Molecular analysis of O6-substituted guanine-induced mutagenesis of ras oncogenes.
We have designed an Ha-ras/thymidine kinase (TK) cassette that permits the incorporation of chemically synthesized adducts within specific domains of the rat Ha-ras protooncogene. This cassette has been used to evaluate the mutagenicity of O6-substituted guanine residues, including O6-methylguanine and O6-benzylguanine, incorporated within the 12th codon of this locus. Mutations were monitored by the ability of these modified Ha-ras DNAs to transform Rat4 TK-cells. Our results indicate that both types of O6-substituted guanines are substantially mutagenic, although the methyl substituent induced a 2-fold higher percentage of transformed Rat4 TK+ colonies than its bulkier benzyl analogue. Interestingly, the mutagenicity of both O6-substituted guanines was found to be independent of their relative position within codon 12, therefore suggesting that the specific activation of Ha-ras oncogenes by GGA----GAA mutations in tumors induced by methylating carcinogens might be due to differences in the accessibility of these guanine residues to the carcinogen rather than to a differential rate of repair. Molecular analysis of the mutations induced by these O6-substituted guanines indicated that O6-methylguanine exclusively induced G----A transitions. In contrast, O6-benzylguanine produced G----C and G----T transversions in addition to G----A transitions. These results suggest that O6-methylguanine and its bulkier analogue O6-benzylguanine may induce mutagenesis by different mechanisms.
Mitra
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G
Developmental Oncology, National Cancer Institute-Frederick Cancer Research Facility, MD 21701.
Pauly
G T
GT
Kumar
R
R
Pei
G K
GK
Hughes
S H
SH
Moschel
R C
RC
Barbacid
M
M
D000478:Alkylation; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004259:DNA-Directed DNA Polymerase; D004926:Escherichia coli / Q000235:genetics; D005784:Gene Amplification; D011905:Genes, ras; D006147:Guanine / Q000031:analogs & derivatives* / Q000032:analysis / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes; D010957:Plasmids; D051381:Rats; D015183:Restriction Mapping; D014162:Transfection
D015345:Oligonucleotide Probes; D006147:Guanine; D004259:DNA-Directed DNA Polymerase