Mutagenesis by 0⁶meG residues within codon 12 of the human Ha-ras proto-oncogene in monkey cells

Abstract

The first or/and the second guanines of the human Ha-ras codon 12 (normally GGC) were substituted by O6 meG residues and the modified sequence was subsequently introduced into an SV40-based shuttle vector able to replicate in both simian cells and bacteria. After replication in simian COS7 cells (proficient in O6-alkyl-guanine transferase), plasmid DNA was extracted and mutations were screened in E. coli DH5 alpha cells. The vast majority of the mutations induced by O6 meG were G----A transitions. The mutation frequency observed at the second guanine of codon 12 (12G2 position: 3.75% +/- 0.4) was higher than the one observed at the first guanine (12G1 position: 1.09% +/- 0.6). This difference was confirmed by the results obtained when two adjacent O6 meG residues were positioned within codon 12. The higher mutation frequency observed for the 12G2 position could be attributed to differential repair or/and variation in polymerase fidelity. These results are in agreement with animal experiments where alkylating agents gave rise to mutation on G2 position of codon 12.

Mutagenesis by O6-Methyl-, O6-Ethyl-, and O6-Benzylguanine and O4-Methylthymine in Human Cells: Effects of O6-Alkylguanine-DNA Alkyltransferase and Mismatch Repair

“…Aspects of the mutagenicity and repair of these types of modified bases have been studied previously in mammalian cells using the site-specific mutagenesis approach (1)(2)(3)(4)(5)(6)(7)(8)(9). We have used this approach here to examine mutagenesis and repair of O 6 -methyl (m 6 G)-, O 6 -ethyl (e 6 G)-, and O 6 -benzylguanine (b 6 G), as well O 4 -methylthymine (m 4 T) in human cells.…”

Comparative study of mutagenesis by O6-methylguanine in the human Ha-ras oncogene inE.coliandin vitro

“…This is in agreement with the kinetic results of Georgiadis et al (9) on in vitro repair by the ada protein of 06-meG at position 12G2 of the rat oncogene. Slower repair at 12G2 was also proposed as a possible explanation for the higher mutagenesis observed in monkey COS cells using vectors containing the same synthetic sequences as employed here (11). Based on three independent observations, therefore, it appears that production of G:C -~A:T mutations at the second position of codon 12 of the rat and human Ha-ras oncogene may be favoured by slow AGT repair.…”

“…In some cases, where the same oligonucleotide was synthesised by both methods, identical results were obtained in mutagenesis experiments. Oligonucleotides 12G1/PE/M and 12G2/PE/M are the same as those employed in the previously published study using shuttle vectors in mammalian cells (11). All oligonucleotides employed in the construction of mutagenesis vectors were additionally purified and characterised as already described (11).…”

“…Hirani-Hojati et al (10) have shown that, following in vitro methylation of a plasmid containing the human Ha-ras oncogene with methyl-a-acetoxymethylnitrosamine, only mutations at 12G2 could be detected, suggesting that mutagenesis in the human gene may exhibit the same overall sequence specificity as seen in the rat. More recently, using a shuttle vector containing a sequence of the human gene, Pletsa et al (11) showed that more mutations are induced in monkey COS cells by 06-meG when present at position 12G2 than at 12G1, implying a significant role for adduct repair and/or polymerase fidelity in the determination of the observed sequence specificity.…”

Different Mutation Frequencies and Spectra among Organs by N‐Methyl‐N‐nitrosourea in rpsL (strA) Transgenic Mice

“…N-Methyl-N-nitrosourea (MNU) is an alkylating agent, which has been shown to induce G:C to A:T transitions by forming O 6 -methylguanine adducts in prokaryotic and eukaryotic cells, probably through the mismatch pairing of O 6 -methylguanine with thymidine during DNA replication. [17][18][19] It has been reported that mutation frequencies were increased by MNU in a tissue-specific manner by using MNU-treated lacI transgenic mice and that the predominant mutations in MNU-treated mice were G:C to A:T transitions. 20,21) We herein describe MNU-induced mutations in various organs of not only adults but also fetuses of the rpsL transgenic mice.…”