Interaction of the human topoisomerase I-DNA complex with oligo-1,3-thiazolecarboxamides and their oligonucleotide conjugates

Nonnatural thiazole-containing oligopeptides (TCOs) bind to the DNA minor groove and inhibit the reaction catalyzed by human topoisomerase I (TopoI). The effect is directly proportional to the number of thiazole monomers in TCO. Several TCOs with three or four thiazole monomers act 3–10 times more efficiently than distamycin A, a natural antibiotic containing pyrrole rings. Additional groups at the N and C termini only slightly affect TopoI inhibition by TCO. The inhibitory effect of TCOs is higher than that of homo-or heterooligopeptides containing imidazole or pyrrole monomers, and the most potent are oligopeptide-oligonucleotide conjugates. The plausible causes of the different effects of distamycin and the nonnatural peptides on DNA relaxation catalyzed by TopoI are discussed.     

Specific interactions of distamycin A and its analogs with (A-T) rich and (G-C) rich duplex regions of DNA and deoxypolynucleotides.

The specific interaction of distamycin A and analogs with DNA's and synthetic deoxypolynucleotide duplexes were studied in detail by means of circular dichroism and the data were analyzed together with viscosity results of several natural DNA's. At low ligand to nucleotide ratio the previously reported specific binding to (A-T) pairs of DNA is verified by a highly favoured interaction with (A-T)-enriched segments of distamycins containing four and five methylpyrrole carboxamide units. At higher distamycin concentration a second specific binding to (G-C) pairs most probably through hydrogen bonding is established. Viscometric results suggest a distamycin-induced local bending of the helix and could support the idea of a preferential alignment of the ligand molecule along only one strand in the groove which differs from the netropsin interaction mechanism. The possibility of an overlapping binding of the oligopeptides in the small groove is discussed.     

Specific protection of DNA by distamycin A, netropsin and bis-netropsins against the action of DNAse I

Interaction of netropsin, distamycin A and a number of bis-netropsins with DNA fragments of definite nucleotide sequence was studied by footprinting technique. The nuclease protection experiments were made at fixed DNA concentration and varying ligand concentrations. The affinity of ligand for a DNA site was estimated from measurements of ligand concentration that causes 50% protection of the DNA site. Distribution pattern of the protected and unprotected regions along the DNA fragment was compared with the theoretically expected arrangement of the ligand along the same DNA. The comparison led us to the following conclusions: 1. Footprinting experiments show that at high levels of binding the arrangement of netropsin molecules along the DNA corresponds closely to the distribution pattern expected from theoretical calculations based on the known geometry of netropsin--DNA complex. However, the observed differences in the affinity of netropsin for various DNA sequences is markedly greater than that expected from theoretical calculations. 2. Netropsin exhibits a greater selectivity of binding than that expected for a ligand with three specific reaction centers associated with the antibiotic amide groups. It binds preferentially to DNA regions containing four or more successive AT pairs. Among 13 putative binding sites for netropsin with four or more successive AT pairs there are 11 strong binding sites and two weaker sites which are occupied at 2 D/P less than or equal to 1/9 and 2 D/P = 1/4, respectively. 3. The extent of specificity manifested by distamycin A is comparable to that shown by netropsin although the molecule of distamycin A contains four rather than three amide groups. At high levels of binding distamycin A occupies the same binding sites on DNA as netropsin does. 4. The binding specificity of bis-netropsins is greater than that of netropsin. Bis-netropsins can bind to DNA in such a way that the two netropsin-like fragments are implicated in specific interaction with DNA base pairs. However, the apparent affinity of bis-netropsins estimated from footprinting experiments is comparable with that of netropsin for the same DNA region. 5. At high levels of binding bis-netropsins and distamycin A (but not netropsin) can occupy any potential site on DNA irrespectively of the DNA sequence. 6. Complex formation with netropsin increases sensitivity to DNase I at certain DNA sites along with the protection effect observed at neighboring sites.     

