Tallimustine lesions in cellular DNA are AT sequence-specific but not region-specific.

Tallimustine (FCE 24517) is an AT-specific alkylating antitumor derivative of distamycin. This study examined levels of tallimustine lesions in intracellular DNA, their sequence- and region-specificity, and the long-range distribution of the drug binding motif. Tallimustine adducts in DNA converted to strand breaks by heating allowed the quantitation of drug lesions. In bulk DNA of intact human leukemia CEM cells, tallimustine formed 0.15 +/- 0.04 and 0.64 +/- 0.18 lesions/kbp at 5 and 50 microM, respectively. These lesions represent monoadducts as no interstrand cross-links or DNA-protein cross-links were detected. Tallimustine adducts in intracellularly treated DNA showed a general preference for sequences with T-tracts, suggesting a propensity for intrinsically bent motifs. Major drug-adducted sites identified by repetitive primer extension, included 5'-TTTTGPu-3' and 5'-TTTTGC-3' motif. Despite the high specificity at the nucleotide level, tallimustine did not differentiate among bulk DNA and three discrete AT-rich regions of genomic DNA examined by quantitative PCR stop assay with lesion frequencies ranging from 0.23 to 0.39 lesions/kbp at 25 microM drug. In comparisons of lesion frequencies and cytotoxicity, tallimustine adducts are approximately 50 times more lethal than relatively nonsequence specific cisplatin adducts but are >100 times less lethal than lesions by an unrelated AT-specific drug, bizelesin. However, the 5'-TTTTGPu-3' motifs targeted by tallimustine are relatively infrequent and scattered throughout the genome. In contrast, the motifs 5'-T(A/T)(4)A-3' motifs targeted by bizelesin, while also infrequent, cluster in defined AT-rich islands. The lack of region-specificity may be the reason tallimustine adducts, despite high AT-specificity at the nucleotide level, are less lethal than region-specific bizelesin adducts.     

A molecular understanding of mitoxantrone-DNA adduct formation: effect of cytosine methylation and flanking sequences

When mitoxantrone is activated by formaldehyde it can form adducts with DNA. These occur preferentially at CpG and CpA sequences and are enhanced 2-3-fold at methylated CpG sequences compared with non-methylated sites. We sought to understand the molecular factors involved in enhanced adduct formation at these methylated sites. This required, first, clarification of factors that contributed to the formation of adducts at CpG sites. For this purpose mass spectrometry of an oligonucleotide duplex (containing a single CpG adduct site) was used to confirm the presence of an additional carbon atom (derived from formaldehyde) on the drug-DNA complex. The effect of 3'-flanking sequences was revealed by electrophoretic analysis of oligonucleotide-drug adducts, and the preferred adduct-forming site was identified as 5'-CGG-3'. Radiolabeled studies of drug-DNA adducts confirmed that the site of attachment involved the exocyclic amino of guanine. Molecular modeling analysis of the relative stability of the intercalated form of mitoxantrone was consistent with observed adduct-forming potential of CG sites with varying flanking sequences. The known preference for adduct formation at methylated CG sites was confirmed by energetics calculations and shown to be due to a shift of equilibrium of the intercalated form of the drug from the major groove (at CG sites) to the minor groove (at methylated CG sites). This increases the relative amount of drug that is located adjacent to the N-2 exocyclic amino of guanine in the minor groove, where covalent linkage is facilitated. These results account for the enhanced covalent binding of mitoxantrone to methylated CG sequences and provide a molecular model of the interactions.     

7-Azido-actinomycin D: a photoaffinity probe of the sequence specificity of DNA binding by actinomycin D

Actinomycin D (ActD) is a DNA-binding antitumor antibiotic that appears to act in vivo by inhibiting RNA polymerase. The mechanism of DNA binding of ActD has attracted much attention because of its strong preference for 5'-dGpdC-3' sequences. Binding is thought to involve intercalation of the tricyclic aromatic phenoxazone ring into a GC step, with the two equivalent cyclic pentapeptide lactone substituents lying in the minor groove and making hydrogen bond contacts with the 2-amino groups of the nearest neighbor guanines. Recent studies have indicated, however, that binding is also influenced by next-nearest neighboring bases. We have examined this higher order specificity using 7-azido-actinomycin-D as a photoaffinity probe, and DNA sequencing techniques to quantitatively monitor sites of covalent photoaddition. We found that GC doublets were strongly preferred only if the 5'-flanking base was a pyrimidine and the 3'-flanking base was not cytosine. In addition we observed a previously unreported preference for binding at a GG doublet in the sequence 5'-TGGG-3'.     

