Out-of-shape DNA minor groove binders: induced fit interactions of heterocyclic dications with the DNA minor groove

DB921 and DB911 are benzimidazole-biphenyl isomers with terminal charged amidines. DB911 has a central meta-substituted phenyl that gives it a shape similar to those of known minor groove binding compounds. DB921 has a central para-substituted phenyl with a linear conformation that lacks the appropriate radius of curvature to match the groove shape. It is thus expected that DB911, but not DB921, should be an effective minor groove binder, but we find that DB921 not only binds in the groove but also has an unusually high binding constant in SPR experiments (2.9 x 10(8) M(-)(1), vs 2.1 x 10(7) M(-)(1) for DB911). ITC thermodynamic analysis with an AATT sequence shows that the stronger binding of DB921 is due to a more favorable binding enthalpy relative to that of DB911. CD results support minor groove binding for both compounds but do not provide an explanation for the binding of DB921. X-ray crystallographic analysis of DB921 bound to AATT shows that an induced fit structural change in DB921 reduces the twist of the biphenyl to complement the groove, and places the functional groups in position to interact with bases at the floor of the groove. The phenylamidine of DB921 forms indirect contacts with the bases through a bound water. The DB921-water pair forms a curved binding module that matches the shape of the minor groove and provides a number of strong interactions that are not possible with DB911. This result suggests that traditional views of compound curvature required for minor groove complex formation should be reevaluated.     

Crystal structures of nucleosome core particles in complex with minor groove DNA-binding ligands

We determined the crystal structures of three nucleosome core particles in complex with site-specific DNA-binding ligands, the pyrrole-imidazole polyamides. While the structure of the histone octamer and its interaction with the DNA remain unaffected by ligand binding, nucleosomal DNA undergoes significant structural changes at the ligand-binding sites and in adjacent regions to accommodate the ligands. Our findings suggest that twist diffusion occurs over long distances through tightly bound nucleosomal DNA. This may be relevant to the mechanism of ATP-dependent and spontaneous nucleosome translocation, and to the effect of bound factors on nucleosome dynamics.     

New complex of post-activated neocarzinostatin chromophore with DNA: bulge DNA binding from the minor groove

Neocarzinostatin (NCS-chrom), a natural enediyne antitumor antibiotic, undergoes either thiol-dependent or thiol-independent activation, resulting in distinctly different DNA cleavage patterns. Structures of two different post-activated NCS-chrom complexes with DNA have been reported, revealing strikingly different binding modes that can be directly related to the specificity of DNA chain cleavage caused by NCS-chrom. The third structure described herein is based on recent studies demonstrating that glutathione (GSH) activated NCS-chrom efficiently cleaves DNA at specific single-base sites in sequences containing a putative single-base bulge. In this structure, the GSH post-activated NCS-chrom (NCSi-glu) binds to a decamer DNA, d(GCCAGAGAGC), from the minor groove. This binding triggers a conformational switch in DNA from a loose duplex in the free form to a single-strand, tightly folded hairpin containing a bulge adenosine embedded between a three base pair stem. The naphthoate aromatic moiety of NCSi-glu intercalates into a GG step flanked by the bulge site, and its substituent groups, the 2-N-methylfucosamine carbohydrate ring and the tetrahydroindacene, form a complementary minor groove binding surface, mostly interacting with the GCC strand in the duplex stem of DNA. The bulge site is stabilized by the interactions involving NCSi-glu naphthoate and GSH tripeptide. The positioning of NCSi-glu is such that only single-chain cleavage via hydrogen abstraction at the 5'-position of the third base C (which is opposite to the putative bulge base) in GCC is possible, explaining the observed single-base cleavage specificity. The reported structure of the NCSi-glu-bulge DNA complex reveals a third binding mode of the antibiotic and represents a new family of minor groove bulge DNA recognition structures. We predict analogue structures of NCSi-R (R = glu or other substituent groups) may be versatile probes for detecting the existence of various structures of nucleic acids. The NMR structure of this complex, in combination with the previously reported NCSi-gb-bulge DNA complex, offers models for specific recognition of DNA bulges of various sizes through binding to either the minor or the major groove and for single-chain cleavage of bulge DNA sequences.     

The role of minor groove functional groups in DNA hydration

Here we describe the crystal structure of modified [d(CGCGAATTCGCG)]₂ refined to 2.04 Å. The modification, which affects only the two thymines at the central ApT step, involves isosteric removal of the 2-keto oxygen atoms and substitution of the N1 nitrogen with carbon. The crystal structure reveals the ability of this modified thymine to effectively base pair with adenine in [d(CGCGAAtTCGCG)]₂. The structure also suggests that the minor groove ‘spine of hydration’ is destabilized but essentially intact.     

