Abstract
The binding of the iron complex of the antineoplastic glycopeptide bleomycin A2 (Fe-BLM) to calf thymus DNA and the self-complementary oligonucleotides d(CGCGCG) and d(ATATAT) has been studied using optical, EPR, and resonance Raman spectroscopies. An increase in the intensity of the bands at 365 and 384 nm is observed in the optical spectrum of Fe(III)-BLM when the drug binds to either oligonucleotide. However, in the presence of phosphate, this increase is observed only with d(CGCGCG) and not with d(ATATAT). In addition, the gmax feature in the EPR spectrum of low spin Fe(III)-BLM is narrowed in a way suggesting a reduction of possible conformers that the drug can achieve when it is bound to d(CGCGCG) or to calf thymus DNA but not when bound to d(ATATAT). When Fe(III)-BLM is bound to d(CGCGCG), changes in the resonance Raman spectrum of the metal drug complex suggest conformational changes in three of the ligands to iron: the beta-hydroxyhistidyl amide, the pyrimidine, and the axial hydroxide. In addition, the Fe-OH band undergoes narrowing, again consistent, with the reduction of conformers of the drug. No such resonance Raman changes are observed upon binding to d(ATATAT). The changes in the pyrimidine modes upon binding d(CGCGCG) to the drug are consistent with a recently proposed model (Wu, W., Vanderwall, D. E., Turner, C. J., Kozarich, J. W., and Stubbe, J. (1996) J. Am. Chem. Soc. 118, 1281-1294) of DNA recognition by activated bleomycin, HOO-Fe(III)-BLM, in which the pyrimidine moiety of the drug is important for the preferential cleavage of 5'-GpPy-3' sequences.
Highlights
Bleomycin A2 (BLM,1 Fig. 1A; for reviews, see Refs. 1– 4) is a glycopeptide antibiotic used clinically in the treatment of various cancers
We report changes in the metal binding site of Fe-BLM induced by complexation with DNA, using optical, EPR, and resonance Raman spectroscopy
We have extended our studies to Fe-BLM bound to the self-complementary hexanucleotides, d(CGCGCG) and d(ATATAT), with the intent of detecting structural changes in the metal binding site that correlate with the sequence specificity of DNA cleavage
Summary
Bleomycin A2 (BLM, Fig. 1A; for reviews, see Refs. 1– 4) is a glycopeptide antibiotic used clinically in the treatment of various cancers. There have been reports implicating almost every region of the Fe-BLM molecule, including the C-terminal bithiazole and dimethylsulfonium groups (15–17), the N-terminal metal binding site (18 –20), the primary amine of the -aminoalanine residue (21), and the “linker region” that connects the metal binding site with the bithiazole group (22, 23), as being responsible, at least in part, for the sequence selectivity of Fe-BLM-mediated DNA degradation. While many of these reports appear to provide conflicting results, the one requirement from all but one (24) of these studies is that for sequencespecific cleavage of DNA to occur, both the metal binding site and bithiazole moiety must be intact. We investigated the effect of phosphate on Fe(III)-BLM bound to DNA, since it has been shown that phosphate increases the efficiency of DNA cleavage by Fe-BLM and that phosphate converts Fe(III)-BLM from a low to a high spin complex, an effect that can be negated by the addition of DNA (28)
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