Effects of DNA methylation on topoisomerase I and II cleavage activities.
DNA methylation is deregulated during oncogenesis. Since several major anti-cancer drugs act on topoisomerases, we investigated the effects of cytosine methylation on topoisomerase cleavage activities. Both topoisomerase I and II cleavage patterns were modified by CpG methylation in c-myc gene DNA fragments. Topoisomerase II changes, mainly cleavage reduction, occurred for methylation sites within 7 base pairs from the topoisomerase II breaks and were different for VM-26 and azatoxin. For topoisomerase I, cleavage enhancement as well as suppression were observed. Using synthetic methylated oligonucleotides, we show that hemimethylation is sufficient to alter topoisomerase I activity. Cytosine methylation on the scissile strand within the topoisomerase I consensus sequence had strong effects. Cleavage was stimulated by methylation at position -4 and was strongly inhibited by methylation at position -3 (with position -1 being the enzyme-linked nucleotide). This inhibitory effect was attributed to the presence of a methyl group in the major groove, since the transition uracil to thymine also inhibited cleavage. Altogether these results suggest an interaction of topoisomerase I with the DNA major grove at positions -3 and -4. In addition, DNA methylation may have profound effects on the activity of topoisomerases and may alter the distribution of cleavage sites produced by anticancer drugs in chromatin.
Leteurtre
F
F
Laboratory of Molecular Pharmacology, National Cancer Institute, Bethesda, Maryland 20892.
Kohlhagen
G
G
Fesen
M R
MR
Tanizawa
A
A
Kohn
K W
KW
Pommier
Y
Y
D001483:Base Sequence; D003596:Cytosine; D004247:DNA / Q000138:chemical synthesis / Q000378:metabolism*; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D004250:DNA Topoisomerases, Type II / Q000378:metabolism*; D016259:Genes, myc; D006801:Humans; D007700:Kinetics; D008745:Methylation; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000378:metabolism; D016133:Polymerase Chain Reaction; D013379:Substrate Specificity; D013941:Thymine
D009838:Oligodeoxyribonucleotides; D003596:Cytosine; D004247:DNA; D004264:DNA Topoisomerases, Type I; D004250:DNA Topoisomerases, Type II; D013941:Thymine