Efficient conjugation and characterization of distamycin-based peptides with selected oligonucleotide stretches

Selected sequences of oligodeoxyribonucleotides (ODNs) have been conjugated efficiently with distamycin-based peptides containing reactive cysteine and oxyamine functionalities at the C-terminus. The conjugation was performed easily within 30-60 min, using individual modified oligonucleotide stretches having sequences of 5'-d(GCTTTTTTCG)-3', 5'-d(GCTATATACG)-3', and 5'-AGCGCGCGCA-3'. Two types of linkages were used for making the covalent connection: (i) a five-membered thiazolidine ring and (ii) an oxime. These distamycin-like polyamide-ODN conjugates were then converted to the corresponding DNA duplexes using complementary oligonucleotide sequences. To elucidate the binding specificity of the distamycin-oligonucleotide conjugates, UV-melting temperature measurements were performed. These studies indicated that the distamycin-ODN conjugate favored binding with the duplex with sequence 5'-d(GCTTTTTTCG)-3' rather than 5'-d(GCTATATACG)-3'. On the other hand, no stabilization of the duplex with sequence 5'-d(AGCGCGCGCA)-3' was observed. UV results also suggest that the thiazolidine and oxime linkages do not significantly influence the process of distamycin binding to the minor groove surface of the DNA duplex. The results obtained from duplex UV-melting studies were further corroborated by a temperature-dependent study of the circular dichroism spectra of the conjugates and a fluorescence displacement titration assay using Hoechst 33258 fluorophore as a competitive binder for the minor groove. All these studies reinforce the fact that the specific stabilization of A/T rich DNA-DNA duplexes by distamycin was preserved upon conjugation with oligonucleotide stretches.     

Enhancement and alteration of bleomycin-catalyzed site-specific DNA cleavage by distamycin A and some minor groove binders.

The effects of compounds which bind in the DNA minor groove of A.T rich sequences, on bleomycin-catalyzed site-specific DNA cleavage were investigated by a DNA sequencing technique. Distamycin A enhanced bleomycin-catalyzed DNA cleavage in G.C rich sequences such as 5'-GGGGC-3' (under scoring; the cleaved nucleotide). The cleavage in such a sequence in the presence of distamycin A was greater than that in the absence of distamycin A by as much as about 100 times. Neither Hoechst 33258, 4',6-diamidino-2-phenylindole (DAPI) nor berenil caused extensive enhancement. The results suggest that the distamycin-induced conformational changes of DNA through interactions other than the DNA minor groove binding in A.T-rich sequences are specifically suitable for the bleomycin action.     

Synthesis of nonlinear DNA-binding peptide with binding specificity determinants close to those of 434 Cro-repressor

