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Topoisomerase
I-mediated cytotoxicity of N-methyl-N'-nitro-N-nitrosoguanidine: trapping of
topoisomerase I by the O6-methylguanine
Pourquier P, Waltman JL, Urasaki Y, Loktionova NA, Pegg AE, Nitiss JL,
Pommier Y
Laboratory of Molecular Pharmacology, Division of Basic Sciences, National
Cancer Institute, NIH, Bethesda, Maryland 20892-4255, USA
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.
PMID: 11196197 [PubMed - indexed for MEDLINE]
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