Figure 4 - uploaded by Richard Giegé
Content may be subject to copyright.
Structures of yeast tRNA Asp (left) and yeast tRNA Phe (right). The sites of Fe(II)·BLM A 2 -mediated cleavage are indicated by arrows.
Source publication
Two crystallographically defined tRNAs, yeast tRNAAsp and tRNAPhe, were used as substrates for oxidative cleavage by Fe·bleomycin to facilitate definition at high resolution of the structural
elements in RNAs conducive to bleomycin binding and cleavage. Yeast tRNAAsp underwent cleavage at G45 and U66; yeast tRNAPhe was cleaved at four sites, namely...
Context in source publication
Citations
... 56 Likewise, the cleavage of tRNAs by Fe·BLM occurred disproportionately at the junction between single-and double-strand regions, where the minor groove-like structures might be expected to be relatively wide, 57 and proved to be very sensitive to even small changes in tRNA structure. 58 As noted in Tables 1 and 2, the methylation of cytidine nucleobases in the major groove of hairpin DNAs 7, 10, and 11 led in each case to decreased Fe·BLM binding affinity and a decrease in DNA cleavage products. Because the chemistry of DNA cleavage involves initial abstraction of a H atom from the C-4′ position of deoxyribose in the DNA minor groove, the diminution of DNA binding and cleavage could not have resulted from a direct steric interaction between the methyl group of 5-methylcytidine and Fe·BLM. ...
In a recent study, we have described the enhanced double-strand cleavage of hairpin DNAs by Fe•bleomycin (Fe•BLM) which accompanies increasingly strong binding of this antitumor agent, and suggested that this effect may bear relevance to the mechanism by which BLM mediates its antitumor effects. Because the DNA in tumor cells is known to be hypomethylated on cytidine relative to that in normal cells, it seemed of interest to study the possible effects of methylation status on BLM-induced double-strand DNA cleavage. Three hairpin DNAs found to bind strongly to bleomycin, and their methylated counterparts, were used to study the effect of methylation on bleomycin-induced DNA degradation. Under conditions of limited DNA cleavage, there was a significant overall decrease in the cleavage of methylated hairpin DNAs. Cytidine methylation was found to result in decreased BLM induced cleavage at the site of methylation, and to result in enhanced cleavage at adjacent non-methylated sites. For two of the three hairpin DNAs studied, methylation was accompanied by a dramatic decrease in the binding affinity for Fe•BLM, suggesting the likelihood of diminished double-strand cleavage. The source of the persistent binding of BLM by the third hairpin DNA was identified. Also identified was the probable molecular mechanism for diminished binding and cleavage of the methylated DNAs by BLM. The possible implications of these findings for the antitumor selectivity of bleomycin are discussed.
... 56 Likewise, the cleavage of tRNAs by Fe·BLM occurred disproportionately at the junction between single-and double-strand regions, where the minor groove-like structures might be expected to be relatively wide, 57 and proved to be very sensitive to even small changes in tRNA structure. 58 As noted in Tables 1 and 2, the methylation of cytidine nucleobases in the major groove of hairpin DNAs 7, 10, and 11 led in each case to decreased Fe·BLM binding affinity and a decrease in DNA cleavage products. Because the chemistry of DNA cleavage involves initial abstraction of a H atom from the C-4′ position of deoxyribose in the DNA minor groove, the diminution of DNA binding and cleavage could not have resulted from a direct steric interaction between the methyl group of 5-methylcytidine and Fe·BLM. ...
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 effect 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 ten 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 with which 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 different from those reported previously.
... It is widely used for several malignant tumor types including squamous cell carcinoma, malignant lymphoma, testicular cancer, ovarian cancer, and in combination with other antitumor agents (1). The mechanism of its action is mediated by the generation of reactive oxygen species (ROS), and then degraded DNA and RNA in a selective manner (2,3). The advantage of BLM is that it does not cause suppression of bone marrow and immune system. ...
NC0604, the new antibiotic of the bleomycin family, was isolated from the fermentation broth of Streptomyces verticillus var. pingyangensis n.sp. In this study, we investigated its mechanisms of antitumor action in vitro and in vivo. The results showed that the colony formation inhibition of NC0604 was 2-10 times higher than that of bleomycin to several tumor cell types. In comparison to bleomycin, a better antitumor efficacy of NC0604 was observed in the BALB/c mice loading mouse hepatoma H22 tumors. Flow cytometry assay detected an increase of intracellular reactive oxygen species and arrest at G2/M phase in human hepatoma HepG2 cells after exposure to NC0604. The comet of DNA damage was also observed in the NC0604-treated cells using single cell gel electrophoresis assay. The chromatin condensation appeared in the NC0604-induced apoptotic cells. The activation of apoptotic caspase pathway and increase of apoptosis-modulated protein expression, such as p53, Bax, were also detected by Western blot analysis. Taken together, the anti-tumor actions of NC0604 involve activation of the apoptotic pathway and it is valuable for further drug development.