Accomodation of S-cis-tamoxifen-N(2)-guanine adduct within a bent and widened DNA minor groove

The non-steroidal anti-estrogen tamoxifen [TAM] has been in clinical use over the last two decades as a potent adjunct chemotherapeutic agent for treatment of breast cancer. It has also been given prophylactically to women with a strong family history of breast cancer. However, tamoxifen treatment has also been associated with increased endometrial cancer, possibly resulting from the reaction of metabolically activated tamoxifen derivatives with cellular DNA. Such DNA adducts can be mutagenic and the activities of isomeric adducts may be conformation-dependent. We therefore investigated the high resolution NMR solution conformation of one covalent adduct (cis-isomer, S-epimer of [TAM]G) formed from the reaction of tamoxifen [TAM] to N(2)-of guanine in the d(C-[TAM]G-C).d(G-C-G) sequence context at the 11-mer oligonucleotide duplex level. Our NMR results establish that the S-cis [TAM]G lesion is accomodated within a widened minor groove without disruption of the Watson-Crick [TAM]G. C and flanking Watson-Crick G.C base-pairs. The helix axis of the bound DNA oligomer is bent by about 30 degrees and is directed away from the minor groove adduct site. The presence of such a bulky [TAM]G adduct with components of the TAM residue on both the 5'- and the 3'-side of the modified base could compromise the fidelity of the minor groove polymerase scanning machinery.     

Proton equilibria in the minor groove of DNA.

Poisson-Boltzmann calculations by Pack and co-workers suggest the presence of regions of increased hydrogen ion density in the grooves of DNA. As an experimental test of this prediction, we have attached proton-sensitive probes, with variable linker lengths, to random-sequence DNA at G sites in the minor groove. The amino groups of beta-alanine, gamma-aminobutyric acid (GABA), and epsilon-aminocaproic acid have been coupled at pH 5, via a formaldehyde link, to the exocyclic amino group of guanine, utilizing a reaction that has been extensively investigated by Hanlon and co-workers. The resulting adducts at pH 5 retained duplex B form but exhibited typical circular dichroism (CD) changes previously shown to be correlated with the presence of a net positive charge in the minor groove. Increases in the solvent pH reversed the CD spectral changes in a manner suggesting deprotonation of the carboxylic acid group of the adduct. These data were used to calculate an apparent pK(a) for the COOH. The pK(a) was increased by 2.4 units for beta-alanine, by 1.7 units for GABA, and by 1.5 units for epsilon-amino caproic acid, relative to their values in the free amino acid. This agrees well with Poisson-Boltzmann calculations and the energy minimization of the structures of the adducts that place the carboxyl groups in acidic domains whose hydrogen ion density is approximately 2 orders of magnitude greater than that of bulk solvent.     

Effects of the minor groove pyrimidine nucleobase functional groups on the stability of duplex DNA: the impact of uncompensated minor groove amino groups

DNA sequences containing four types of analog nucleosides are described. All four are pyridine derivatives constructed as C-nucleosides so that they mimic the pyrimidine derivatives 2'-deoxyuridine, thymidine or 2'-deoxycytidine, but in all cases the analogs lack the corresponding O2-carbonyls that in duplex DNA are located in the minor groove. In place of the O2-carbonyl is a hydrogen atom, a polar fluorine atom, or a nonpolar methyl group. The described C-nucleosides have native-like bidentate Watson-Crick hydrogen-bonding faces and can form essentially normal W-C base pairs of varying stability with A or G. In each modified base pair, two inter-residue hydrogen bonds should be present. In spite of a common number of interstrand hydrogen bonds, the thermodynamic stabilities of the prepared duplexes, each containing two analog base pairs, vary dramatically. Most notably, base pairs containing uncompensated purine amino groups (those lacking a hydrogen-bonding partner) in the minor groove exhibit the most dramatic reductions in thermodynamic stability. Removal of such uncompensated amino groups results in increased duplex stability. Base pairs containing fluorine in the minor groove positioned adjacent to an amino group seem to enhance duplex stability marginally (relative to --H or --CH(3)), but there is little evidence to suggest that fluorine is an effective hydrogen-bonding partner in these systems. The presence of minor groove methyl groups results in the least stable duplexes in each series of sequences.     

