Interaction of the DNA-binding antitumor antibiotics, chromomycin and mithramycin with erythroid spectrin.
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The aureolic acid group of antitumor antibiotics, chromomycin A3 and mithramycin, are well established as transcription inhibitors, which bind reversibly to DNA at and above physiological pH, in the presence of divalent metal ions such as Mg2+. As part of our broad objective to elucidate their intracellular mode of action, other than association with DNA, we studied their interactions with the erythrocyte cytoskeletal protein, spectrin, in the absence and presence of magnesium. Different spectroscopic studies, such as absorbance, fluorescence and CD, have shown that both free chromomycin and mithramycin and their Mg2+ complexes bind to spectrin with an affinity higher than that reported for DNA. The affinity constants for the association of chromomycin and mithramycin (or their Mg2+ complexes) with spectrin are comparable with those for the association of spectrin with other cytoskeletal proteins, for example F-actin, ankyrin, protein 4.1, etc. The nature of the binding of the two antibiotics to spectrin is different. The mode of binding of the antibiotics with spectrin also changes in the presence of Mg2+. The interaction leads to a change in the tertiary structure of the protein. The relevance of the results to our understanding of the mode of action of the antibiotics is discussed. (+info)
A high-resolution structure of a DNA-chromomycin-Co(II) complex determined from pseudocontact shifts in nuclear magnetic resonance.
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BACKGROUND: The drug chromomycin-A(3) binds to the minor groove of DNA and requires a divalent metal ion for complex formation. (1)H, (31)P and (13)C pseudocontact shifts occurring in the presence of a tightly bound divalent cobalt ion in the complex between d(TTGGCCAA)(2) and chromomycin-A(3) have been used to determine the structure of the complex. The accuracy of the structure was verified by validation with nuclear Overhauser enhancements (NOEs) and J-coupling constants not used in the structure calculation. RESULTS: The final structure was determined to 0.7 A resolution. The structure was compared with a structure obtained in an earlier study using NOEs, in order to assess the accuracy of NOEs in giving global structural information for a DNA complex. Although some basic features of the structures agreed, they differed substantially in the fine structural details and in the DNA axis curvature generated by the drug. The distortion of base-pair planarity that was observed in the NOE structure was not seen in our structure. Differences in drug orientation and hydrogen bonding also occurred. The curvature and elongation of the DNA that was obtained previously was not found to occur in our study. CONCLUSIONS: The use of pseudocontact shifts has enabled us to obtain a high-precision global structure of the chromomycin-DNA complex, which provides an accurate template on which to consider targeting minor groove binding drugs. The effect of such binding is not propagated far along the helix but is restricted to a local kink in the axis that reverts to its original direction within four base pairs. (+info)
Biospecific interaction analysis (BIA) of low-molecular weight DNA-binding drugs.
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DNA-binding drugs have been reported to be able to interfere with the activity of transcription factors in a sequence-dependent manner, leading to alteration of transcription. This and similar effects could have important practical applications in the experimental therapy of many human pathologies, including neoplastic diseases and viral infections. The analysis of the biological activity of DNA-binding drugs by footprinting, gel retardation, polymerase chain reaction, and in vitro transcription studies does not allow a real time study of binding to DNA and dissociation of the generated drugs/DNA complexes. The recent development of biosensor technologies for biospecific interaction analysis (BIA) enables monitoring of a variety of molecular reactions in real-time by surface plasmon resonance (SPR). In this study, we demonstrate that molecular interactions between DNA-binding drugs (chromomycin, mithramycin, distamycin, and MEN 10567) and biotinylated target DNA probes immobilized on sensor chips is detectable by SPR technology using a commercially available biosensor. The target DNA sequences were synthetic oligonucleotides mimicking the Sp1, NF-kB, and TFIID binding sites of the long terminal repeat of the human immunodeficiency type 1 virus. The results obtained demonstrate that mithramycin/DNA complexes are less stable than chromomycin/DNA complexes; distamycin binds to both NF-kB and TATA box oligonucleotides, but distamycin/(NF-kB)DNA complexes are not stable; the distamycin analog MEN 10567 binds to the NF-kB mer and the generated drug/DNA complexes are stable. The experimental approach described in this study allows fast analysis of molecular interactions between DNA-binding drugs and selected target DNA sequences. Therefore, this method could be used to identify new drugs exhibiting differential binding activities to selected regions of viral and eukaryotic gene promoters. (+info)
SAF-Box, a conserved protein domain that specifically recognizes scaffold attachment region DNA.
