Determination of the number and location of the manganese binding sites of DNA quadruplexes in solution by EPR and NMR

The paramagnetic metal ion Mn2+ has been used to probe the electrostatic potentials of a DNA quadruplex that has two quartets with an overall fold of the chair type. A quadruplex with a basket type structure has also been examined. The binding of the paramagnetic ion manganese to these quadruplex DNAs has been investigated by solution state electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) spectroscopies. The EPR results indicate that the DNA aptamer, d(GGTTGGTGTGGTTGG), binds two manganese ions and that the binding constants for each of these sites is approximately 10(5) M-1. The NMR results indicate that the binding sites of the manganese are in the narrow grooves of this quadruplex DNA. The binding sites of the DNA quadruplex formed by dimers of d(GGGGTTTTGGGG) which forms a basket structure are also in the narrow groove. These results indicate that the close approach of phosphates in the narrow minor grooves of the quadruplex structures provide strong binding sites for the manganese ions and that EPR and NMR monitoring of manganese binding can be used to distinguish between the different types of quadruplex structures.     

Enzymatic and chemical footprinting of anthracycline antitumor antibiotics and related saccharide side chains

DNase I and three DNA chemical footprinting agents were used to compare the DNA binding properties of the anthracycline antitumor antibiotics daunomycin, aclacinomycin A, and ditrisarubicin B. These anthracyclines contain a tetracyclic chromophore which intercalates into DNA and a monosaccharide, trisaccharide, and two trisaccharide side chains, respectively. These side chains consist of between one and three 2,6-dideoxy, 1,4-diaxially linked sugars. Three chemical probes, fotemustine, dimethyl sulfate, 4-(2'-bromoethyl)phenol, and the enzymic probe DNase I were used in the footprinting experiments. The chemical probes provided a clear picture of the binding pattern at 37 degrees C and more detailed information than that obtained using the standard DNase I footprinting assay. All three anthracyclines showed preferred binding to 5'-GT-3' sequences in both the chemical and enzymatic footprinting. DNase I footprinting showed that the number of base pairs of DNA protected from cleavage increased with the number of saccharide groups present at particular sites and is consistent with DNA binding of the saccharide side chains. Alkylation of runs of guanine by fotemustine was inhibited by all three anthracyclines, while alkylation by dimethyl sulfate was enhanced for most guanines. The probe 4-(2'-bromoethyl)phenol showed that all three anthracyclines completely protected all of the adenines in the minor groove from alkylation, and enhanced major groove guanine alkylation was observed with aclacinomycin A, daunomycin, and, to a much lesser extent, ditrisarubicin B. These results are consistent with intercalation of the aglycone ring and binding of the rigid, hydrophobic saccharide side chains in the minor groove. Footprinting of four methyl glycosides related to the anthracyclines showed no evidence of DNA binding with any of the agents studied.     

Altering the DNA-binding specificity of Mu transposase in vitro

We describe the isolation of a variant of Mu transposase (MuA protein) which can recognize altered att sites at the ends of Mu DNA. No prior knowledge of the structure of the DNA binding domain or its mode of interaction with att DNA was necessary to obtain this variant. Protein secondary structure programs initially helped target mutations to predicted helical regions within a subdomain of MuA demonstrated to harbor att DNA binding activity. Of the 54 mutant positions examined, only two showed decreased affinity for att DNA, while eight others affected assembly of the Mu transpososome. A variant impaired in DNA binding [MuA(R146V)], and predicted to be in the recognition helix of an HTH motif, was challenged with altered att sites created from degenerate oligonucleotides to select for novel DNA binding specificity. DNA sequences bound to MuA(R146V) were detected by gel-retardation, and following several steps of PCR amplification/enrichment, were identified by cloning and sequencing. The strategy allowed recovery of an altered att site for which MuA(R146V) showed higher affinity than for the wild-type site, although this site was bound by wild-type MuA as well. The altered association between MuA(R146V) and an altered att site target was competent in transposition. We discuss the strengths and limitations of this methodology, which has applications in dissecting the functional role of specific protein-DNA associations.     

