Use of yeast transformation by oligonucleotides to study DNA lesion bypass in vivo

Chie Otsuka, Keita Kobayashi, Naho Kawaguchi, Nozomu Kunitomi, Kei Moriyama, Yoshihiro Hata, Shigenori Iwai, David Loakes, Vladimir N. Noskov, Youri Pavlov, Kazuo Negishi

Research output: Contribution to journalArticle

18 Scopus citations

Abstract

We have studied mutagenic specificities of DNA lesions in vivo in yeast CYC1 oligonucleotide transformation assay. We introduced two lesions into oligonucleotides. One was a nucleoside analog, 3,4-dihydro-6H,8H-pyrimido[4,5-c][1,2]oxazin-7-one 2′-deoxyriboside (dP), which is highly mutagenic to bacteria. It is supposed to be a miscoding, but otherwise good template for DNA polymerases. The other lesion was the TT pyrimidine(6-4)pyrimidone photoproduct, one of the typical UV lesions, which blocks DNA replication. These oligonucleotides were used to transform yeast cyc1 mutants with ochre nonsense mutation to Cyc1+. As expected from its templating properties in vitro, the transforming activity of dP-containing oligonucleotides was similar to those of unmodified oligonucleotides. Results indicated that dP may direct incorporation of guanine and adenine at a ratio of 1:20 or more in vivo. An oligonucleotide containing the photoproduct showed the transforming activity of as low as 3-5% of that of the corresponding unmodified oligonucleotide. This bypass absolutely required REV1 gene. The sequence analysis of the transformants has shown that the lesion was read as TT and TC at a ratio of 3:7, indicating its high mutagenic potential.

Original languageEnglish (US)
Pages (from-to)53-60
Number of pages8
JournalMutation Research - Fundamental and Molecular Mechanisms of Mutagenesis
Volume502
Issue number1-2
DOIs
Publication statusPublished - May 22 2002

    Fingerprint

Keywords

  • (6-4)Photoproduct
  • Nucleoside analog
  • Oligonucleotide transformation
  • REV1
  • Saccharomyces cerevisiae

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics
  • Health, Toxicology and Mutagenesis

Cite this