoxyuridine- and 2-aminopurine-induced mutagenesis. Proc Natl Acad Sci USA 77:1801–1805
Howard BD, Tessman I (1964) Identification of the altered bases in mutated single-stranded DNA. J Mol Biol 9:364–371
Kaufman ER, Davidson RL (1978) Bromodeoxyuridine mutagenesis in mammalian cells: Mutagenesis is independent of the amount of bromouracil in DNA. Proc Natl Acad Sci USA 75:4982–4986
Lasken RS, Goodman MF (1984) The biochemical basis of 5-bromouracil-induced mutagenesis. J Biol Chem 259:11491–11495
Mathews CK (1972) Biochemistry of DNA-defective amber mutants of bacteriophage T4. III. Nucleotide Pools. J Biol Chem 247:7430–7438
Mathews CK (1985) Enzymatic channeling of DNA precursors. In: DeSerres FJ (ed) Genetic Consequences of Nucleotide Imbalance. Plenum Press, New York, pp 47–56
Mathews CK, Allen JR (1983) DNA precursor biosynthesis. In: Mathews CK, Kutter EM, Mosig G, Berget P (eds) Bacteriophage T4. American Society for Microbiology, Washington DC, pp 59–70
Mathews CK, Sinha NK (1982) Are DNA precursors concentrated at replication sites?. Proc Natl Acad Sci USA 79:302–306
Moen LK, Howell ML, Lasser GW, Mathews CK (1988) Deoxyribonucleoside triphosphate synthetase of bacteriophage T4 J Mol Recognition 1: 48–57
Platz A, Sjöberg B-M (1980) Construction and characterization of hybrid plasmids containing the Escherichia coli nrd region. J Bacteriol 143:561–568
Platz A, Karlsson M, Hahne S, Eriksson S, Sjöberg B-M (1985) Alterations in intracellular deoxyribonucleotide levels of mutationally altered ribonucleotide reductases in Escherichia coli. J Bacteriol 164:1194–1199
Sargent RG (1987) Deoxyribonucleotides as determinants of DNA replication fidelity in bacteriophage T4. Thesis, Oregon State University, Corvaliis
Sargent RG, Mathews CK (1987) Imbalanced dNTP pools and spontaneous mutation rates determined during dCMP deaminase-defective bacteriophage T4 infections. J Biol Chem 262:5546–5553
Sinha NK, Goodman MF (1983) Fidelity of DNA replication. In: Mathews CK, Kutter EM, Mosig G, Berget P (eds) Bacteriophage T4. American Society for Microbiology, Washington DC, pp 131–137
Skopek TR, Hutchinson F (1982) DNA base sequence changes induced by bromouracil mutagenesis of lambda phage. J Mol Biol 159:19–33
Slabaugh MB, Johnson TL, Mathews CK (1984) Vaccinia virus induces ribonucleotide reductase in primate cells. J Virol 52:507–514
Terzaghi B, Streisinger G, Stahl FW (1962) The mechanism of 5-bromouracil mutagenesis in the bacteriophage T4. Proc Natl Acad Sci USA 48:1519–1524
Thelander L, Reichard P (1979) Reduction of ribonucleotides. Annu Rev Biochem 48: 133–158
Wilson GG, Tanyashin VI, Murray NE (1977) Molecular cloning of fragments of bacteriophage T4. Mol Gen Genet 156:203–214
Author information
Authors and Affiliations
Additional information
Communicated by B.J. Kilbey
Rights and permissions
About this article
Cite this article
Geoffrey Sargent, R., Ji, J., Mun, B. et al. Ribonucleotide reductase: A determinant of 5-bromodeoxyuridine mutagenesis in phage T4. Mol Gen Genet 217, 13–19 (1989). https://doi.org/10.1007/BF00330936
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00330936