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SARs (scaffold attachment regions) are candidate DNA elements for partitioning eukaryotic genomes into independent chromatin loops by attaching DNA to proteins of a nuclear scaffold or matrix. The interaction of SARs with the nuclear scaffold is evolutionarily conserved and appears to be due to specific DNA binding proteins that recognize SARs by a mechanism not yet understood. We describe a novel, evolutionarily conserved protein domain that specifically binds to SARs but is not related to SAR binding motifs of other proteins. This domain was first identified in human scaffold attachment factor A (SAF-A) and was thus designated SAF-Box. The SAF-Box is present in many different proteins ranging from yeast to human in origin and appears to be structurally related to a homeodomain. We show here that SAF-Boxes from four different origins, as well as a synthetic SAF-Box peptide, bind to natural and artificial SARs with high specificity. Specific SAR binding of the novel domain is achieved by an unusual mass binding mode, is sensitive to distamycin but not to chromomycin, and displays a clear preference for long DNA fragments. This is the first characterization of a specific SAR binding domain that is conserved throughout evolution and has DNA binding properties that closely resemble that of the unfractionated nuclear scaffold. (+info)
Effect of the antitumor antibiotic chromomycin A3 on the humoral immune response in rats.
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Chromomycin A3 (250 mug/kg) suppressed the humoral immune response in rats against sheep erythrocytes when administered 48 h or later after antigenic stimulus. The antibiotic at this dose enhanced immunity when given along with or before antigen administration. The natural heterohemagglutinin levels in rabbits and guinea pigs were not affected by the antibiotic (10 mug/kg per day x 7). (+info)
In vivo topoisomerase II cleavage of the Drosophila histone and satellite III repeats: DNA sequence and structural characteristics.
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We have identified two classes of in vivo topoisomerase II cleavage sites in the Drosophila histone gene repeat. One class co-localizes with DNase I-hypersensitive regions and another novel class maps to a subset of consecutive nucleosome linker sites in the scaffold-associated region (SAR) of the histone gene loop. Prominent topoisomerase II cleavage is also observed in one of the linker regions of the two nucleosomes spanning satellite III, a centromeric SAR-like DNA sequence with a repeat length of 359 bp. At the sequence level, in vivo topoisomerase II cleavage is highly site specific. Comparison of 10 nucleosome linker sites defines an in vivo cleavage sequence whose major characteristic is a prominent GC-rich core. These GC-rich cleavage sites are flanked by extensive arrays of oligo(dA).oligo(dT) tracts characteristic of SAR sequences. Treatment of cells with distamycin selectively enhances cleavage at nucleosome linker sites of the SAR and satellite regions, suggesting that AT-rich sequences flanking cleavage sites may be involved in determining topoisomerase II activity in the cell. These observations provide evidence for the association of topoisomerase II with SARS in vivo. (+info)
Aureolic acids: similar antibiotics with different biosynthetic gene clusters.
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In this issue of Chemistry & Biology, Mendez and colleagues describe the sequence and organization of the chromomycin gene cluster. Unexpectedly, the arrangement is starkly different from the mithramycin biosynthetic cluster, despite similarity in the individual genes and the near identical structures of the two antibiotic aureolic acids. (+info)
Variation in chromosome numbers, CMA bands and 45S rDNA sites in species of Selaginella (Pteridophyta).
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BACKGROUND AND AIMS: Selaginella is the largest genus of heterosporous pteridophytes, but karyologically the genus is known only by the occurrence of a dysploid series of n=7-12, and a low frequency of polyploids. Aiming to contribute to a better understanding of the structural chromosomal variability of this genus, different staining methods were applied in species with different chromosome numbers. METHODS: The chromosome complements of seven species of Selaginella were analysed and, in four of them, the distribution of 45S rDNA sites was determined by fluorescent in situ hybridization. Additionally, CMA/DA/DAPI and silver nitrate staining were performed to investigate the correlation between the 45S rDNA sites, the heterochromatic bands and the number of active rDNA sites. KEY RESULTS: The chromosome numbers observed were 2n=18, 20 and 24. The species with 2n=20 exhibited chromosome complement sizes smaller and less variable than those with 2n=18. The only species with 2n=24, S. convoluta, had relatively large and asymmetrical chromosomes. The interphase nuclei in all species were of the chromocentric type. CMA/DA/DAPI staining showed only a weak chromosomal differentiation of heterochromatic bands. In S. willdenowii and S. convoluta eight and six CMA+ bands were observed, respectively, but no DAPI+ bands. The CMA+ bands corresponded in number, size and location to the rDNA sites. In general, the number of rDNA sites correlated with the maximum number of nucleoli per nucleus. Ten rDNA sites were found in S. plana (2n=20), eight in S. willdenowii (2n=18), six in S. convoluta (2n=24) and two in S. producta (2n=20). CONCLUSIONS: The remarkable variation in chromosome size and number and rDNA sites shows that dramatic karyological changes have occurred during the evolution of the genus at the diploid level. These data further suggest that the two putative basic numbers of the genus, x=9 and x=10, may have arisen two or more times independently. (+info)