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Research has been conducted on the bleomycin and metallobleomycins. The interaction properties of these agents with site-specific and nonspecific DNA oligomers have been investigated.

Research has been conducted on the Co-bleomycin coordination chemistry. Nuclear magnetic resonance spectroscopy has been used in investigating Co(II) in Co(II)-bleomycin coordination sphere.

A study on the 455-amino-acid cysteine proteinase called bleomycin hydrolase (BH) demonstrated two-hybrid and in vitro binding of human BH (hBH) with ribosomal proteins L11 and L29. The experiment also illustrated hBH association with the ribosomal subcellular fraction from Chinese hamster ovary cells and binding of recombinant His-tagged hBH to isolated CHO ribosomes. The study strongly suggests that hBH exists in both free cytosolic and ribosome-bound forms.

The intrinsic endopeptidase activity related to human bleomycin hydrolase (hBH) was described by using artificial substrates and by implementing intracellular dimerization of hBH. Two independent locations of homomeric interactions were identified via deductive domain analysis. The results revealed that 8 C-terminal amino acids, called bleomycin hydrolase-like domain, were not critical for aminopeptidase activity and but were important for bleomycin deamidation.

Analysis of bleomycin (BLM) activity in the beta-globin gene cluster indicates that intensity of BLM damage sites differs in purified DNA and intact cells. The chromatin structure probably affects BLM activity at the beta-globin promoter sequence. BLM activity is low at the site of protein-DNA interactions in intact cells. It is affected at nuclear factor binding sites in the beta-globin locus control region hypersensitive site 2 of intact cells. The activity of BLM is lowest in K562 cells with a ten-fold reduction in damage at the tandem nuclear factor-erythroid 2/AP1 DNA sequence element.

13C spectra indicating 113Cd-13C spin-spin couplings show that nitrogen atoms from the primary amine, pyrimidine and imidazole are bound to 113Cd in 113Cd-bleomycin. Bound and free forms of the secondary amine nitrogen may be in equilibrium. There are at least two differently coordinated forms of cadmium in the molecule and there are at most four nitrogen atoms bound to Cd at low temperature. These anions can bind to major and minor forms of the structure and there is a minor species which does not associate with the chloride.

The cleavage of a DNA triple helix by Fe(II).bleomycin (BLM) occurs on both strands of the 32-base pair duplex at the duplex-triplex junction. Cleavage is highest at the 5'-duplex-triplex junction which is the favored binding site of intercalating agents. Molecular-modeling analysis of the triplex region reveals significant alterations in the minor groove width. The preference of BLM for the duplex-triplex junctions appears to be due to the shape of the minor groove.

HO2-Co(III)bleomcyin A2 and Co(III)bleomycin A2 are synthesized during the oxidation of Co(III) bleomycin A2 which is performed by dioxygen compounds. Two dimensional nuclear magnetic resonance (NMR) spectral methods determine the H1 NMR chemical shift assignments for the protons of the two products. The proton chemical shift is different for both the products, indicating the presence of numerous metal-binding domains and peptide linkers in the products. The variation in connecting the products to DNA by the domains and linkers produces the difference in chemical shift assignments.

Chromatin reconstituted from a supercoiled plasmid and core histone was studied with respect to the stability of the abasic sites induced by bleomycin and neocarzinostatin. Spontaneous cleavage in chromatin was observed in all the drug-induced abasic sites. The mutagenic potential of bleomycin-induced abasic sites was determined by transfecting bleomycin-treated shuttle vectors into mammalian cells. The results suggest that the abasic sites have only a slight potential to produce base substitutions in mammalian cells and that a substantial fraction of the double-stranded breaks induced by bleomycin and neocarzinostatin result from spontaneous cleavage of abasic sites.

The sequence specific isotope effects of activated iron bleomycin (Fe BLM) on DNA cleavage at thymidine residues was studied after (4'-2H)thymidine was incorporated into DNA. A phosphorus-32 end label was used as a remote label for quantifying these effects. Results showing no isotope effect for DNA deuteriated at other positions demonstrates the high regiospecificity of the BLM-mediated carbon-hydrogen bond cleavage. For Fe BLM, the 4'-isotope effects were found to have a magnitude of close to 2-7 and were shown to be sensitive to changes in BLM structure and in the cleavage mechanism.

The lability of oxygen in activated bleomycin (BLM) was investigated with the use of electron paramagnetic resonance spectroscopy to measure the exchange of 17O between activated BLM and solvent. It was demonstrated that exchange does not occur between oxygen bound to activated BLM and that of solvent. It was postulated that the radical rebound mechanism of BLM-mediated DNA degradation may not be operative.

The reaction of ferric bleomycin, Fe(super III)BLM, with DNA in the presence of ascorbate was studied to resolve the BLM paradox. BLM is a chemotherapeutic agent causing DNA-breakage when activated by reducing agents. However, these same agents protect cellular DNA from BLM damage. Results suggest that activation of BLM and generation of redox-inactive BLM is facilitated by a single reaction. Moreover, BLM-induced DNA damage results only when BLM-DNA reaction occurs ahead of BLM reduction.

The structure of 4'-oxidized abasic site (X) is determined by two-dimensional nuclear magnetic resonance (NMR) methods, molecular modeling and molecular dynamics simulations. Molecular dynamics simulations have shown that the normal abasic site is more flexible than the 4'-oxidized abasic site.

A study on bleomycin-DNA interactions describes the use of oligonucleotides incorporating a site-specific minor groove-blocking base analogue, N2-isobutyrylguanine in mapping the DNA binding site occupied by bleomycin during strand cleavage. The findings suggest that N2-isobutyrylguanine may be a generally useful reagent for mapping bindings sites of minor groove ligands. Experimental materials, methodology and results are presented.