Human 170 kDa and 180 kDa Topoisomerases II Bind Preferentially to Curved and Left-Handed Linear DNA

Abstract

The binding activities of the 170 kDa and the 180 kDa human topoisomerases II (topo IIa and topo IIβ) to linear DNA fragments with different degrees of curvature were characterized. In gel retardation experiments it was shown that both forms of the enzyme bind preferentially to a curved 287 bp fragment, forming a detectable stable complex. The affinity for straight DNA fragments of similar length is significantly lower. Both a commercially available topo IIa, isolated from placenta, and topo IIα and topo IIβ purified from nuclear extracts of the Namahva lymphoma tissue culture line gave similar results. The effects of double-stranded poly[d(A-T)], poly[d(G-C)], supercoiled plasmid DNA and linear Z-DNA on the topo II- complex with curved DNA were analyzed in competition experiments. The hierarchy of affinities of the 180 kDa topo IIβ for these DNAs has the order: linear left-handed DNA > supercoiled DNA ? curved DNA ? poly[d(A-T)] ? poly[d(G-C)]. The 170 kDa topo IIa binds with similar affinity to curved DNA and linear Z-DNA ? supercoiled DNA ? linear B- DNA The data imply that human topoisomerase II binding is more sensitive to DNA secondary structure than to DNA sequence per se. The ability of the enzyme to preferentially recognize a wide variety of sequences in unusual secondary structures suggests a mode of targeting the enzyme in vivo to regions of high negative supercoiling.     

Synthesis and DNA binding properties of saturated distamycin analogues

A series of saturated heterocyclic analogues of distamycin were prepared and examined. A fluorescent intercalator displacement (FID) assay conducted on p[dA]-p[dT] DNA to obtain C(50) values and a hairpin deoxyoligonucleotide containing an A/T-rich binding site was used to evaluate DNA binding affinity. It is observed that saturated heterocycles greatly reduce the DNA binding relative to distamycin.     

DNA structural variations produced by actinomycin and distamycin as revealed by DNAase I footprinting.

The technique of DNAase I footprinting has been used to investigate preferred binding sites for actinomycin D and distamycin on a 160-base-pair DNA fragment from E. coli containing the tyr T promoter sequence. Only sites containing the dinucleotide step GpC are protected by binding of actinomycin, and all such sites are protected. Distamycin recognizes four major regions rich in A + T residues. Both antibiotics induce enhanced rates of cleavage at certain regions flanking their binding sites. These effects are not restricted to any particular base sequence since they are produced in runs of A and T by actinomycin and in GC-rich sequences by distamycin. The observed increases in susceptibility to nuclease attack are attributed to DNA structural variations induced in the vicinity of the ligand binding site, most probably involving changes in the width of the helical minor groove.     

DNA sequence recognition of thiazole-containing cross-linked polyamides can be favored

The binding ability of cross-linked thiazolated polyamides (containing the base sequence-reading elements thiazole(Th)-pyrrole(Py)-pyr-role(Py) and thiazole(Th)-imidazole(Im)-pyrrol(Py) to various DNA dodecamers has been investigated. CD titration experiments at high salt concentration demonstrate that the dimers with a heptanediyl linker (C7 dimer) show a significantly higher sequence specificity than their corresponding monomers. The dimer of Th-Py-Py primarily prefers binding to pure AT sequences and that of Th-Im-Py to the dodecamer sequences containing a GC pair within the central sequence (e.g. AACGTT). Surprisingly, the sequence binding ability is strongly influenced by the presence of a T-A step: e.g. Th-Py-Py has a similar affinity to the sequences TTTAAA and ATCGTA; likewise Th-Im-Py shows a preference for these sequences. The CD results correlate with footprinting data. Related biochemical studies on the effect of polyamides on DNA gyrase activity in vitro show that the C7 dimers most effectively inhibit the enzyme activity compared with the monomers and the natural reference minor groove binder distamycin. The highest inhibitory potency is observed for the Th-Py-Py-dimer. The role of the T-A step in binding of the cross-linked dimer to the minor groove is discussed in light of the sequence recognition of the TATA box binding protein.     

