Abstract
The major DNA cytosine methyltransferase isoform in mouse erythroleukemia cells, Dnmt1, exhibits potent dead-end inhibition with a single-stranded nucleic acid by binding to an allosteric site on the enzyme. The previously reported substrate inhibition with double-stranded substrates also involves binding to an allosteric site. Thus, both forms of inhibition involve ternary enzyme-DNA-DNA complexes. The inhibition potency of the single-stranded nucleic acid is determined by the sequence, length, and most appreciably the presence of a single 5-methylcytosine residue. A single-stranded phosphorothioate derivative inhibits DNA methylation activity in nuclear extracts. Mouse erythroleukemia cells treated with the phosphorothioate inhibitor show a significant decrease in global genomic methylation levels. Inhibitor treatment of human colon cancer cells causes demethylation of the p16 tumor suppressor gene and subsequent p16 re-expression. Allosteric inhibitors of mammalian DNA cytosine methyltransferases, representing a new class of molecules with potential therapeutic applications, may be used to elucidate novel epigenetic mechanisms that control development.
Highlights
The patterns of DNA cytosine methylation in mammals evolve throughout development [1]
Poly(dI1⁄7dC) and hemimethylated (CGG/CCG)12, a 36-bp fragment containing the triplet repeat sequence in which expansion in the FMR1 gene causes fragile-X syndrome, show complex kinetics consistent with DNA binding to an allosteric site, no such behavior is observed with unmethylated
The relative velocities versus S/Km plots conveniently normalize the data between the two Allosteric DNA Cytosine Methyltransferase Inhibitor diverse DNA substrates
Summary
Materials—S-Adenosyl-L-[methyl-3H]methionine (75 Ci/mmol, 1 mCi/ ml ϭ 37 GBq) was purchased from Amersham Biosciences. The poly(dI1⁄7dC) concentrations were ϳ2.0, 4.0, 8.0, 16, 35, 80, 160, 250, 400, 700, and 1000 picomolar in duplex DNA; Dnmt was 3.0 nM. Inhibition with Single-stranded Oligonucleotides—Initial velocity data were collected with poly(dI1⁄7dC), AdoMet, GC-boxb, and GC-boxbMET at the concentrations indicated in Fig. 2 legend. IC50 determinations with GC-boxbMET, GC-boxpMET, GC-boxcMET, GC-boxdMET, GC-boxeMET, and CRE used 4 nM Dnmt and 50 pM poly(dI1⁄7dC). The reaction was stopped after 60 min, and label incorporation into DNA was determined as described [41]. Mouse Erythroleukemia Cell Studies—MEL cells, prepared as described [41] to a density of 106/ml, were treated with inhibitors (7.7 M GC-boxpMET, 1.5 M 5AC, or 18 M antisense oligonucleotide) along with LipofectAMINE as described by the manufacturer. Amplification was the same as p16 with the exception of using an annealing temperature of 60 °C and 27 amplification cycles
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