Summary
The survival of M13 DNA was studied after partial replacement of thymine by uracil in the bacteriophage. Uracils carry the same genetic information as the thymines. Nevertheless in Escherichia coli wild-type cells, uracils in DNA are replaced by thymines by excision repair initiated by uracil-DNA glycosylase (UDG). Thus inactivation of uracil-containing phage DNA is solely due to repair initiated by UDG. Incorporation of uracils was achieved in one or in both strands, either randomly or site-specifically using differently uracylated oligonucleotides. The results show that up to 580 uracils can be repaired without a significant decrease in survival if the uracils are localized in the (−) strand only. Incorporation of 246 uracils into the (+) strand leads to ∼ 30% or ∼ 10% survival when expressed in Escherichia coli strains CMK and JM103, respectively. However, when uracils are distributed over both strands a sharp decrease in survival occurs. This shows that the repair of two uracils localized in close proximity on opposite strands of the DNA by the excision repair mechanism is difficult, whereas uracils occurring in one strand are repaired more efficiently, irrespective of their number.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Bambara RA, Uyemura D, Choi T (1978) On the processive mechanism of Escherichia coli DNA polymerase I. Quantitative assessment of processivity. J Biol Chem 253:413–423
Bayne ML, Dumas LB (1979) Initiation of DNA synthesis on the isolated strands of bacteriophage f1 replication-form DNA. J Virol 29:1014–1022
Bien M, Steffen H, Schulte-Frohlinde D (1988) Repair of plasmid pBR322 damaged by γ-irradiation or by restriction endonucleases using different recombination-proficient E. coli strains. Mutat Res 194:193–205
Bonura T, Smith KC (1975a) Enzymatic production of deoxyribonucleic acid double-strand breaks after ultraviolet irradiation of E. coli K-12. J Bacteriol 121:511–517
Bonura T, Smith KC (1975b) Quantitative evidence for enzymatically-induced DNA double-strand breaks as lethal lesions in UV irradiated pol + and polA1 strains of E. coli K-12. Photochem Photobiol 22:243–248
Bresler SE (1975) Theory of misrepair mutagenesis. Mutat Res 29:467–472
Dianov GL, Timchenko TV, Sinitsina OI, Kuzminov AV, Medvedev OA, Salganik RI (1991) Repair of uracil residues closely spaced on the opposite strands of plasmid DNA results in double-strand break formation. Mol Gen Genet 225:448–452
Dower WJ, Miller JF, Ragsdale CW (1988) High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res 16:6127–6145
Gates FT, Linn S (1977) Endonuclease V of Escherichia coli. J Biol Chem 252:1647–1653
Grollman AP (1990) Site specific mutagenesis. In: Mendelsohn ML, Albertini RJ (eds) Mutation and the Environment, part A: Basic Mechanisms. Wiley-Liss, Inc, pp 61–70
Hartke A, Schulte-Frohlinde D (1991) Survial of M13mp18 gapped duplex DNA as a function of gap length. Mutat Res 255:39–48
Hutchinson F, Stein J (1981) Mutagenesis of lambda phage: Weigle mutagenesis is induced by coincident lesions in the double helical DNA of the host cell genome. Mol Gen Genet 181:458–463
Koffel-Schwartz N, Maenhaut-Michel G, Fuchs RPP (1987) Specific strand loss in N-2-acetylaminofluorene-modified DNA. J Mol Biol 193:651–659
Kow YW (1989) Mechanism of action of Escherichia coli exonuclease III. Biochemistry 28:3280–3287
Kow YW, Faundez G, Melamede RJ, Wallace SS (1991) Processing of model single-strand breaks in ΦX174 RF transfecting DNA by Escherichia coli. Radiat Res 126:357–366
Kunkel TA (1985) Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci USA 82:488–492
Kunkel TA, Roberts JD, Zakour RA (1987) Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymmol 154:367–382
Lindahl T (1974) An N-glycosidase from Escherichia coli that releases free uracil from DNA containing deaminated cytosine residues. Proc Natl Acad Sci USA 71:3649–3653
Messing J (1983) New M13 vectors for cloning. Methods Enzymol 101:20–78
Nakamaye KL, Eckstein F (1986) Inhibition of restriction endonuclease Ncil cleavage by phosphorothioate groups and its application to oligonucleotide-directed mutagenesis. Nucleic Acids Res 14:9679–9698
Rosamond J, Telander KM, Linn S (1979) Modulation of the action of the recBC enzyme of Escherichia coli K12 by Ca2+. J Biol Chem 254:8646–8652
Sagher D, Strauss B (1983) Insertion of nucleotides opposite apurinic/apyrimidinic sites in deoxyribonucleic acid during in vitro synthesis: uniqueness of adenine nucleotides. Biochemistry 22:4518–4526
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning, a laboratory manual. Cold Spring Harbour Laboratory Press, Cold Spring Harbor, New York
Schulte-Frohlinde D, Bothe E, Klimczak U, Ludwig DC, Mark F, Rettberg P, Schünemann S, Ventur Y, Worm K-H (1992) Radiation-induced damage and biological inactivation of plasmid DNA in water-alcohol mixtures: What is the lethal damage? In: Cadwick KH, Moschini G, Varma MN (eds) Biophysical Modelling of Radiation Effects. Adam Hilger, Bristol, pp 253–260
Schünemann S, Schulte-Frohlinde D (1990) Effect of deoxyuracilincorporation on the activity of M13-DNA. Int J Radiat Biol 57:1262
Sedgwick SG (1976) Misrepair of overlapping daughter strand gaps as a possible mechanism for UV-induced mutagenesis in uvr strains of E. coli. Mutat Res 41:185–200
Shenoy S, Ehrlich KC, Ehrlich M (1987) Repair of thymine guanine and uracil-guanine mismatched base-pairs in bacteriophage M13mp18 DNA heteroduplices. J Mol Biol 197:617–626
Shlomai J, Kornberg A (1978) Deoxyuridine triphosphatase of Escherichia coli. J Biol Chem 253:3305–3312
Smith KC, Sharma RC (1987) A model for the recA-dependent rapair of excision gaps in UV-irradiated Escherichia coli. Mutat Res 183:1–9
Taylor AF, Weiss B (1982) Role of exonuclease III in the base excision repair of uracil-containing DNA. J Bacteriol 151:351–357
Taylor JW, Ott J, Eckstein F (1985) The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA. Nucleic Acids Res 13:8765–8785
Tye BK, Lehman IR (1977) Excision repair of uracil incorporated in DNA as a result of a defect in dUTPase. J Biol Chem 117:293–306
Tye BK, Chien J, Lehman IR, Duncan BK, Warner HR (1978) Uracil incorporation: A source of pulse-labeled DNA fragments in the replication of the Escherichia coli chromosome. Proc Natl Acad Sci USA 75:233–237
Wang TCV, Smith KC (1986) Postreplicational formation and repair of DNA double-strand breaks in UV-irradiated Escherichia coli uvrB cells. Mutat Res 165:39–44
Worm K-H, Schulte-Frohlinde D (1991) Comparative study of transformation by the calcium chloride method and by electroporation using linearized plasmid-DNA. Int J Radiat Biol 59:573
Author information
Authors and Affiliations
Additional information
Communicated by R. Devoret
Rights and permissions
About this article
Cite this article
Schünemann, S., Schulte-Frohlinde, D. Survival of phage M13 with uracils on one or both DNA strands. Molec. Gen. Genet. 233, 348–354 (1992). https://doi.org/10.1007/BF00265430
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00265430