Site-specific cleavage of RNA by Fe(II).bleomycin.

Abstract

Bleomycin is an antitumor agent whose activity has long been thought to derive from its ability to degrade DNA. Recent findings suggest that cellular RNA may be a therapeutically relevant locus. At micromolar concentrations, Fe(II)-bleomycin readily cleaved a Bacilus subtilis tRNAHis precursor in a highly selective fashion, but Escherichia coli tRNATYr precursor was largely unaffected even under more forcing conditions. Other substrates included an RNA transcript encoding a large segment of the reverse transcriptase from human immunodeficiency virus 1. RNA cleavage was oxidative, %10-fold more selective than DNA cleavage, and largely unaffected by nonsubstrate RNAs. RNA sequence analysis suggested recognition of RNA tertiary structure, rather than recognition of specific sequences; subsets of nucleotides at the junction of single-and double-stranded regions were especially susceptible to cleavage. The ready accessibility of cellular RNAs to xenobiotic agents, the high selectivity of bleomycin action on RNAs, and the paucity ofmechanisms for RNA repair suggest that RNA may be a therapeutically relevant target for bleomycin.The bleomycins (BLMs) are antitumor antibiotics believed to function by DNA degradation (1, 2). DNA degradation is sequence-selective, occurring primarily at a subset of all 5'-GT-3' and 5'-GC-3' sequences (3, 4). A wealth of information exists concerning the mechanism of BLM-mediated DNA degradation in cell-free (1, 2, 5) and cellular (5, 6) systems.In contrast, few studies have dealt with RNA as a substrate for BLM. Further, in spite of the compartmentalization of DNA and RNA in nucleated cells, most studies of RNA cleavage have been carried out with DNA present. For example, BLM degraded only the poly(dT) strand of a poly(rA)-poly(dT) hybrid (7,8). Hori (9) showed that relaxation of simian virus 40 form I DNA was hardly affected by even a large excess of Escherichia coli tRNA. Those experiments that employed RNA alone were carried out in the absence of added metal ions (10, 11). Magliozzo et al. (12) have reported degradation of yeast tRNAPhe by high concentrations of Fe(II)-BLM; product bands corresponding to <5-10% of the starting tRNA were observed by methylene blue staining of a polyacrylamide gel. Thin layer chromatographic analysis indicated products that comigrated with adenine and uracil.Although none ofthe foregoing studies suggested that RNA might constitute an efficient substrate for BLM, ongoing studies in this laboratory of BLM-mediated DNA oligonucleotide degradation have demonstrated that alteration of DNA conformation can significantly change the pattern and extent of cleavage normally obtained with B-form DNA (13,14). On the chance that the limited RNA cleavage observed (vide supra) might actually have resulted from highly efficient cleavage at a few conformationally unique sites, we studied several RNAs as substrates. Presently, we show that Fe(II)-BLM does mediate RNA degradation and that the process is highly site-selective.

Crystal structure of DNA-bound Co(III)·bleomycin B 2 : Insights on intercalation and minor groove binding

“…Of the DNA lesions formed, double-strand DNA cleavage is thought to be most relevant to the cytotoxicity of bleomycin. Although DNA is the generally accepted locus of drug activity, O 2 -activated Fe(II)⅐bleomycin also mediates the selective cleavage of RNA (10), and Cu(I)⅐bleomycin is capable of cleaving DNA (11); however, the in vivo relevance of this alternative nucleic acid target and metal ion remain to be fully determined.…”

Violaceols Function as Actin Inhibitors Inducing Cell Shape Elongation in Fibroblast Cells

“…We hypothesized that the 2,2 0 -dihydroxy moiety in violaceol-I and -II might be chelated with a metal ion, as with ethylenediaminetetraacetic acid, coenzyme Q10, or Bleomycin. 33,34) As shown in Fig. 2, we observed that violaceol-I and -II increased the emission intensity of Ca 2þ more than 50 times as compared to the levels of in living cells (Fig.…”

Iron regulatory elements (IREs): a family of mRNA non-coding sequences

“…In general, the organic ligands in the transition-metal complexes bind to specific sites on nucleic acids and the metal serves as a reporter for the binding interaction by producing free radicals which cleave the nucleic acid at, or near, the site of binding. The same chemistry has been exploited for many years in the use of Fe-bleomycin, which cleaves DNA and RNA, as an anti-tumour agent (Carter et al, 1990). Since the transition-metal complexes are intermediate in size between the bulky protein nucleases and the small alkylating reagents, they access sites in the IRE and FL for which no information could be obtained with more classical probes.…”

“…However, it is possible to imagine that the IRP regulator binds preferentially to one conformation and eIFs bind preferentially to another conformation, with competition for binding one or other of the proteins being a crucial component of regulation in the cell. Fe-bleomycin and Rh(phen)2phi3+ are bulky complexes which appear to target higher-order interactions (Carter et al, 1990;Chow et al, 1992). Both have been useful in understanding the effect of FL mutation on IRE function.…”