Minor groove-directed and intercalative ligand-DNA interactions in the poisoning of human DNA topoisomerase I by protoberberine analogs

Spectroscopic, calorimetric, DNA cleavage, electrophoretic, and computer modeling techniques have been employed to characterize the DNA binding and topoisomerase poisoning properties of three protoberberine analogs, 8-desmethylcoralyne (DMC), 5,6-dihydro-8-desmethylcoralyne (DHDMC), and palmatine, which differ in the chemical structures of their B- and/or D-rings. DNA topoisomerase-mediated cleavage assays revealed that these compounds were unable to poison mammalian type II topoisomerase. By contrast, the three protoberberine analogs poisoned human topoisomerase I according to the following hierarchy: DHDMC > DMC > palmatine. DNA binding by all three protoberberine analogs induced negative flow linear dichroism signals as well as unwinding of the host duplex. These two observations are consistent with an intercalative mode of protoberberine binding to duplex DNA. However, a comparison of the DNA binding properties for DMC and DHDMC, which differ only by the state of saturation at the 5,6 positions of the B-ring, revealed that the protoberberine analogs do not "behave" like classic DNA intercalators. Specifically, saturation of the 5-6 double bond in the B-ring of DMC, thereby converting it to the DHDMC molecule, was associated with enhanced DNA unwinding as well as a reversal of DNA binding preference from a DNA duplex with an inaccessible or occluded minor groove {poly[d(G-C)]2} to DNA duplexes with accessible or unobstructed minor grooves {poly[d(A-T)]2 and poly[d(I-C)]2}. In addition, a comparison of the DNA binding properties for DHDMC and palmatine revealed that transferring the 11-methoxy moiety on the D-ring of DHDMC to the 9 position, thereby converting it to palmatine, was associated with a reduction in binding affinity for both duplexes with unobstructed minor grooves as well as for duplexes with occluded minor grooves. These DNA binding properties are consistent with a "mixed-mode" DNA binding model for protoberberines in which a portion of the ligand molecule intercalates into the double helix, while the nonintercalated portion of the ligand molecule protrudes into the minor groove of the host duplex, where it is thereby available for interactions with atoms lining the floor and/or walls of the minor groove. Furthermore, saturation at the 5,6 positions of the B-ring, which causes the A-ring to be tilted relative to the plane formed by rings C and D, appears to stabilize the interaction between the host duplex and the minor groove-directed portion of the protoberberine ligand. Computer modeling studies on the DHDMC-poly[d(A-T)]2 complex suggest that this interaction may involve van der Waals contacts between the ligand A-ring and backbone sugar atoms lining the minor groove of the host duplex. The hierarchy of topoisomerase I poisoning noted above suggests that this minor groove-directed interaction may play an important role in topoisomerase I poisoning by protoberberine analogs. In the aggregate, our results presented here, coupled with the recent demonstration of topoisomerase I poisoning by minor groove-binding terbenzimidazoles [Sun, Q., Gatto, B., Yu, C., Liu, A. , Liu, L. F., & LaVoie, E. J. (1995) J. Med. Chem. 38, 3638-3644], suggest that minor groove-directed ligand-DNA interactions may be of general importance in the poisoning of topoisomerase I.     