Design, synthesis and DNA binding activity of a nonlinear 102 residue peptide are reported. The peptide contains four sequence-specific DNA binding domains of 434 Cro protein. These four domains were linked covalently to a symmetrical carboxyterminal crosslinker that contains four arms each ending with an aliphatic aminogroup. From CD studies we have found that in aqueous buffer in the presence of 20% trifluoroethanol the peptide residues assume alpha helical, beta-sheet and random coiled conformations with an alpha helical content of about 16% at room temperature. The alpha helicity is increased up to 40% in the presence of 40% trifluoroethanol. Upon complex formation between the peptide and DNA a change in the peptide conformation takes place which is consistent with an alpha-beta transition in the DNA binding, helix-turn-helix motif of 434 Cro repressor. Evidently residues present in helices alpha(2) and alpha(3) form a beta hairpin which is inserted in the minor DNA groove. The latter inference is supported by our observations that the peptide can displace minor groove binding antibiotic distamycin A from a complex with poly(dA).poly(dT). As revealed from DNase protection studies the peptide exhibits preferences for binding to operator and pseudooperator sites recognized by 434 Cro repressor. It binds strongly to operator sites OR1, OR2 and OR3 and exhibits a greater affinity for pseudooperator site Op1. From analysis of nucleotide sequences in the strong affinity binding sites for the peptide on DNA a conclusion is drawn that it binds to pseudosymmetrical nucleotide sequences 5'-ACAA(W)nCTGT-3', where W is an arbitrary nucleotide. n is equal to six or seven. In the strongest affinity binding site for the peptide on DNA (Op1) motif 5'-ACAA-3' is replaced by sequence 5'-ACCA-3'. A difference in binding specificity shown by the peptide and 434 Cro protein could be attributed to a flexibility of the connecting chains between DNA-binding domains in the peptide molecule as well as to a replacement of Thr - Ala in the alpha 2 helix. Removal of two residues from the N-terminal end of helix alpha 2 in each of the four DNA binding domains of 434 Cro present in the peptide leads to a loss of binding specificity, although the modified peptide binds to DNA unspecifically.     

Lesion selectivity in blockage of lambda exonuclease by DNA damage.

Various kinds of DNA damage block the 3' to 5' exonuclease action of both E. coli exonuclease III and T4 DNA polymerase. This study shows that a variety of DNA damage likewise inhibits DNA digestion by lambda exonuclease, a 5' to 3' exonuclease. The processive degradation of DNA by the enzyme is blocked if the substrate DNA is treated with ultraviolet irradiation, anthramycin, distamycin, or benzo[a]-pyrene diol epoxide. Furthermore, as with the 3' to 5' exonucleases, the enzyme stops at discrete sites which are different for different DNA damaging agents. On the other hand, digestion of treated DNA by lambda exonuclease is only transiently inhibited at guanine residues alkylated with the acridine mustard ICR-170. The enzyme does not bypass benzo[a]-pyrene diol epoxide or anthramycin lesions even after extensive incubation. While both benzo[a]-pyrene diol epoxide and ICR-170 alkylate the guanine N-7 position, only benzo[a]-pyrene diol epoxide also reacts with the guanine N-2 position in the minor groove of DNA. Anthramycin and distamycin bind exclusively to sites in the minor groove of DNA. Thus lambda exonuclease may be particularly sensitive to obstructions in the minor groove of DNA; alternatively, the enzyme may be blocked by some local helix distortion caused by these adducts, but not by alkylation at guanine N-7 sites.     

7-Azido-Actinomycin D: A Photoaffinity Probe of the Sequence Specificity of DNA Binding by Actinomycin D

Abstract

Actinomycin D (ActD) is a DNA-binding antitumor antibiotic that appears to act in vivo by inhibiting RNA polymerase. The mechanism of DNA binding of ActD has attracted much attention because of its strong preference for 5′-dGpdC-3′ sequences. Binding is thought to involve intercalation of the tricyclic aromatic phenoxazone ring into a GC step, with the two equivalent cyclic pentapeptide lactone substituents lying in the minor groove and making hydrogen bond contacts with the 2-amino groups of the nearest neighbor guanines. Recent studies have indicated, however, that binding is also influenced by next-nearest neighboring bases. We have examined this higher order specificity using 7-azido-actinomycin-D as a photoaffinity probe, and DNA sequencing techniques to quantitatively monitor sites of covalent photoaddition. We found that GC doublets were strongly preferred only if the 5′- flanking base was a pyrimidine and the 3′-flanking base was not cytosine. In addition we observed a previously unreported preference for binding at a GG doublet in the sequence 5′- TGGG-3′.     

Drug-DNA sequence-dependent interactions analysed by electric linear dichroism.