Assembly of the replication initiation complex on SV40 origin DNA

The assembly of the complex that forms over the simian virus 40 origin to initiate DNA replication is not well understood. This complex is composed of the virus-coded T antigen and three cellular proteins, replication protein A (RPA), DNA polymerase α/primase (pol/prim) and topoisomerase I (topo I) in association with the origin. The order in which these various proteins bind to the DNA was investigated by performing binding assays using biotinylated origin DNA. We demonstrate that in the presence of all four proteins, pol/prim was essential to stabilize the initiation complex from the disruptive effects of topo I. At the optimal concentration of pol/prim, topo I and RPA bound efficiently to the complex, although pol/prim itself was not detected in significant amounts. At higher concentrations less topo I was recruited, suggesting that DNA polymerase is an important modulator of the binding of topo I. Topo I, in turn, appeared to be involved in recruiting RPA. RPA, in contrast, seemed to have little or no effect on the recruitment of the other proteins to the origin. These and other data suggested that pol/prim is the first cellular protein to interact with the double-hexameric T antigen bound to the origin. This is likely followed by topo I and then RPA, or perhaps by a complex of topo I and RPA. Stoichiometric analysis of the topo I and T antigen present in the complex suggested that two molecules of topo I are recruited per double hexamer. Finally, we demonstrate that DNA has a role in recruiting topo I to the origin.     

Influence of template inactivators on the binding of DNA polymerase to DNA

The agents daunomycin, ethidium bromide, distamycin A and cytochrome c inhibit DNA dependent DNA polymerase I (E. coli) reaction competitively to DNA. The influence of these template inactivators on the binding of DNA polymerase to native as well as denatured DNA has been determined by affinity chromatography. Cytochrome c blocks the binding of the enzyme to double-stranded and to single-stranded DNA Sepharose. In contrast to these results daunomycin, ethidium bromide or distamycin A reduce the binding affinity only with denatured DNA Sepharose as matrix. These data are discussed with respect to the modification by template inactivators of the affinity of DNA to the different binding sites of the DNA polymerase.     

Preparation of yuanhuacine and relative daphne diterpene esters from Daphne genkwa and structure-activity relationship of potent inhibitory activity against DNA topoisomerase I

Two new daphne diterpene esters Yuanhuajine (2) and Yuanhuagine (4), together with three known daphne diterpene esters yuanhuacine (1), yuanhuadine (3), and yuanhuapine (5), were isolated and identified from Daphne genkwa, a traditional Chinese medicine. Their structures were elucidated by a combination of UV, IR, MS and NMR ((1)H NMR, (13)C NMR, HSQC, and HMBC) spectra. In order to explore the structure-activity relationship, three compounds 6, 7, and 8 were prepared as three derivatives of 1. Inhibitory activities against DNA topoisomerase I (topo I) were assessed for the compounds 1-8. These compounds, except for 8, exhibited potent inhibitory activities against DNA topo I at IC(50) levels of 11.1-53.4 microM and they are new type of topo I inhibitors bearing different structures compared with the known topo I inhibitors. The agarose-gel electrophoresis experiments showed that the orthoester group of daphne diterpene esters was necessary for the inhibitory activity against DNA topo I, and the inhibition against DNA topo I is probably one of the anti-tumor mechanisms of daphne diterpene esters.     

The binding of topoisomerase I to T antigen enhances the synthesis of RNA-DNA primers during simian virus 40 DNA replication

Topoisomerase I (topo I) is required for the proper initiation of simian virus 40 (SV40) DNA replication. This enzyme binds to SV40 large T antigen at two places, close to the N-terminal end and near the C-terminal end of the helicase domain. We have recently demonstrated that the binding of topo I to the C-terminal site is necessary for the stimulation of DNA synthesis by topo I and for the formation of normal amounts of completed daughter molecules. In this study, we investigated the mechanism by which this stimulation occurs. Contrary to our expectation that the binding of topo I to this region of T antigen provides the proper unwound DNA substrate for initiation to occur, we demonstrate that binding of topo I stimulates polymerase alpha/primase (pol/prim) to synthesize larger amounts of primers consisting of short RNA and about 30 nucleotides of DNA. Topo I binding also stimulates the production of large molecular weight DNA by pol/prim. Mutant T antigens that fail to bind topo I normally do not participate in the synthesis of expected amounts of primers or large molecular weight DNAs indicating that the association of topo I with the C-terminal binding site on T antigen is required for these activities. It is also shown that topo I has the ability to bind to human RPA directly, suggesting that the stimulation of pol/prim activity may be mediated in part through RPA in the DNA synthesis initiation complex.     