The platelet cytoskeleton contains elements of the prothrombinase complex

Triton-insoluble cytoskeletons prepared from thrombin-activated platelets were found to potentiate the activation of prothrombin (prothrombinase activity). Cytoskeletons prepared from red cells or lymphoblasts contained no prothrombinase activity. The platelet prothrombinase activity was dependent on cytoskeletal-associated Factor Va, and exogenously added Factor Xa and prothrombin. Cytoskeletons contained 38% of the total platelet prothrombinase activity. Both platelets and cytoskeletons displayed half-maximal activities at similar prothrombin concentrations. The role of lipids in the cytoskeletal prothrombinase activity was investigated. Cytoskeletons were found to contain 3.8% of the total platelet phospholipids, consisting of the following lipids expressed as percentage of total present in platelets: 6.0% sphingomyelin, 3.8% phosphatidylcholine, 2.9% phosphatidyl-ethanolamine, 4.4% phosphatidylinositol, and 2.2% phosphatidylserine. The cytoskeletal prothrombinase activity and the lipid phosphorus content of cytoskeletons decreased after treatment of cytoskeletons with various doses of phospholipase C. Incubation of cytoskeletons with the highest concentrations tested (10 micrograms/ml) resulted in a 72% loss of phosphatidylserine and 84% loss of cytoskeletal prothrombinase activity. Cytoskeletal prothrombinase activity destroyed by phospholipase C treatment could be restored to control levels by treatment of hydrolyzed cytoskeletons with total cytoskeletal lipid or mixtures of phosphatidylserine/phosphatidylcholine (25:75% by weight). These results suggest that the cytoskeletal prothrombinase complex in addition to containing Factor Va, as has been previously shown (15), contains a lipid cofactor activity consisting in part of phosphatidylserine.     

DNA sequence recognition in the minor groove by hairpin microgonotropens

Two novel microgonotropens (MGTs) comprised of hairpin N-propylaminepyrrole polyamides linked to a Hoechst 33258 (Ht) analogue (3 and 4) were synthesized on solid phase by adopting an Fmoc technique using a series of HOBt mediated coupling reactions. The dsDNA-binding properties of MGTs 3 and 4 were determined by thermal denaturation experiments. Both MGTs were found to be selective for their nine-bp match dsDNA sequence 9 and were less tolerant of G/C bp substitutions in the binding region than linear progenitor MGT 1. MGT 3 was intolerant of a G/C substitution located in the middle of the binding region and did not bind to sequences 13 and 14. MGT 4 also did not bind to sequence 13, and its linker-bound Ht moiety was found to be more sensitive to a G/C substitution in the Ht-binding target, as demonstrated by the lack of binding to sequence 16.     

Protein and drug interactions in the minor groove of DNA

Interactions between proteins, drugs, water and B-DNA minor groove have been analyzed in crystal structures of 60 protein–DNA and 14 drug–DNA complexes. It was found that only purine N3, pyrimidine O2, guanine N2 and deoxyribose O4′ are involved in the interactions, and that contacts to N3 and O2 are most frequent and more polar than contacts to O4′. Many protein contacts are mediated by water, possibly to increase the DNA effective surface. Fewer water-mediated contacts are observed in drug complexes. The distributions of ligands around N3 are significantly more compact than around O2, and distributions of water molecules are the most compact. Distributions around O4′ are more diffuse than for the base atoms but most distributions still have just one binding site. Ligands bind to N3 and O2 atoms in analogous positions, and simultaneous binding to N3 and N2 in guanines is extremely rare. Contacts with two consecutive nucleotides are much more frequent than base–sugar contacts within one nucleotide. The probable reason for this is the large energy of deformation of hydrogen bonds for the one nucleotide motif. Contacts of Arg, the most frequent amino acid ligand, are stereochemically indistinguishable from the binding of the remaining amino acids except asparagine (Asn) and phenylalanine (Phe). Asn and Phe bind in distinct ways, mostly to a deformed DNA, as in the complexes of TATA-box binding proteins. DNA deformation concentrates on dinucleotide regions with a distinct deformation of the δ and backbone torsion angles for the Asn and δ, , ζ and χ for the Phe-contacted regions.     

Binding of Hoechst 33258 to the minor groove of B-DNA

An X-ray crystallographic structure analysis has been carried out on the complex between the antibiotic and DNA fluorochrome Hoechst 33258 and a synthetic B-DNA dodecamer of sequence C-G-C-G-A-A-T-T-C-G-C-G. The drug molecule, which can be schematized as: phenol-benzimidazole-benzimidazole-piperazine, sits within the minor groove in the A-T-T-C region of the DNA double helix, displacing the spine of hydration that is found in drug-free DNA. The NH groups of the benzimidazoles make bridging three-center hydrogen bonds between adenine N-3 and thymine O-2 atoms on the edges of base-pairs, in a manner both mimicking the spine of hydration and calling to mind the binding of the auti-tumor drug netropsin. Two conformers of Hoechst are seen in roughly equal populations, related by 180 degrees rotation about the central benzimidazole-benzimidazole bond: one form in which the piperazine ring extends out from the surface of the double helix, and another in which it is buried deep within the minor groove. Steric clash between the drug and DNA dictates that the phenol-benzimidazole-benzimidazole portion of Hoechst 33258 binds only to A.T regions of DNA, whereas the piperazine ring demands the wider groove characteristic of G.C regions. Hence, the piperazine ring suggests a possible G.C-reading element for synthetic DNA sequence-reading drug analogs.     

