DNA methylation diminishes bleomycin-mediated strand scission

Abstract

Three DNA duplexes differing substantially in sequence were derived from pBR322 plasmid DNA and supercoiled SV40 DNA by digestion with appropriate restriction endonucleases. Following treatment with the restriction methylase HhaI (recognition sequence: GCGC) or HhaI and HpaII (CCGG), the unmethylated and methylated DNAs were compared as substrates for the antitumor agent bleomycin. Bleomycin-mediated strand scission was shown to diminish substantially at a number of sites in proximity to the methylated cytidine moieties, especially where multiple sites had been methylated within a DNA segment of limited size. Detailed analysis of the DNA substrates revealed that both strands of DNA within a methylated region became more refractory to cleavage by bleomycin and that the protective effect could extend as many as 14 base pairs in proximity to the 5-methylcytidine moieties. Among the methylated DNA segments that became more resistant to bleomycin cleavage was a HpaII site of SV40 DNA, methylation of which has previously been shown to diminish the synthesis of the major late viral capsid protein following microinjection into Xenopus laevis oocytes. Study of the cleavage reaction at varying salt levels suggested that diminished bleomycin strand scission may be due, at least in part, to local conformational changes of the DNA to Z form (or other non-B-form structures). The results are generally consistent with the hypothesis that one mechanism for the expression of selective therapeutic action by certain DNA damaging agents could involve the recognition of specific methylation patterns.

Characterization of the calcium‐binding sites of calcineurin B

“…CaN, calcineurin; CaNA, calcineurin A; CaNB, calcineurin B; rCaNB, recombinant CaNB; CaM, calmodulin; CsA, cyclosporin A; CyP, cyclophilin; FKBP, FK506 binding protein; NFAT, nuclear factor of activated T cells; Tris, tris(hydroxymethyl)aminomethane; PIPES, piperazine-N,N'-bis(2-ethanesulfonic acid); HEPES, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; TnC, troponin C; DTT, dithiothreitol; NMR, nuclear magnetic resonance. basis of its amino acid sequence, CaNB is postulated to harbor four calcium-binding sites of the EF hand type [5], similar to the helix-loop-helix motif of other calcium-binding proteins such as troponin C (TnC) and CaM [6]. Metal-binding sites of Cd2+-substituted metalloproteins can be probed by H3Cd NMR, thus taking advantage of the favorable characteristics of this spin-l/2 nucleus [7][8][9].…”

Pyrrole-imidazole hairpin polyamides with high affinity at 5'-CGCG-3' DNA sequence; influence of cytosine methylation on binding

“…However, it is known that the reactivities of some DNA-interacting drugs are affected by cytosine methylation. For example, benzo[ a ]pyrene and mitomycin C show increased covalent adduct formation ( 27–30 ), while bleomycin gives a decreased cleavage reaction ( 31 ), although these drugs act in the DNA minor groove. The precise reason for the stronger binding of polyamide to the methylated DNA is not clear, however, hydrophobic and electronic factors would contribute to this phenomenon.…”

Genome Instability and Bleomycin Sensitivity Test

“…Stvaranje lomova u molekuli DNA pod utjecajem bleomicina smanjuje se u blizini 5-metilcitidina, a taj se utjecaj može primijetiti čak i 14 parova baza dalje od takvog mjesta (76,77).…”