The interactions between 20 drugs and a variety of synthetic DNA polymers and natural DNAs were studied by electric linear dichroism (ELD). All compounds tested, including several clinically used antitumour agents, are thought to exert their biological activities mainly by virtue of their abilities to bind to DNA. The selected drugs include intercalating agents with fused and unfused aromatic structures and several groove binders. To examine the role of base composition and base sequence in the binding of these drugs to DNA, ELD experiments were carried out with natural DNAs of widely differing base composition as well as with polynucleotides containing defined alternating and non-alternating repeating sequences, poly(dA).poly(dT), poly(dA-dT).poly(dA-dT),poly(dG).poly(dC) and poly(dG-dC).poly(dG-dC). Among intercalating agents, actinomycin D was found to be by far the most GC-selective. GC selectivity was also observed with an amsacrine-4-carboxamide derivative and to a lesser extent with methylene blue. In contrast, the binding of amsacrine and 9-aminoacridine was practically unaffected by varying the GC content of the DNAs. Ethidium bromide, proflavine, mitoxantrone, daunomycin and an ellipticine derivative were found to bind best to alternating purine-pyrimidine sequences regardless of their nature. ELD measurements provided evidence for non-specific intercalation of amiloride. A significant AT selectivity was observed with hycanthone and lucanthone. The triphenyl methane dye methyl green was found to exhibit positive and negative dichroism signals at AT and GC sites, respectively, showing that the mode of binding of a drug can change markedly with the DNA base composition. Among minor groove binders, the N-methylpyrrole carboxamide-containing antibiotics netropsin and distamycin bound to DNA with very pronounced AT specificity, as expected. More interestingly the dye Hoechst 33258, berenil and a thiazole-containing lexitropsin elicited negative reduced dichroism in the presence of GC-rich DNA which is totally inconsistent with a groove binding process. We postulate that these three drugs share with the trypanocide 4',6-diamidino-2-phenylindole (DAPI) the property of intercalating at GC-rich sites and binding to the minor groove of DNA at other sites. Replacement of guanines by inosines (i.e., removal of the protruding exocyclic C-2 amino group of guanine) restored minor groove binding of DAPI, Hoechst 33258 and berenil. Thus there are several cases where the mode of binding to DNA is directly dependent on the base composition of the polymer. Consequently the ELD technique appears uniquely valuable as a means of investigating the possibility of sequence-dependent recognition of DNA by drugs.     

Reaction of the antitumor antibiotic CC-1065 with DNA. Location of the site of thermally induced strand breakage and analysis of DNA sequence specificity

CC-1065 is a unique antitumor antibiotic produced by Streptomyces zelensis. The potent cytotoxic effects of this drug are thought to be due to its ability to form a covalent adduct with DNA through N3 of adenine. Thermal treatment of CC-1065-DNA adducts leads to DNA strand breakage. We have shown that the CC-1065 structural modification of DNA that leads to DNA strand breakage is related to the primary alkylation site on DNA. The thermally induced DNA strand breakage occurs between the deoxyribose at the adenine covalent binding site and the phosphate on the 3' side. No residual modification of DNA is detected on the opposite strand around the CC-1065 lesion. Using the early promoter element of SV40 DNA as a target, we have examined the DNA sequence specificity of CC-1065. A consensus sequence analysis of CC-1065 binding sites on DNA reveals two distinct classes of sequences for which CC-1065 is highly specific, i.e., 5'PuNTTA and 5'AAAAA. The orientation of the DNA sequence specificity relative to the covalent binding site provides a basis for predicting the polarity of drug binding in the minor groove. Stereo drawings of the CC-1065-DNA adduct are proposed that are predictive of features of the CC-1065-DNA adduct elucidated in this investigation.     

Use of monoacetyl-4-hydroxyaminoquinoline 1-oxide to probe contacts between guanines and protein in the minor and major grooves of DNA. Interaction of Escherichia coli integration host factor with its recognition site in the early promoter and transposition enhancer of bacteriophage Mu.

