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

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Abstract
Double-stranded and gapped shuttle vectors were used to study mutagenesis in human cells by O6-methyl (m6G)-, O6-ethyl (e6G)-, and O6-benzylguanine (b6G), and O4-methylthymine (m4T) when these bases were incorporated site-specifically in the ATG initiation codon of a lacZ‘ gene. Vectors were transfected into either human kidney cells (293) or colon tumor cells (SO) or into mismatch repair defective human colon tumor cells (H6 and LoVo). Cellular O6-alkylguanine-DNA alkyltransferase (alkyltransferase) was optionally inactivated by treating cells with O6-benzylguanine prior to transfection. In alkyltransferase competent cells, the mutagenicity of all the modified bases was substantially higher in gapped plasmids than in double-stranded plasmids. Alkyltransferase inactivation increased mutagenesis by the three O6-substituted guanines in both double-stranded and gapped plasmids but did not affect m4T mutagenesis. In the absence of alkyltransferase, mutagenesis by m6G and to a lesser extent e6G in double-stranded vectors was higher in the mismatch repair defective H6 and LoVo cells than in SO or 293 cells indicating that e6G as well as m6G were subject to mismatch repair processing in these cells. The level of mutagenesis by m4T and b6G was not affected by mismatch repair status. When incorporated in gapped plasmids and in the absence of alkyltransferase, the order of mutagenicity for the modified bases was m4T > e6G ≅ m6G > b6G. The O6-substituted guanines primarily produced G→A transitions while m4T primarily produced T→C transitions. However, m4T also produced a significant number of T→A transversion mutations in addition to T→C transitions in mismatch repair deficient LoVo cells.