Analogue versus digital recognition of DNA by bleomycin: an effect of the carbohydrate moiety

Abstract

We have sought to determine the influence of the carbohydrate moiety of the antitumour antibiotic hleomycin on the sequence-specific cleavage of DNA. Both bleomycin A2 and deglycobleomycin A2 produce different cleavage patterns with DNA in which the 2-amino group has been removed from guanine, added to adenine, or both, as well as on a designed DNA fragment containing a few defined cleavage sites. Although each drug cleaves DNA primarily at GpT and GpC sites, the cleavage at these sites is frequently found to be stronger with deglycobleomycin compared with bleomycin A 2. Conversely, in most cases the cleavage at secondary sites, in particular at ApT steps, is significantly reduced or even abolished with deglycobleomycin. The results indicate that the gulose-mannose moiety of bleomycin A2 plays a significant role in the recognition of preferred nucleotide sequences and confirm the view that both secondary structure and interaction with guanine are involved in determining sequencespecific cleavage of DNA by bleomycin.Key words." Bleomycin; DNA recognition; DNA cleavage; Sequence specificity to DNA. In addition, NMR studies suggest that the carbohydrate portion could contribute to the interaction since intermolecular NOEs between the gulose and the central thymidine in the sequence d(CGCTAGCG)2 have been detected [12]. We have explored this idea by comparing the patterns of cleavage by bleomycin A2 and deglycobleomycin A2. We report that the sugar moiety of bleomycin is not mandatory for cutting at primary GpT and GpC sites but it plays a decisive role in cutting at secondary sites, particularly ApT. Materials and methods Bleomycin A2 and deglycobleomycinA pure sample of bleomycin A z was obtained from Roger-Bellon Laboratories (Neuilly-sur-Seine, France). The gulose-mannose moiety of bleomycin A2 was specifically cleaved by HF solvolysis according to a published protocol [13]. Deglycobleomycin A2 was purified by HPLC and the integrity of the deglycosylated antibiotic was checked by [~H]NMR and HR-FAB mass spectrometry. Stock solutions were prepared in 10 mM Tris-HCl buffer containing 10 mM NaC1, pH 7.0, divided into 250-gtM aliquots and stored at -20°C.

Deglyco-peplomycin metal complexes on DNA fibers: a role of the sugar moiety for the stability and the orientation of the complexes

“…As to the site specificity of the cleaving reaction, Bailly et al reported that the sugar moiety is not mandatory for cutting at primary GpT and GpC sites, but it plays a decisive role in cutting at secondary sites, particularly ApT [10,11]. NMR studies of Co(III)BLM and Co(III)PEP bound to double helical oligonucleotide revealed that the binding mode of the complex is not affected by the sugar moiety [12,13,14].…”

Glycosylated Natural Products

“…The role of the carbohydrates remains controversial. Early comparative studies between deglycosyl-3 and 3 suggested a role in DNA recognition [29,30]. However, more recent efforts suggest that the sugars do not contribute to DNA affinity or selectivity, but do enhance the DNA cleavage efficiency by approximately fivefold [28].…”

The ratio of single- to double-strand DNA breaks and their absolute values determine cell death pathway

“…In spite of minor differences in their physicochemical properties (Kenani et al, 1988a), deglyco-BLM-A 2 and BLM-A 2 share the same mechanism of nucleophilic attack at the same DNA sequences (Bailly et al, 1995). However, on intact cells, deglyco-BLM-A 2 is much less toxic than BLM-A 2 Deglyco-BLM-A 2 cytotoxicity was also determined in transiently and reversibly electropermeabilized cells, in which the plasma membrane does not restrict hydrophilic molecule cytotoxicity.…”

“…Taking into account that deglyco-BLM-A 2 and BLM-A 2 generate SSB and DSB by the same chemical reaction on DNA (Bailly et al, 1995), it seems interesting to compare the intrinsic toxic capacities of SSB and DSB. At their lowest cytotoxic concentrations in electropermeabilized cells, BLM (10 -9 M) generates 500 DSB (Tounekti et al, 1993) and, deduced from the calculations reported here, deglyco-BLM-A 2 (10 -7 M) should generate 150 000 SSB.…”

“…It has been shown recently that, like all BLMs, deglyco-BLM-A 2 has retained the ability to preferentially recognize the GC sequences on DNA and to produce breaks at this position (Bailly et al, 1995). Thus, deglyco-BLM-A 2 and BLM-A 2 share an identical mechanism of action at the DNA level.…”