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 Citations (Scopus)

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
StatePublished - May 22 2002

Fingerprint

Oligonucleotides
Yeasts
DNA
Pyrimidinones
Nonsense Codon
Guanine
Adenine
DNA-Directed DNA Polymerase
DNA Replication
Nucleosides
Sequence Analysis
Bacteria
Genes

Keywords

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

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics
  • Health, Toxicology and Mutagenesis

Cite this

Use of yeast transformation by oligonucleotides to study DNA lesion bypass in vivo. / Otsuka, Chie; Kobayashi, Keita; Kawaguchi, Naho; Kunitomi, Nozomu; Moriyama, Kei; Hata, Yoshihiro; Iwai, Shigenori; Loakes, David; Noskov, Vladimir N.; Pavlov, Youri; Negishi, Kazuo.

In: Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis, Vol. 502, No. 1-2, 22.05.2002, p. 53-60.

Research output: Contribution to journalArticle

Otsuka, C, Kobayashi, K, Kawaguchi, N, Kunitomi, N, Moriyama, K, Hata, Y, Iwai, S, Loakes, D, Noskov, VN, Pavlov, Y & Negishi, K 2002, 'Use of yeast transformation by oligonucleotides to study DNA lesion bypass in vivo', Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis, vol. 502, no. 1-2, pp. 53-60. https://doi.org/10.1016/S0027-5107(02)00023-4
Otsuka, Chie ; Kobayashi, Keita ; Kawaguchi, Naho ; Kunitomi, Nozomu ; Moriyama, Kei ; Hata, Yoshihiro ; Iwai, Shigenori ; Loakes, David ; Noskov, Vladimir N. ; Pavlov, Youri ; Negishi, Kazuo. / Use of yeast transformation by oligonucleotides to study DNA lesion bypass in vivo. In: Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis. 2002 ; Vol. 502, No. 1-2. pp. 53-60.
@article{4683402345604a04babbba97590b1b91,
title = "Use of yeast transformation by oligonucleotides to study DNA lesion bypass in vivo",
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.",
keywords = "(6-4)Photoproduct, Nucleoside analog, Oligonucleotide transformation, REV1, Saccharomyces cerevisiae",
author = "Chie Otsuka and Keita Kobayashi and Naho Kawaguchi and Nozomu Kunitomi and Kei Moriyama and Yoshihiro Hata and Shigenori Iwai and David Loakes and Noskov, {Vladimir N.} and Youri Pavlov and Kazuo Negishi",
year = "2002",
month = "5",
day = "22",
doi = "10.1016/S0027-5107(02)00023-4",
language = "English (US)",
volume = "502",
pages = "53--60",
journal = "Mutation Research",
issn = "1386-1964",
publisher = "Elsevier BV",
number = "1-2",

}

TY - JOUR

T1 - Use of yeast transformation by oligonucleotides to study DNA lesion bypass in vivo

AU - Otsuka, Chie

AU - Kobayashi, Keita

AU - Kawaguchi, Naho

AU - Kunitomi, Nozomu

AU - Moriyama, Kei

AU - Hata, Yoshihiro

AU - Iwai, Shigenori

AU - Loakes, David

AU - Noskov, Vladimir N.

AU - Pavlov, Youri

AU - Negishi, Kazuo

PY - 2002/5/22

Y1 - 2002/5/22

N2 - 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.

AB - 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.

KW - (6-4)Photoproduct

KW - Nucleoside analog

KW - Oligonucleotide transformation

KW - REV1

KW - Saccharomyces cerevisiae

UR - http://www.scopus.com/inward/record.url?scp=0037157229&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0037157229&partnerID=8YFLogxK

U2 - 10.1016/S0027-5107(02)00023-4

DO - 10.1016/S0027-5107(02)00023-4

M3 - Article

C2 - 11996972

AN - SCOPUS:0037157229

VL - 502

SP - 53

EP - 60

JO - Mutation Research

JF - Mutation Research

SN - 1386-1964

IS - 1-2

ER -