Determination of the number and location of the manganese binding sites of DNA quadruplexes in solution by EPR and NMR in the presence and absence of thrombin

The interaction of a DNA quadruplex with thrombin has been studied by first determining the sites of manganese binding to the quadruplex in the absence of thrombin. This has been followed by determining if the interactions with thrombin displace the bound manganese. A different DNA quadruplex has also been studied as a control. The refined solution structures of two DNA quadruplexes have been used to predict the electrostatic potentials of these DNAs. The calculated electrostatic potentials have been used to predict the locations of the binding sites of the paramagnetic ion manganese to these DNAs. The enhanced relaxation of DNA protons due to the binding of the paramagnetic metal ion Mn2+ has been used to experimentally determine the locations of the binding sites. The NMR results and the predictions based on the electrostatic potentials both place the binding sites of the manganese in the narrow grooves of these quadruplex DNAs. The predicted locations are spatially close to those experimentally observed, and the predicted and experimental locations also have similar electrostatic potential energy. These results have allowed a validation of the predictions of electrostatic potentials from structure. The 15mer quadruplex has two strong Mn2+ binding sites with one in each narrow groove. Both Mn2+ are released when the 15mer is complexed with thrombin, indicating that both narrow grooves are involved in the 15mer-thrombin interactions. The dimer quadruplex has a different structural motif than the 15mer and the presence of thrombin does not appreciably affect its interactions with Mn2+.     

Mono- and dysfunctional nitrogen mustard analogues of the DNA minor groove binder pibenzimol. Synthesis, cytotoxicity and interaction with DNA

Two series of mono- and dysfunctional aniline mustards linked to a bisbenzimidazole minor groove binder have been prepared using a new method (polyphosphate ester-mediated direct coupling of appropriate mustard acids with a preformed advanced phenylenediamine intermediate). As the linker chain attaching the mustard was lengthened the binding site size of the compounds to calf thymus DNA remained essentially constant at 2.6 nucleotides, but reversible binding strength declined by a factor of 2. Analogues with longer linker chains alkylated DNA much more rapidly than those with shorter chains, consistent with the electronic factors. The short chain analogues also failed to alkylate a 120 bp HindIII to Bg/II fragment of the gpt gene, as measured by gel electrophoresis cleavage assays. The longer chain analogues (both mono- and dysfunctional mustards) showed patterns of DNA alkylation that varied with chain length. In particular, while most compounds showed substantial N7 alkylation at many guanine residues, the analogue with a (CH2)3 linker chain showed strong alkylation at adenine sites in poly-AT regions. For the longer chain analogues, the bifunctional mustards were substantially (10- to 20-fold) more cytotoxic than the corresponding monofunctional analogues.     

Minor groove binding of a bis-quaternary ammonium compound: the crystal structure of SN 7167 bound to d(CGCGAATTCGCG)2

The X-ray crystal structure of the complex between the synthetic antitumour and antiviral DNA binding ligand SN 7167 and the DNA oligonucleotide d(CGCGAATTCGCG)2 has been determined to an R factor of 18.3% at 2.6 A resolution. The ligand is located within the minor groove and covers almost 6 bp with the 1-methylpyridinium ring extending as far as the C9-G16 base pair and the 1-methylquinolinium ring lying between the G4-C21 and A5-T20 base pairs. The ligand interacts only weakly with the DNA, as evidenced by long range contacts and shallow penetration into the groove. This structure is compared with that of the complex between the parent compound SN 6999 and the alkylated DNA sequence d(CGC[e6G]AATTCGCG)2. There are significant differences between the two structures in the extent of DNA bending, ligand conformation and groove binding.     

Analysis of co-crystal structures to identify the stereochemical determinants of the orientation of TBP on the TATA box

Possible stereochemical determinants of the orientation of TBP on the TATA box are discussed using the crystal coordinates of TBP-TATA complexes, which have been determined by other groups. The C-terminal half of the TBP beta-sheet interacts with the TATA site of the DNA, and the N-terminal half with the A-rich site, so that the two sites with distinct curvatures produce a unique fit. Although chemical contacts take place between one side of the beta-sheet and the DNA minor groove, the interaction seems to be facilitated indirectly by the characteristics of the other side of the beta-sheet and the DNA major groove. Thus, Ala71, Leu162 and Pro190 differentiate the curvature of the beta-sheet in the N- and C-halves. The methyl positions in the DNA major groove modulate the bendability of the two DNA sites by using differences in the rolling capacity of TA and AT compared with PyT, and in the shifting capacity of AT compared with TT. The deformations of the first steps (TA and PyT) in the two sites are the largest and thus are important for the overall bending of the DNA. The differences between the two DNA sites are greatest at the second steps (AT and TT) and so these are important for determining the orientation of TBP.     

