Mutagenesis by O(6)-methyl-, O(6)-ethyl-, and O(6)-benzylguanine and O(4)-methylthymine in human cells: effects of O(6)-alkylguanine-DNA alkyltransferase and mismatch repair.

Double-stranded and gapped shuttle vectors were used to study mutagenesis in human cells by O(6)-methyl (m(6)G)-, O(6)-ethyl (e(6)G)-, and O(6)-benzylguanine (b(6)G), and O(4)-methylthymine (m(4)T) 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 O(6)-alkylguanine-DNA alkyltransferase (alkyltransferase) was optionally inactivated by treating cells with O(6)-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 O(6)-substituted guanines in both double-stranded and gapped plasmids but did not affect m(4)T mutagenesis. In the absence of alkyltransferase, mutagenesis by m(6)G and to a lesser extent e(6)G in double-stranded vectors was higher in the mismatch repair defective H6 and LoVo cells than in SO or 293 cells indicating that e(6)G as well as m(6)G were subject to mismatch repair processing in these cells. The level of mutagenesis by m(4)T and b(6)G 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 m(4)T > e(6)G congruent with m(6)G > b(6)G. The O(6)-substituted guanines primarily produced G-->A transitions while m(4)T primarily produced T-->C transitions. However, m(4)T also produced a significant number of T-->A transversion mutations in addition to T-->C transitions in mismatch repair deficient LoVo cells.