Cleavage of a 23S rRNA pseudoknot by phenanthroline-Cu(II).
Studying the intricate folding of rRNA within the ribosome remains a complex problem. Phenanthroline-Cu(II) complexes cleave phosphodiester bonds in rRNA in specific regions, apparently especially where the rRNA structure is constrained in some fashion. We have introduced phenanthroline-copper complexes into 50S Escherichia coli ribosomal subunits and shown specific cleavages in the regions containing nucleotides 60-66 and 87-100. This specificity of cleavage is reduced when the ribosome is heated to 80 degrees C and reduced to background when the ribosomal proteins are extracted and the cleavage repeated on protein-free 23S rRNA. It has been suggested that nucleotides 60-66 and 87-95 in E.coli 23S rRNA are involved in a putative pseudoknot structure, which is supported by covariance data. The paired cleavages of nearly equal intensity of these two regions, when in the ribosome, may further support the existence of a pseudoknot structure in the 100 region of 23S rRNA. (+info)
DNA triple helix stabilisation by a naphthylquinoline dimer.
We have used DNase I footprinting to examine the effect of a novel naphthylquinoline dimer, designed as a triplex-specific bis-intercalator, on the stability of intermolecular DNA triplexes. We find that this compound efficiently promotes triplex formation between the 9-mer oligonucleotide 5'-TTTTTTCTT and its oligopurine duplex target at concentrations as low as 0.1 microM, enhancing the triplex stability by at least 1000-fold. This compound, which is the first reported example of a triplex bis-intercalator, is about 30 times more potent than the simple monofunctional ligand. (+info)
Structural insight into a quinolone-topoisomerase II-DNA complex. Further evidence for a 2:2 quinobenzoxazine-mg2+ self-assembly model formed in the presence of topoisomerase ii.
Quinobenzoxazine A-62176, developed from the antibacterial fluoroquinolones, is active in vitro and in vivo against murine and human tumors. It has been previously claimed that A-62176 is a catalytic inhibitor of mammalian topoisomerase II that does not stabilize the cleaved complex. However, at low drug concentrations and pH 6-7, we have found that A-62176 can enhance the formation of the cleaved complex at certain sites. Using a photocleavage assay, mismatched sequences, and competition experiments between psorospermin and A-62176, we pinpointed the drug binding site on the DNA base pairs between positions +1 and +2 relative to the cleaved phosphodiester bonds. A 2:2 quinobenzoxazine-Mg2+ self-assembly model was previously proposed, in which one drug molecule intercalates into the DNA helix and the second drug molecule is externally bound, held to the first molecule and DNA by two Mg2+ bridges. The results of competition experiments between psorospermin and A-62176, as well as between psorospermin and A-62176 and norfloxacin, are consistent with this model and provide the first evidence that this 2:2 quinobenzoxazine-Mg2+ complex is assembled in the presence of topoisomerase II. These results also have parallel implications for the mode of binding of the quinolone antibiotics to the bacterial gyrase-DNA complex. (+info)
Intercalation into DNA is not required for inhibition of topoisomerase I by indolocarbazole antitumor agents.
The DNA-intercalating antitumor drug NB-506 is a potent topoisomerase poison currently undergoing phase I/II clinical trials. It contains a planar indolocarbazole chromophore substituted with a glucose residue. Up until now, it was thought that intercalation of the drug into DNA was essential for the stabilization of topoisomerase I-DNA covalent complexes. But, in the present study, we show that a regio-isomeric form of NB-506 has lost its capacity to intercalate into DNA, but remains an extremely potent topoisomerase I poison. The new analogue contains two hydroxyl groups at positions 2,10 instead of positions 1,11 in NB-506. The relocation of the two OH groups reduces considerably the strength of binding to DNA and prevents the drug from intercalating into the DNA double helix. However, the topoisomerase I inhibition capacity of the new analogue remains very high. The two drug isomers are equally potent at maintaining the integrity of the topoisomerase I-DNA covalent complexes, but stimulate cleavage at different sites on DNA. NB-506 stabilizes topoisomerase I preferentially at sites having a pyrimidine (T or C) and a G on the 5' and 3' sides of the cleaved bond, respectively. The 2,10-isomer induces topoisomerase I-mediated cleavage only at TG sites and, thus, behaves exactly as the reference topoisomerase I poison camptothecin. Finally, cytotoxicity measurements performed with a panel of murine and human cancer cell lines reveal that the newly designed drug is considerably (up to 100-fold) more toxic to tumor cells than the parent drug NB-506. We conclude that the DNA-binding and topoisomerase I poisoning activities of NB-506 can be viewed as two separate mechanisms. (+info)
Linkers designed to intercalate the double helix greatly facilitate DNA alkylation by triplex-forming oligonucleotides carrying a cyclopropapyrroloindole reactive moiety.