... Negative supercoiling in plasmid DNA is known to induce a variety of secondary structures, including A-form, cruciforms, and left-handed DNA (41). BLM has been shown to cleave at the junction of tRNA secondary structures (42,43). Therefore, the results from the two independent cleavage studies support multiple binding modes of BLM that can result in ds-DNA cleavage depending on the structures and conformation of the DNA substrates. ...
The bleomycins (BLMs) are a family of natural glycopeptides used clinically as antitumor agents. In the presence of required cofactors (Fe(2+) and O(2)), BLM causes both single-stranded (ss) and double-stranded (ds) DNA damage with the latter thought to be the major source of cytotoxicity. Previous biochemical and structural studies have demonstrated that BLM can mediate ss cleavage through multiple binding modes. However, our studies have suggested that ds cleavage occurs by partial intercalation of BLM's bithiazole tail 3' to the first cleavage site that facilitates its re-activation and re-organization to the second strand without dissociation from the DNA where the second cleavage event occurs. To test this model, a BLM A5 analog (CD-BLM) with beta-cyclodextrin attached to its terminal amine was synthesized. This attachment presumably precludes binding via intercalation. Cleavage studies measuring ss:ds ratios by two independent methods were carried out. Studies using [(32)P]-hairpin technology harboring a single ds cleavage site reveal a ss:ds ratio of 6.7 +/- 1.2:1 for CD-BLM and 3.4:1 and 3.1 +/- 0.3:1 for BLM A2 and A5, respectively. In contrast with BLM A5 and A2, however, CD-BLM mediates ds-DNA cleavage through cooperative binding of a second CD-BLM molecule to effect cleavage on the second strand. Studies using the supercoiled plasmid relaxation assay revealed a ss:ds ratio of 2.8:1 for CD-BLM in comparison with 7.3:1 and 5.8:1, for BLM A2 and A5, respectively. This result in conjunction with the hairpin results suggest that multiple binding modes of a single BLM can lead to ds-DNA cleavage and that ds cleavage can occur using one or two BLM molecules. The significance of the current study to understanding BLM's action in vivo is discussed.
... DNA strand scission is initiated by the selective abstraction of the C4´-H at pyrimidine residues, mainly in 5¢-GC or 5¢-GT sequences [8]. Additionally, RNA cleavage and damage to other cell particles may contribute to their toxicity [9,10]. ...
The solution structure of the Ga(III)-bleomycin A2 complex (GaBLM) has been determined using 2D NMR methods in combination with molecular dynamics calculations. Complete assignment of the amide and amine protons, observation of 80 NOEs and measurement of 15 (3)JH(-H) coupling constants provided us with a well-defined structure using a restrained simulated annealing protocol. On the basis of distance and dihedral angle constraints agreement, along with potential energy considerations, the favored model is a five-coordinate complex with the primary amine of beta-aminoalanine holding the axial position of a distorted tetragonal pyramid. The disaccharide moiety of GaBLM is not a ligand, sharing the same side of the equatorial plane with the axial amine ligand. Titration of the self-complementary oligonucleotide d(CCAGGCCTGG) with GaBLM results in the formation of only one 1:1 complex in slow exchange on the NMR time scale. Our data indicate that the bithiazole moiety intercalates between the C6*G15 and C7*G14 base pairs, in a similar mode to that reported by earlier studies. Structural implications and comparisons to other metallo-bleomycins are discussed.
The tRNA molecules, in addition to translating the genetic code into protein and defining the second genetic code via their aminoacylation by aminoacyl‐tRNA synthetases, act in many other cellular functions and dysfunctions. This article, illustrated by personal souvenirs, covers the history of ~60 years tRNA research in Strasbourg. Typical examples point up how the work in Strasbourg was a two‐way street, influenced by and at the same time influencing investigators outside of France. All along, research in Strasbourg has nurtured the structural and functional diversity of tRNA. It produced massive sequence and crystallographic data on tRNA and its partners, thereby leading to a deeper physicochemical understanding of tRNA architecture, dynamics, and identity. Moreover, it emphasized the role of nucleoside modifications and in the last two decades, highlighted tRNA idiosyncrasies in plants and organelles, together with cellular and health‐focused aspects. The tRNA field benefited from a rich local academic heritage and a strong support by both university and CNRS. Its broad interlinks to the worldwide community of tRNA researchers opens to an exciting future.
