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
N1 - Funding Information:
We are grateful to Prof. Hikoya Hayatsu for his valuable advice. We thank Mrs. Asako Kawakami for technical assistance. This work is partially supported by a Grant-in-aid for Encouragement of Young Scientists (A) from the Ministry of Education, Culture, Sport, Science and Technology (no. 12771401).
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
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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 - Fundamental and Molecular Mechanisms of Mutagenesis
JF - Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis
SN - 0027-5107
IS - 1-2
ER -