Binding of symmetrical cyanine dyes into the DNA minor groove

Optical methods, such as fluorescence, circular dichroism and linear flow dichroism, were used to study the binding to DNA of four symmetrical cyanine dyes, each consisting of two identical quinoline, benzthiazole, indole, or benzoxazole fragments connected by a trimethine bridge. The ligands were shown to form a monomer type complex into the DNA minor groove. The complex of quinoline-containing ligand with calf thymus DNA appeared to be the most resistant to ionic strength, and it did not dissociate completely even in 1 M NaCl. Binding of cyanine dyes to DNA could also be characterized by possibility to form ligand dimers into the DNA minor groove, by slight preference of binding to AT pairs, as well as by possible intercalation between base pairs of poly(dG)-poly(dC). The correlation found between the binding constants to DNA and the extent of cyanine dyes hydrophobicity estimated as the n-octanol/water partition coefficient is indicative of a significant role of hydrophobic interactions for the ligand binding into the DNA minor groove.     

Enhanced uranyl photocleavage across the minor groove of all (A/T)4 sequences indicates a similar narrow minor groove conformation

The uranyl(VI)-mediated photocleavage of a Drew–Dickerson sequence oligonucleotide (5′-dGATCACGCGAATTCGCGT) either as the (self-complementary) duplex or cloned into the BamH1 site of pUC19 has been studied. At pH 6.5 in acetate buffer relatively enhanced photocleavage is observed at the 3′-end of the AATT sequence corresponding to maximum cleavage across the minor groove in the A/T tract. Thus maximum cleavage correlates with minimum minor groove width in the crystal structure and also with the largest electronegative potential according to computations. Using plasmid constructs with cloned inserts of the type [CGCG(A/T₄)]_n, we also analysed all possible sequence combinations of the (A/T)₄ tract and in all cases we observed maximum uranyl-mediated photocleavage across the minor groove in the (A/T)₄ tract without any significant differences between the various sequences. From these results we infer that DNA double helices of all (A/T)₄ sequences share the same narrow minor groove helix conformation.     

A precursor to a minor species of yeast tRNASer contains an intervening sequence.

Certain tRNAs in S. cerevisiae (tRNATyr and tRNAPhe) arise via precursor molecules which are mature at the 5' and 3' termini but contain intervening sequences adjacent to the anticodon (Knapp et al., 1978; O'Farrell et al., 1978). In addition to these molecules, precursors to several other tRNAs accumulate in a temperature-sensitive mutant (ts136) at the nonpermissive temperature. We have analyzed one of these species and shown that it is a precursor to a minor species of tRNASer. This precursor is also mature at both termini and contains an intervening sequence of 19 nucleotides adjacent to the hypermodified A residue 3' to the anticodon. The sequence can be arranged in a secondary structure in which the anticodon stem is extended by additional base-pairing, and contains the sites of excision and ligation within two looped regions. Support for this structure was provided by analysis of the products of limited digestion with RNAase T1. recently Piper (1978) reported the isolation of a minor species of tRNASer which decodes UCG. He found this species to be structurally heterogeneous and determined that the less abundant form corresponds to the tRNA which is altered in the recessive lethal SUP-RL1 amber suppressor. Our data now suggest that the more abundant form may be restricted to reading UCA in vivo; thus mutation of the minor species would result in complete loss of UCG-decoding ability and explain the recessive lethality of SUP-RL1. We have shown that the precursor which accumulates in ts136 corresponds exclusively to this minor tRNASerUCG species. Our results suggest that this may be the only gene for tRNASer in yeast which contains an intervening sequence.     

DNA minor groove interactions and the biological activity of 2,5-bis

2,5-Bis-[4-(N-cyclobutyl-amidino)phenyl] furan and 2,5-bis-[4-(N-cyclohexyl-amidino)phenyl] furan have activity against Pneumocystis carinii and also show cytotoxicity against several tumour cell lines. These activities are correlated with DNA-binding abilities; the crystal structures of complexes with the DNA sequence d(CGCGAATTCGCG) is reported here. Interactions with, and effects on, the DNA minor groove, are found to be factors in the biological properties of these compounds.