Abstract
The triphosphate of the nucleoside deoxyribosyl dihydropyrimido[4,5-c][1,2]oxazin-7-one (dP) is known to be incorporated into DNA efficiently by Taq polymerase and is a useful tool for polymerase-mediated in vitro mutagenesis. It is shown here that dP is a potent mutagen in Escherichia coli and Salmonella typhimurium . In E.coli , this deoxycytidine analog induces both GC-->AT and AT-->GC transitions. No induced transversions are observed. It is highly mutagenic in wild-type E.coli, but this is much reduced in a strain lacking thymidine kinase. Mutagenesis induced by dP is efficiently inhibited by the addition of thymidine. Partially purified thymidine kinase from E.coli catalyzes phosphorylation of dP to its 5'-monophosphate. When E.coli was grown in the presence of dP, the nucleoside analog was incorporated into its DNA. The content of dP in DNA was dependent on the concentration of dP added to the medium. The incorporation characteristics of the 5'-triphosphate of dP (dPTP) were also studied using E.coli DNA polymerase I large fragment. The results confirm that this triphosphate can be incorporated opposite A and G in the template with similar efficiencies. This indicates that dP is metabolized as a thymidine analog and that the resulting triphosphate induces a high rate of mutagenesis through replicational errors.
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Selected References
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- Cupples C. G., Miller J. H. A set of lacZ mutations in Escherichia coli that allow rapid detection of each of the six base substitutions. Proc Natl Acad Sci U S A. 1989 Jul;86(14):5345–5349. doi: 10.1073/pnas.86.14.5345. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lin P. K., Brown D. M. Synthesis and duplex stability of oligonucleotides containing cytosine-thymine analogues. Nucleic Acids Res. 1989 Dec 25;17(24):10373–10383. doi: 10.1093/nar/17.24.10373. [DOI] [PMC free article] [PubMed] [Google Scholar]
- McCann J., Spingarn N. E., Kobori J., Ames B. N. Detection of carcinogens as mutagens: bacterial tester strains with R factor plasmids. Proc Natl Acad Sci U S A. 1975 Mar;72(3):979–983. doi: 10.1073/pnas.72.3.979. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nedderman A. N., Stone M. J., Williams D. H., Lin P. K., Brown D. M. Molecular basis for methoxyamine-initiated mutagenesis: 1H nuclear magnetic resonance studies of oligonucleotide duplexes containing base-modified cytosine residues. J Mol Biol. 1993 Apr 5;230(3):1068–1076. doi: 10.1006/jmbi.1993.1219. [DOI] [PubMed] [Google Scholar]
- Negishi K., Harada C., Ohara Y., Oohara K., Nitta N., Hayatsu H. N4-aminocytidine, a nucleoside analog that has an exceptionally high mutagenic activity. Nucleic Acids Res. 1983 Aug 11;11(15):5223–5233. doi: 10.1093/nar/11.15.5223. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Negishi K., Tamanoi K., Ishii M., Kawakami M., Yamashita Y., Hayatsu H. Mutagenic nucleoside analog N4-aminocytidine: metabolism, incorporation into DNA, and mutagenesis in Escherichia coli. J Bacteriol. 1988 Nov;170(11):5257–5262. doi: 10.1128/jb.170.11.5257-5262.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Negishi K., Williams D. M., Inoue Y., Iimura T., Katahira M., Kanazawa H., Uesugi S., Hayatsu H., Brown M. D. Mechanism of mutagenesis induced by cytosine analogs bearing N(4)-substitutions. Nucleic Acids Symp Ser. 1995;(34):231–232. [PubMed] [Google Scholar]
- OKAZAKI R., KORNBERG A. DEOXYTHYMIDINE KINASE OF ESCHERICHIA COLI. I. PURIFICATION AND SOME PROPERTIES OF THE ENZYME. J Biol Chem. 1964 Jan;239:269–274. [PubMed] [Google Scholar]
- Stone M. J., Nedderman A. N., Williams D. H., Lin P. K., Brown D. M. Molecular basis for methoxyamine initiated mutagenesis. 1H nuclear magnetic resonance studies of base-modified oligodeoxynucleotides. J Mol Biol. 1991 Dec 5;222(3):711–723. doi: 10.1016/0022-2836(91)90507-3. [DOI] [PubMed] [Google Scholar]
- Tanaka K., Yoshioka A., Tanaka S., Wataya Y. An improved method for the quantitative determination of deoxyribonucleoside triphosphates in cell extracts. Anal Biochem. 1984 May 15;139(1):35–41. doi: 10.1016/0003-2697(84)90386-5. [DOI] [PubMed] [Google Scholar]
- Zaccolo M., Williams D. M., Brown D. M., Gherardi E. An approach to random mutagenesis of DNA using mixtures of triphosphate derivatives of nucleoside analogues. J Mol Biol. 1996 Feb 2;255(4):589–603. doi: 10.1006/jmbi.1996.0049. [DOI] [PubMed] [Google Scholar]