Monoacetyl-4-hydroxyaminoquinoline 1-oxide (Ac-HAQO) reacts with DNA to form adducts at the C8- and N2-positions of guanine and with the N6-position of adenine. Only the N2-guanine adduct blocks the 3'-5' exonuclease action of phage T4 DNA polymerase. Piperidine treatment cleaves the DNA at sites bearing C8-guanine adducts. The N2-position of guanine lies in the minor groove of DNA, whereas the C8-position of guanine occupies the major groove. We have taken advantage of these characteristics to employ Ac-HAQO in conjunction with either T4 DNA polymerase or piperidine in a footprinting technique to probe the interaction of the Escherichia coli integration host factor (IHF) with its binding site. We show that when IHF binds to its recognition site both the N2- and C8-positions of guanines are protected from modification by AcHAQO. In addition, the binding of IHF to DNA was prevented when either an N2- or a C8-AQO adduct was present in the binding site. When dimethylsulfate was used as the footprinting reagent, IHF protected against methylation of the N3 position of adenine in the minor groove but not the N7 position of guanine in the major groove. The difference in results obtained with the two reagents is ascribed to their relative sizes. Both DMS and AcHAQO are excluded by IHF from the minor groove, but only the larger AcHAQO molecule is excluded from the major groove.     

Distamycin-induced inhibition of homeodomain-DNA complexes.

The mobility shift assay was used to study the competition of the minor groove binder distamycin A with either an Antennapedia homeodomain (Antp HD) peptide or derivatives of a fushi tarazu homeodomain (ftz HD) peptide for their AT-rich DNA binding site. The results show that distamycin and the homeodomain peptides compete under the conditions: (i) preincubation of DNA with distamycin and subsequent addition of HD peptide; (ii) simultaneous incubation of DNA with distamycin and HD peptide; and (iii) preincubation of DNA with HD peptide and subsequent addition of distamycin. There is also competition when using a peptide which lacks the N-terminal arm of ftz HD that is involved in contacts in the minor groove. It is proposed that the protein's binding affinity is diminished by distamycin-induced conformational changes of the DNA. The feasibility of the propagation of conformational changes upon binding in the minor groove is also shown for the inhibition of restriction endonucleases differing in the AT content of their recognition site and of their flanking DNA sequences. Thus, it is demonstrated that minor groove binders can compete with the binding of proteins in the major groove, providing an experimental indication for the influence of biological activities exerted by DNA ligands binding in the minor groove.     

Assignment of DNA binding sites for 4',6-diamidine-2-phenylindole and bisbenzimide (Hoechst 33258). A comparative footprinting study

DNA binding sites for the minor groove-binding ligands DAPI (4',6-diamidine-2-phenylindole) and Hoechst 33258 (bisbenzimide) have been analysed using DNAase I and micrococcal nuclease footprinting techniques. Both drugs appear to bind to AT-rich regions containing at least four such basepairs. Hoechst 33258 seems to bind relatively poorly to nucleotide sequences containing the alternating step TpA. However, in contrast to DAPI, it can more readily accommodate the presence of guanosine residues at the end of the binding site. We compare the DNA binding sites for DAPI and Hoechst 33258 with those determined for the related minor groove-binding ligands, berenil, netropsin and distamycin A, under comparable conditions, and discuss the importance of using different footprinting probes when analysing drug-DNA interactions.     

Minor groove to major groove, an unusual DNA sequence-dependent change in bend directionality by a distamycin dimer

DNA sequence-dependent conformational changes induced by the minor groove binder, distamycin, have been evaluated by polyacrylamide gel electrophoresis. The distamycin binding affinity, cooperativity, and stoichiometry with three target DNA sequences that have different sizes of alternating AT sites, ATAT, ATATA, and ATATAT, have been determined by mass spectrometry and surface plasmon resonance to help explain the conformational changes. The results show that distamycin binds strongly to and bends five or six AT base pair minor groove sites as a dimer with positive cooperativity, while it binds to ATAT as a weak, slightly anticooperative dimer. The bending direction was evaluated with an in phase A-tract reference sequence. Unlike other similar monomer minor groove binding compounds, such as netropsin, the distamycin dimer changes the directionality of the overall curvature away from the minor groove to the major groove. This distinct structural effect may allow designed distamycin derivatives to have selective therapeutic effects.     