Triplex-forming oligonucleotides (TFOs) bind sequence-specifically in the major groove of double-stranded DNA. Cyclopropapyrroloindole (CPI), the electrophilic moiety that comprises the reactive subunit of the antibiotic CC-1065, gives hybridization-triggered alkylation at the N-3 position of adenines when bound in the minor groove of double-stranded DNA. In order to attain TFO-directed targeting of CPI, we designed and tested linkers to 'thread' DNA from the major groove-bound TFO to the minor groove binding site of CPI. Placement of an aromatic ring in the linker significantly enhanced the site-directed reaction, possibly due to a 'threading' mechanism where the aromatic ring is intercalated. All of the linkers containing aromatic rings provided efficient alkylation of the duplex target. The linker containing an acridine ring system, the strongest intercalator in the series, gave a small but clearly detectable amount of non-TFO-specific alkylation. An equivalent-length linker without an aromatic ring was very inefficient in DNA target alkylation. (+info)
Bisanthracycline WP631 inhibits basal and Sp1-activated transcription initiation in vitro.
An in vitro transcription assay was used to compare the capacity of the bisintercalating anthracycline WP631 (which displays a remarkably high DNA-binding affinity) and the monointercalating anthracycline daunomycin to inhibit transcription initiation of the adenovirus major late promoter linked to a G-less transcribed DNA template. Both drugs inhibit basal RNA synthesis in a concentration-dependent way, and the drug concentrations required to inhibit transcription initiation are similar. However, in this study WP631 was around 15 times more efficient at inhibiting transcription initiation when used with an adenovirus promoter containing an upstream Sp1-protein binding site under experimental conditions in which the Sp1 protein acted as a transactivator in vitro. The differences in the ability of each drug to inhibit transcription initiation were related to the competition between Sp1 and the drugs for the same binding site. Concentrations of WP631 as low as 60 nM could inhibit the Sp1-activated transcription initiation in vitro. In contrast, the concentration of daunomycin required to inhibit Sp1-activated transcription by 50% was almost the same as the concentration required to inhibit basal transcription. The efficiency of WP631 at displacing Sp1 from its putative binding site was confirmed using gel retardation and footprinting assays. These results are the first unequivocal example of a direct effect of an intercalator on activated transcription initiation. (+info)
Selective nucleosome disruption by drugs that bind in the minor groove of DNA.
Previous studies have shown that drugs which bind in the DNA minor groove reduce the curvature of bent DNA. In this article, we examined the effects of these drugs on the nucleosome assembly of DNA molecules that display different degrees of intrinsic curvature. DAPI (4,6-diamidino-2-phenylindole) inhibited the assembly of a histone octamer onto a 192-base pair circular DNA fragment from Caenorhabditis elegans and destabilized a nucleosome that was previously assembled on this segment. The inhibitory effect was highly selective since it was not seen with nonbent molecules, bent molecules with noncircular shapes, or total genomic DNA. This marked template specificity was attributed to the binding of the ligand to multiple oligo A-tracts distributed over the length of the fragment. A likely mechanism for the effect is that the bound ligand prevents the further compression of the DNA into the minor groove which is required for assembly of DNA into nucleosomes. To further characterize the effects of the drug on chromatin formation, a nucleosome was assembled onto a 322-base pair DNA fragment that contained the circular element and a flanking nonbent segment of DNA. The position of the nucleosome along the fragment was then determined using a variety of nuclease probes including exonuclease III, micrococcal nuclease, DNase I, and restriction enzymes. The results of these studies revealed that the nucleosome was preferentially positioned along the circular element in the absence of DAPI but assembled onto the nonbent flanking sequence in the presence of the drug. DAPI also induced the directional movement of the nucleosome from the circular element onto the nonbent flanking sequence when a nucleosome preassembled onto this template was exposed to the drug under physiologically relevant conditions. (+info)
Protamine-induced condensation and decondensation of the same DNA molecule.
The DNA in sperm and certain viruses is condensed by arginine-rich proteins into toroidal subunits, a form of packaging that inactivates their entire genome. Individual DNA molecules were manipulated with an optical trap to examine the kinetics of torus formation induced by the binding of protamine and a subset of its DNA binding domain, Arg6. Condensation and decondensation experiments with lambda-phage DNA show that toroid formation and stability are influenced by the number of arginine-rich anchoring domains in protamine. The results explain why protamines contain so much arginine and suggest that these proteins must be actively removed from sperm chromatin after fertilization. (+info)