1H NMR study of [d(GCGATCGC)]2 and its interaction with minor groove binding 4',6-diamidino-2-phenylindole

Two-dimensional NMR spectroscopy has been applied to study the solution binding of 4',6-diamidino-2-phenylindole (DAPI) to synthetic DNA duplex [d(GCGATCGC)]2. The structure of the complex at a molar ratio of 1:1 drug:duplex has been investigated. NMR results indicate that DAPI binds selectively in the minor groove of the DNA region containing only two A:T base pairs. The results disagree with conclusions drawn from footprinting experiments and show that the presence of the G3NH2 group in the minor groove does not prevent the binding. A molecular model is proposed that closely resembles the crystal structure previously published for the interaction of DAPI with the dodecamer [d(CGCGAATTCGCG)]2, containing four A:T base pairs in the binding site. In this model, DAPI lies in the minor groove, nearly isohelical, with its aromatic rings adjacent to H4' protons of T5 and C6 deoxyribose and the NH indole group oriented toward the DNA axis. The binding does not perturb the B-type conformation of the duplex, and the DNA oligomer conserves its 2-fold symmetry, indicating that fast exchange dynamics exist between the two stereochemically equivalent binding sites of the palindromic sequence. The binding constant and the exchange rate between free and bound species were also measured by NMR spectroscopy.     

Synthesis, cytotoxicity and antitumor activity of some new simplified pyrazole analogs of the antitumor agent CC-1065. Effect of an hydrophobic group on antitumor activity

Three simplified pyrazole analogs (7-9) of the antitumor agents CC-1065, were synthesized. In in vitro assays, against L1210 cell lines all derivatives showed a cytotoxicity in a pM range, values close to the natural target compound (+)-CC-1065. In in vivo tests, against disseminate L1210 leukemia cells, synthesized compounds showed a good potency (O.D. 300 micrograms/Kg) but no activity. These observations further validate the effect of the hydrophilic and/or hydrophobic characteristics of the substituents present on the molecules, confirming the relevance of this phenomena on in vivo activity. In fact in this case the increase of hydrophobic characteristics of the molecules produce the loss of activity, probably due to a worse bioavailability of the drugs in animals.     

Structural characterization of a DNA duplex modeled on a DNA:RNA hybrid of the polypurine tract recognized by a reverse transcriptase

The structure of d(TTAAAAGAAAAGGG):d(CCCTTTTCTTTTAA) has been characterized by NMR. The minor grooves of the two dA-tracts are suggested to be rather narrow, and the portion linking the two dA tracts exhibits a slightly deviated structure from a standard B DNA, in order to maintain the narrowness of the minor groove. The structure of the dG-tract is also slightly deviated. Additionally, specific broadening of resonances is observed for the residues at or near the junction between the dA-tract and the dG-tract, suggesting local structural polymorphology.     

On the importance of van der Waals interaction in the groove binding of DNA with ligands: restrained molecular dynamics study

Comparison of interaction energy between an oligonucleotide and a DNA-binding ligand in the minor and major groove modes was made by use of restrained molecular dynamics. Distortion in DNA was found for the major groove mode whereas less significant changes for both ligand and DNA were detected for the minor groove binding after molecular dynamics simulation. The conformation of the ligand obtained from the major groove modes resembles that computed with the ligand soaked in water. The van der Waals contact energy was found to be as significant as electrostatic energy and more important for difference in binding energy between these two binding modes. The importance of van der Waals force in groove binding was supported by computations on the complex formed by the repressor peptide fragment from the bacteriophage 434 and its operator oligonucleotide.     