Combinatorial determination of sequence specificity for nanomolar DNA-binding hairpin polyamides

Development of sequence-specific DNA-binding drugs is an important pharmacological goal, given the fact that numerous existing DNA-directed chemotherapeutic drugs rely on the strength and selectivity of their DNA interactions for therapeutic activity. Among the DNA-binding antibiotics, hairpin polyamides represent the only class of small molecules that can practically bind any predetermined DNA sequence. DNA recognition by these ligands depends on their side-by-side amino acid pairings in the DNA minor groove. Extensive studies have revealed that these molecules show extremely high affinity for sequence-directed, minor groove interaction. However, the specificity of such interactions in the presence of a large selection of sequences such as the human genome is not known. We used the combinatorial selection method restriction endonuclease protection, selection, and amplification (REPSA) to determine the DNA binding specificity of two hairpin polyamides, ImPyPyPy-gamma-PyPyPyPy-beta-Dp and ImPyPyPy-gamma-ImPyPyPy-beta-Dp, in the presence of more than 134 million different sequences. These were verified by restriction endonuclease protection assays and DNase I footprinting analysis. Our data showed that both hairpin polyamides preferentially selected DNA sequences having consensus recognition sites as defined by the Dervan pairing rules. These consensus sequences were rather degenerate, as expected, given that the stacked pyrrole-pyrrole amino acid pairs present in both polyamides are unable to discriminate between A.T and T.A base pairs. However, no individual sequence within these degenerate consensus sequences was preferentially selected by REPSA, indicating that these hairpin polyamides are truly consensus-specific DNA-binding ligands. We also discovered a preference for overlapping consensus binding sites among the sequences selected by the hairpin polyamide ImPyPyPy-gamma-PyPyPyPy-beta-Dp, and confirmed by DNase I footprinting that these complex sites provide higher binding affinity. These data suggest that multiple hairpin polyamides can cooperatively bind to their highest-affinity sites.     

Formaldehyde cross-links daunorubicin and DNA efficiently: HPLC and X-ray diffraction studies

Formaldehyde (HCHO) cross-links the anticancer drug daunorubicin (DAU) to DNA efficiently. When DAU is mixed with DNA hexamers, d(CGCGCG) and d(CGTDCG), in the presence of HCHO, stable covalent adducts of DNA are formed, as shown by the HPLC analyses. The major adducts are identical with the materials in the respective crystals which can be readily obtained from the 1:1 mixture of DAU-d(CGCGCG) and DAU-d(CGTDCG) plus HCHO, but not from the solution without HCHO. The high-resolution (1.5 A) X-ray crystal structure of those adducts shows unambiguously that they contain a covalent methylene bridge between the N3' of daunosamine and the N2 of the guanine or 2-aminoadenine. The perfect juxtaposition of the two amino groups in the minor groove of the complex provides a template for an efficient addition of HCHO. The methylene bridge does not perturb the conformation of the drug-DNA complex, when compared to the structure of DAU-d(CGTACG). The results suggest new approaches for synthesizing a new type of potential anticancer drug by attaching a reactive (e.g., alkylating) functional group at the N3' amino position of daunorubicin/doxorubicin. The stable drug-DNA adduct may be useful as probes for other biological studies.     

Distamycin A affects the stability of NF-kappaB p50-DNA complexes in a sequence-dependent manner

The effect of two different DNA minor groove binding molecules, Hoechst 33258 and distamycin A, on the binding kinetics of NF-kappaB p50 to three different specific DNA sequences was studied at various salt concentrations. Distamycin A was shown to significantly increase the dissociation rate constant of p50 from the sequences PRDII (5'-GGGAAATTCC-3') and Ig-kappa B (5'-GGGACTTTCC-3') but had a negligible effect on the dissociation from the palindromic target-kappaB binding site (5'-GGGAATTCCC-3'). By comparison, the effect of Hoechst 33258 on binding of p50 to each sequence was found to be minimal. The dissociation rates for the protein--DNA complexes increased at higher potassium chloride concentrations for the PRDII and Ig-kappaB binding motifs and this effect was magnified by distamycin A. In contrast, p50 bound to the palindromic target-kappaB site with a much higher intrinsic affinity and exhibited a significantly reduced salt dependence of binding over the ionic strength range studied, retaining a K(D) of less than 10 pM at 150 mM KCl. Our results demonstrate that the DNA binding kinetics of p50 and their salt dependence is strongly sequence-dependent and, in addition, that the binding of p50 to DNA can be influenced by the addition of minor groove-binding drugs in a sequence-dependent manner.