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Effects of DNA methylation on topoisomerase I and II cleavage activities
F Leteurtre1, G Kohlhagen, M R Fesen
1Laboratory of Molecular Pharmacology, National Cancer Institute, Bethesda, Maryland 20892.
Abstract:
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.
Insights
DNA methylation alters cancer drug targets. Cytosine methylation impacts topoisomerase I and II activity, potentially changing how anti-cancer drugs affect DNA cleavage patterns in chromatin.
Area of Science:
- Molecular Biology
- Cancer Research
- Biochemistry
Background:
- DNA methylation is frequently deregulated in cancer.
- Many anti-cancer drugs target topoisomerases, enzymes crucial for DNA replication and repair.
Purpose of the Study:
- To investigate the impact of cytosine methylation on topoisomerase I and II cleavage activities.
- To understand how DNA methylation influences the efficacy of topoisomerase-targeting anti-cancer drugs.
Main Methods:
- Analysis of topoisomerase I and II cleavage patterns on methylated c-myc gene DNA fragments.
- Utilizing synthetic methylated oligonucleotides to assess hemimethylation effects.
- Comparing cleavage activities with different methylation sites and drug treatments (VM-26, azatoxin).
Main Results:
- CpG methylation modified topoisomerase I and II cleavage patterns in c-myc DNA.
- Topoisomerase II cleavage was reduced near methylation sites, with drug-specific differences.
- Topoisomerase I activity showed both cleavage enhancement and suppression, with hemimethylation being sufficient to alter activity.
- Methylation at specific positions (-3, -4) on the scissile strand significantly affected topoisomerase I cleavage, suggesting major groove interaction.
Conclusions:
- DNA methylation profoundly affects topoisomerase activity.
- Methylation patterns can alter the distribution of DNA cleavage sites induced by anti-cancer drugs.
- These findings highlight the interplay between epigenetic modifications and drug-induced DNA damage in cancer therapy.