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Topoisomerase I-mediated Cytotoxicity of N-Methyl-N'-nitro-N-nitrosoguanidine: Trapping of Topoisomerase I by the O⁶-Methylguanine¹

Laboratory of Molecular Pharmacology, Division of Basic Sciences, National Cancer Institute, NIH, Bethesda, Maryland 20892 [P. P., Y. U., Y. P.]; Molecular Pharmacology Department, St. Jude Children’s Hospital, Memphis, Tennessee 38105 [J. L. W., J. L. N.]; and Department of Cellular and Molecular Physiology, Milton S. Hershey Medical Center, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033 [N. A. L., A. E. P.]

Alkylating agents such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) are known to covalently link alkyl groups at the position 6 of guanines (O6MG) in DNA. O⁶-alkylguanine-DNA alkyltransferase (AGT) specifically removes the 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 incorporated 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 bonding pattern, such as in the case of the 8-oxoguanine, whereas inhibition of religation could be attributed to altered base pairing, such as abasic sites or base mismatches, because incorporation of a 6-thioguanine did not affect Top1 activity. Top1-DNA covalent complexes are also induced in MNNG-treated CHO cells constitutively lacking the AGT enzyme. Conversely, no increase could be detected in CHO cells transfected with the wild-type human AGT. Moreover, we show that yeasts overexpressing the human Top1 are more sensitive 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 modifications.

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