Topoisomerase I-mediated cytotoxicity of N-methyl-N '-nitro-N-nitrosoguanidine: Trapping of topoisomerase I by the O-6-methylguanine

Alkylating agents such as N methyl-N'-nitro-N-nitrosoguanidine (MNNG) are k nown to covalently link alkyl groups at the position 6 of guanines (O6hMG) in DNA. O-6-alkylguanine-DNA alkyltransferase (AGT) specifically removes th e methyl group of the O6MG. Using purified human topoisomerase I (Top1), we found an 8-10-fold enhancement of Top1 cleavage complexes when O6MG is inc orporated in oligonucleotides at the +1 position relative to a unique Top1 cleavage site. Top1 poisoning by O6MG is attributable to a decrease of the Top1-mediated DNA religation as well as an increase in the enzyme cleavage step. Increased cleavage is probably linked to a change in the hydrogen bon ding pattern, such as in the ease of the 8-oxoguanine, whereas inhibition o f religation could be attributed to altered base pairing, such as abasic si tes or base mismatches, because incorporation of a 6-thioguanine did not af fect Top1 activity. Top1-DNA covalent complexes are also induced in MNNG-tr eated CHO cells constitutively lacking the AGT enzyme. Conversely, no incre ase could be detected in CHO cells transfected with the wild-type human AGT . Moreover, we show that yeasts overexpressing the human Top1 are more sens itive to MNNG, whereas knock-out Top1 strain cells display some resistance to the drug. Altogether, these results suggest a role for Top1 poisoning by alkylated bases in the antiproliferative activity of alkylating agents as well as in the DNA lesions resulting from endogenous and carcinogenic DNA m odifications.