The p14ARF Alternate Reading Frame Protein Enhances DNA Binding of Topoisomerase I by Interacting with the Serine 506-Phosphorylated Core Domain

In addition to its well-characterized function as a tumor suppressor, p14ARF (ARF) is a positive regulator of topoisomerase I (topo I), a central enzyme in DNA metabolism and a target for cancer therapy. We previously showed that topo I hyperphosphorylation, a cancer-associated event mediated by elevated levels of the protein kinase CK2, increases topo I activity and the cellular sensitivity to topo I-targeted drugs. Topo I hyperphosphorylation also increases its interaction with ARF. Because the ARF−topo I interaction could be highly relevant to DNA metabolism and cancer treatment, we identified the regions of topo I involved in ARF binding and characterized the effects of ARF binding on topo I function. Using a series of topo I deletion constructs, we found that ARF interacted with the topo I core domain, which encompasses most of the catalytic and DNA-interacting residues. ARF binding increased the DNA relaxation activity of hyperphosphorylated topo I by enhancing its association with DNA, but did not affect the topo I catalytic rate. In cells, ARF promoted the chromatin association of hyperphosphorylated, but not basal phosphorylated, topo I, and increased topo I-mediated DNA nicking under conditions of oxidative stress. The aberrant nicking was found to correlate with increased formation of DNA double-strand breaks, which are precursors of many genome destabilizing events. The results suggest that the convergent actions of oxidative stress and elevated CK2 and ARF levels, which are common features of cancer cells, lead to a dysregulation of topo I that may contribute both to the cellular response to topo I-targeted drugs and to genome instability.     

Simian virus 40 DNA replication is dependent on an interaction between topoisomerase I and the C-terminal end of T antigen

Topoisomerase I (topo I) is needed for efficient initiation of simian virus 40 (SV40) DNA replication and for the formation of completed DNA molecules. Two distinct binding sites for topo I have been previously mapped to the N-terminal (residues 83 to 160) and C-terminal (residues 602 to 708) regions of T antigen. By mutational analysis, we identified a cluster of six residues on the surface of the helicase domain at the C-terminal binding site that are necessary for efficient binding to topo I in enzyme-linked immunosorbent assay and far-Western blot assays. Mutant T antigens with single substitutions of these residues were unable to participate normally in SV40 DNA replication. Some mutants were completely defective in supporting DNA replication, and replication was not enhanced in the presence of added topo I. The same mutants were the ones that were severely compromised in binding topo I. Other mutants demonstrated intermediate levels of activity in the DNA replication assay and were correspondingly only partially defective in binding topo I. Mutations of nearby residues outside this cluster had no effect on DNA replication or on the ability to bind topo I. These results strongly indicate that the association of topo I with these six residues in T antigen is essential for DNA replication. These residues are located on the back edges of the T-antigen double hexamer. We propose that topo I binds to one site on each hexamer to permit the initiation of SV40 DNA replication.     

Interaction of dimeric intercalating dyes with single-stranded DNA.

The unsymmetrical cyanine dye thiazole orange homodimer (TOTO) binds to single-stranded DNA (ssDNA, M13mp18 ssDNA) to form a fluorescent complex that is stable under the standard conditions of electrophoresis. The stability of this complex is indistinguishable from that of the corresponding complex of TOTO with double-stranded DNA (dsDNA). To examine if TOTO exhibits any binding preference for dsDNA or ssDNA, transfer of TOTO from pre-labeled complexes to excess unlabeled DNA was assayed by gel electrophoresis. Transfer of TOTO from M13 ssDNA to unlabeled dsDNA proceeds to the same extent as that from M13 dsDNA to unlabeled dsDNA. A substantial amount of the dye is retained by both the M13 ssDNA and M13 dsDNA even when the competing dsDNA is present at a 600-fold weight excess; for both dsDNA and ssDNA, the pre-labeled complex retains approximately one TOTO per 30 bp (dsDNA) or bases (ssDNA). Rapid transfer of dye from both dsDNA and ssDNA complexes is seen at Na+ concentrations > 50 mM. Interestingly, at higher Na+ or Mg2+ concentrations, the M13 ssDNA-TOTO complex appears to be more stable to intrinsic dissociation (dissociation in the absence of competing DNA) than the complex between TOTO and M13 dsDNA. Similar results were obtained with the structurally unrelated dye ethidium homodimer. The dsDNA- and ssDNA-TOTO complexes were further examined by absorption, fluorescence and circular dichroism spectroscopy. The surprising conclusion is that polycationic dyes, such as TOTO and EthD, capable of bis-intercalation, interact with dsDNA and ssDNA with very similar high affinity.     

