O-6-METHYLGUANINE INDUCES INTRACHROMOSOMAL HOMOLOGOUS RECOMBINATION IN HUMAN-CELLS

N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), which alkylates many posi tions in DNA including the O-6 position of guanine, efficiently induce s intrachromosomal homologous recombination in mouse L-cells, To inves tigate the role of O-6-methylguanine in the induction of homologous re combination in human cells, three cell strains containing duplicated c opies of the Herpes simplex virus I thymidine kinase (Htk) gene and th ree cell strains containing duplicated copies of the gene coding for h ygromycin phosphotransferase (hyg) were treated with MNNG. Neither the Htk genes nor the hyg genes' code for a functional enzyme because eac h contains an insertion mutation at a unique site, i.e. 8-bp XhoI link er insertions in the Htk genes and 10-bp HindIII linker insertions in the hyg genes, These cell strains differ in their level of O-6-alkylgu anine-DNA alkyltransferase (AGT), which specifically removes the methy l group from the O-6 position of guanine, Generation of a functional H tk or hyg gene has been shown to require intrachromosomal homologous r ecombination between the two mutant Htk genes or the two mutant hyg ge nes, In all six cell strains, MNNG induced a dose-dependent increase i n the frequency of homologous recombination. In each set, there was an inverse correlation between the frequency of MNNG-induced recombinati on and the level of AGT activity. To further study the role of O-6-met hylguanine in the induction of homologous recombination, we used O-6-b enzylguanine to inactivate AGT in two additional human cell strains co ntaining the hyg recombination substrate. After depletion of AGT activ ity by O-6-benzylguanine, both cell strains showed a significantly ele vated level of MNNG-induced homologous recombination, These results in dicate that O-6-methylguanine is the principal lesion responsible for the induction of homologous recombination in these human cells by this methylating agent.