Characterization of bleomycin-mediated cleavage of a hairpin DNA library.

A study of BLM A5 was conducted using a previously isolated library of hairpin DNAs found to bind strongly to metal-free BLM. The ability of Fe(II)·BLM to affect cleavage on both the 3' and 5' arms of the hairpin DNAs was characterized. The strongly bound DNAs were found to be efficient substrates for Fe·BLM A5-mediated hairpin DNA cleavage. Surprisingly, the most prevalent site of BLM-mediated cleavage was found to be the 5'-AT-3' dinucleotide sequence. This dinucleotide sequence and other sequences generally not cleaved well by BLM when examined using arbitrarily chosen DNA substrates were apparent when examining the library of 10 hairpin DNAs. In total, 132 sites of DNA cleavage were produced by exposure of the hairpin DNA library to Fe·BLM A5. The existence of multiple sites of cleavage on both the 3' and 5' arms of the hairpin DNAs suggested that some of these might be double-strand cleavage events. Accordingly, an assay was developed to test the propensity of the hairpin DNAs to undergo double-strand DNA damage. One hairpin DNA was characterized using this method and gave results consistent with earlier reports of double-strand DNA cleavage but with a sequence selectivity that was different from those reported previously.

Dynamics of bleomycin interaction with a strongly bound hairpin DNA substrate, and implications for cleavage of the bound DNA.

Recent studies involving DNAs bound strongly by bleomycins have documented that such DNAs are degraded by the antitumor antibiotic with characteristics different from those observed when studying the cleavage of randomly chosen DNAs in the presence of excess Fe·BLM. In the present study, surface plasmon resonance has been used to characterize the dynamics of BLM B(2) binding to a strongly bound hairpin DNA, to define the effects of Fe(3+), salt, and temperature on BLM-DNA interaction. One strong primary DNA binding site, and at least one much weaker site, were documented. In contrast, more than one strong cleavage site was found, an observation also made for two other hairpin DNAs. Evidence is presented for BLM equilibration between the stronger and weaker binding sites in a way that renders BLM unavailable to other, less strongly bound DNAs. Thus, enhanced binding to a given site does not necessarily result in increased DNA degradation at that site; i.e., for strongly bound DNAs, the facility of DNA cleavage must involve other parameters in addition to the intrinsic rate of C-4' H atom abstraction from DNA sugars.

Synthesis and evaluation of verticipyrone analogues as mitochondrial complex I inhibitors.

Verticipyrone has recently been isolated from the culture broth of Verticillium sp. and shown to inhibit NADH fumarate reductase, as well as NADH oxidoreductase (complex I) of the mitochondrial electron transport chain. In order to assess the structural elements in verticipyrone essential for complex I inhibitor, 15 structural analogues were prepared and analyzed for their effects on mitochondrial NADH oxidoreductase and NADH oxidase activities. Also measured were the abilities of several of the analogues to inhibit respiration as judged by a shift to glycolysis, and to inhibit the growth of several mammalian cell lines. The nature of the pyrone ring was shown to be important to potency of inhibition, as was the length and nature of substituents in the side chain of the analogues.