Human topoisomerase I promotes initiation of simian virus 40 DNA replication in vitro

Addition of purified human topoisomerase I (topo I) to simian virus 40 T antigen-driven in vitro DNA replication reactions performed with topo I-deficient extracts results in a greater than 10-fold stimulation of completed molecules as well as a more than 3-fold enhancement of overall DNA replication. To further characterize this stimulation, we first demonstrate that bovine topo I but not Escherichia coli topo I can also enhance DNA replication. By using several human topo I mutants, we show that a catalytically active form of topo I is required. To delineate whether topo I influences the initiation or the elongation step of replication, we performed delayed pulse, pulse-chase, and delayed pulse-chase experiments. The results illustrate that topo I cannot promote the completion of partially replicated molecules but is needed from the beginning of the reaction to initiate replication. Competitive inhibition experiments with the topo I binding T antigen fragment 1-246T and a catalytically inactive topo I mutant suggest that part of topo I's stimulation of replication is mediated through a direct interaction with T antigen. Collectively, our data indicate that topo I enhances the synthesis of fully replicated DNA molecules by forming essential interactions with T antigen and stimulating initiation.     

Specific activation of open complex formation at an Escherichia coli promoter by oligo(N-methylpyrrolecarboxamide)s: effects of peptide length and identification of DNA target sites

It has previously been shown that open complex formation at a promoter containing a block substitution of nonalternating A-T sequences in the spacer DNA separating the contacted -10 and -35 regions could be accelerated by distamycin. No stimulation was observed at a promoter with a substitution of alternating A-T base pairs in the same region or at the promoter with wild-type spacer. Here we compare the effect of distamycin [tris(N-methylpyrrolecarboxamide), formally a P3] with that of its extended homologues P4, P5, and P6. It is found that the stimulatory potential of these synthetic oligopeptides which bind in the minor groove of DNA ranks in the order P4 greater than (distamycin, P5) greater than P6. The interaction of these peptides with the three promoters was studied by monitoring the positions of the promoter DNA protected from MPE-Fe(II) cleavage in the presence of different concentrations of ligand. The results suggest that a higher affinity of oligopeptide for the spacer DNA than for the -10 and/or -35 region is a necessary, but not sufficient condition for stimulation. Different patterns of protected DNA regions are seen with each of the three promoters; with distamycin, P4, and P5, a unique arrangement of protected regions is observed for the variant containing nonalternating A-T base pairs in its spacer DNA. These data support the hypothesis that differences in the ways the minor-groove binders interact with each of the promoter variants account for the observed differential stimulation. We further postulate that it is a ligand-induced structural change in the nonalternating A-T DNA which is responsible for the activation of open complex formation at the promoter containing this substitution.     

Use of the antibiotics actinomycin D and distamycin A to limit the action of restriction endonucleases and to map DNA

It is shown that distamycin A and actinomycin D protect the recognition sites of certain restriction endonucleases from the attack by these nucleases due to specific interaction of these antibiotics with double-stranded DNA. Distamycin A protects A-T containing sites and actinomycin G-C rich sites. Among Hind II recognition sites which have alternative structure (GTPyPuAC) distamycin A protects only Hpa I similar sites (GTTAAC). It is shown with several restriction endonucleases that antibiotic action depends on the nucleotide sequences in the recognition sites and in their closest environment. Proper concentrations of antibiotic give rise to larger fragments. Use of both distamycin A and actinomycin D allows to obtain a set of overlapping fragments. The data obtained with various DNAs and restriction endonucleases allow to conclude that these antibiotics may be useful for DNA mapping and for preparation of large functional fragments of DNA.