O6-methylguanine induces intrachromosomal homologous recombination in human cells

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Abstract
N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), which alkylates many positions in DNA including the O6 position of guanine, efficiently induces intrachromosomal homologous recombination in mouse L-cells. To investigate the role of O6-methylguanine in the induction of homologous recombination in human cells, three cell strains containing duplicated copies of the Herpes simplex virus I thymidine kinase (Htk) gene and three cell strains containing duplicated copies of the gene coding for hygromycin phosphotransfer-ase (hyg) were treated with MNNG. Neither the Htk genes nor the hyg genes code for a functional enzyme because each contains an insertion mutation at a unique site, i.e. 8-bp XhoI linker insertions in the Htk genes and 10-bp HindIII linker insertions in the hyg genes. These cell strains differ in their level of O6-alkylguanine-DNA alkyltrans-ferase (AGT), which specifically removes the methyl group from the O6 position of guanine. Generation of a functional Htk or hyg gene has been shown to require intrachromo-somal homologous recombination between the two mutant Htk genes or the two mutant hyg genes. In all six cell strains, MNNG induced a dose-dependent increase in the frequency of homologous recombination. In each set, there was an inverse correlation between the frequency of MNNG-induced recombination and the level of AGT activity. To further study the role of O6-methylguanine in the induction of homologous recombination, we used O6-benzylguanine to inactivate AGT in two additional human cell strains containing the hyg recombination substrate. After depletion of AGT activity by O6-benzylguanine, both cell strains showed a significantly elevated level of MNNG-induced homologous recombination. These results indicate that O6-methylguanine is the principal lesion responsible for the induction of homologous recombination in these human cells by this methylating agent.

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