Synthesis, DNA binding, cytotoxicity and sequence specificity of a series of imidazole-containing analogs of the benzoic acid mustard distamycin derivative tallimustine containing an alkylating group at the C-terminus

In an attempt to produce additional alkylation and crosslinking in the minor groove of DNA, imidazole-containing analogs of distamycin were synthesized with benzoic acid mustard (BAM) and methoxyaziridinyl moieties present at the N- and C-termini, respectively. Analogs 1a-c differed in the number of methylene units (2-4 respectively) between the C-terminal carbonyl group and the methoxyaziridinyl moiety. DNA binding affinity to several polynucleotides decreased with increasing linker length, whereas DNA interstrand crosslinking ability, as measured by a plasmid gel based assay, increased. The in vitro cytotoxicity in human chronic myeloid leukemia K562 cells and the panel of human tumor cell lines at the National Cancer Institute decreased with increasing number of methylene units, and no increase in cytotoxicity was observed over compound AR-1-122 which did not contain the methoxyaziridinyl moiety. 1a-c had the same sequence selectivity of alkylation as AR-1-122, showing alkylation only at 5'-TTTTGPu sequences. The relative binding to these sequences decreased with increasing number of methylene units. The addition of a methoxyaziridinyl moiety in this group of imidazole and BAM-containing compounds can, therefore, increase crosslinking ability to naked DNA but this does not result in an increase in cytotoxicity. In contrast the cytotoxicity was related to their ability to produce sequence specific alkylation at 5'-TTTTGPu sequences.     

A mechanism for the entrapment of DNA at an air-water interface

Addition of the intercalating dye quinacrine to a low ionic strength solution of DNA in quantities sufficient to saturate the high affinity sites in the DNA will result in the accumulation of the DNA at the solution interface. This entrapment of DNA at the air-water interface has been assayed by the adsorption of DNA to untreated carbon-coated electron microscope grids touched to the solution surface. Other intercalating dyes can also bring about this entrapment, if they possess a side arm large enough to occupy one of the DNA grooves when the dye is intercalated into the DNA. The extension and unwinding of the DNA helix brought about by the intercalating chromophore of the dye molecules are not requirements for the entrapment process. Spermidine, a simple polyamine that will bind to the DNA minor groove but that has no intercalating chromophore, was found to bring about this entrapment. Even simple mono- and divalent cations in the absence of the above ligands were found to promote a low level of surface entrapment. A model for the entrapment of DNA at the air-water interface is proposed in which one (or both) of the hydrophobic grooves of the DNA becomes a surface-active agent as a consequence of the association of various ligands and charge neutralization.     

Role of protein-induced bending in the specificity of DNA recognition: crystal structure of EcoRV endonuclease complexed with d(AAAGAT) + d(ATCTT)

The crystal structure of EcoRV endonuclease has been determined at 2. 1 A resolution complexed to two five-base-pair DNA duplexes each containing the cognate recognition half-site. The highly localized 50 degrees bend into the major groove seen at the center TA-step of the continuous GATATC site is preserved in this discontinuous DNA complex lacking the scissile phosphates. Thus, this crystal structure provides evidence that covalent constraints associated with a continuous target site are not essential to enzyme-induced DNA bending, even when these constraints are removed directly at the locus of the bend. The scissile phosphates are also absent in the crystal structure of EcoRV bound to the non-specific site TCGCGA, which shows a straight B-like conformation. We conclude that DNA bending by EcoRV is governed only by the sequence and is not influenced by the continuity of the phosphodiester backbone. Together with other data showing that cleavable non-cognate sites are bent, these results indicate that EcoRV bends non-cognate sites differing by one or two base-pairs from GATATC, but does not bend non-specific sites that are less similar. Structural and thermodynamic considerations suggest that the sequence-dependent energy cost of DNA bending is likely to play an important role in determining the specificity of EcoRV. This differential cost is manifested at the binding step for bent non-cognate sequences and at the catalytic step for unbent non-specific sequences.