Molecular details of quinolone-DNA interactions: solution structure of an unusually stable DNA duplex with covalently linked nalidixic acid residues and non-covalent complexes derived from it

Quinolones are antibacterial drugs that are thought to bind preferentially to disturbed regions of DNA. They do not fall into the classical categories of intercalators, groove binders or electrostatic binders to the backbone. We solved the 3D structure of the DNA duplex (ACGCGU-NA)2, where NA denotes a nalidixic acid residue covalently linked to the 2'-position of 2'-amino-2'-deoxyuridine, by NMR and restrained torsion angle molecular dynamics (MD). In the complex, the quinolones stack on G:C base pairs of the core tetramer and disrupt the terminal A:U base pair. The displaced dA residues can stack on the quinolones, while the uracil rings bind in the minor groove. The duplex-bridging interactions of the drugs and the contacts of the displaced nucleotides explain the high UV-melting temperature for d(ACGCGU-NA)2 of up to 53 degrees C. Further, non-covalently linked complexes between quinolones and DNA of the sequence ACGCGT can be generated via MD using constraints obtained for d(ACGCGU-NA)2. This is demonstrated for unconjugated nalidixic acid and its 6-fluoro derivative. The well-ordered and tightly packed structures thus obtained are compatible with a published model for the quinolone-DNA complex in the active site of gyrases.     

Structure of a photoactive rhodium complex intercalated into DNA

Intercalating complexes of rhodium(III) are strong photo-oxidants that promote DNA strand cleavage or electron transfer through the double helix. The 1.2 A resolution crystal structure of a sequence-specific rhodium intercalator bound to a DNA helix provides a rationale for the sequence specificity of rhodium intercalators. It also explains how intercalation in the center of an oligonucleotide modifies DNA conformation. The rhodium complex intercalates via the major groove where specific contacts are formed with the edges of the bases at the target site. The phi ligand is deeply inserted into the DNA base pair stack. The primary conformational change of the DNA is a doubling of the rise per residue, with no change in sugar pucker from B-form DNA. Based upon the five crystallographically independent views of an intercalated DNA helix observed in this structure, the intercalator may be considered as an additional base pair with specific functional groups positioned in the major groove.     

Engineering a structure switching mechanism into a steroid-binding aptamer and hydrodynamic analysis of the ligand binding mechanism

The steroid binding mechanism of a DNA aptamer was studied using isothermal titration calorimetry (ITC), NMR spectroscopy, quasi-elastic light scattering (QELS), and small-angle X-ray spectroscopy (SAXS). Binding affinity determination of a series of steroid-binding aptamers derived from a parent cocaine-binding aptamer demonstrates that substituting a GA base pair with a GC base pair governs the switch in binding specificity from cocaine to the steroid deoxycholic acid (DCA). Binding of DCA to all aptamers is an enthalpically driven process with an unfavorable binding entropy. We engineered into the steroid-binding aptamer a ligand-induced folding mechanism by shortening the terminal stem by two base pairs. NMR methods were used to demonstrate that there is a transition from a state where base pairs are formed in one stem of the free aptamer, to where three stems are formed in the DCA-bound aptamer. The ability to generate a ligand-induced folding mechanism into a DNA aptamer architecture based on the three-way junction of the cocaine-binding aptamer opens the door to obtaining a series of aptamers all with ligand-induced folding mechanisms but triggered by different ligands. Hydrodynamic data from diffusion NMR spectroscopy, QELS, and SAXS show that for the aptamer with the full-length terminal stem there is a small amount of structure compaction with DCA binding. For ligand binding by the short terminal stem aptamer, we propose a binding mechanism where secondary structure forms upon DCA binding starting from a free structure where the aptamer exists in a compact form.     

Determinants of precatalytic conformational transitions in the DNA cytosine methyltransferase M.HhaI

The DNA methyltransferase M.HhaI is an excellent model for understanding how recognition of a nucleic acid substrate is translated into site-specific modification. In this study, we utilize direct, real-time monitoring of the catalytic loop position via engineered tryptophan fluorescence reporters to dissect the conformational transitions that occur in both enzyme and DNA substrate prior to methylation of the target cytosine. Using nucleobase analogues in place of the target and orphan bases, the kinetics of the base flipping and catalytic loop closure rates were determined, revealing that base flipping precedes loop closure as the rate-determining step prior to methyl transfer. To determine the mechanism by which individual specific hydrogen bond contacts at the enzyme-DNA interface mediate these conformational transitions, nucleobase analogues lacking hydrogen bonding groups were incorporated into the recognition sequence to disrupt the major groove recognition elements. The consequences of binding, loop closure, and catalysis were determined for four contacts, revealing large differences in the contribution of individual hydrogen bonds to DNA recognition and conformational transitions on the path to catalysis. Our results describe how M.HhaI utilizes direct readout contacts to accelerate extrication of the target base that offer new insights into the evolutionary history of this important class of enzymes.     

Stereochemical complementarity of progesterone and cavities between base pairs in partially unwound double stranded DNA using computer modeling and energy calculations to determine degree of fit

Computer modeling was applied for the first time to investigate previously reported complementarity of progesterone and cavities formed between base pairs in partially unwound double stranded DNA. Computer graphics enabled a more objective assessment of complementarity; energy calculations provided a rigorous method to evaluate degree of fit. Graphics confirmed that the complementarity was virtually "lock and key", i.e. close contacts were formed between van der Waals surfaces in the progesterone/DNA complexes and hydrogen bonds were formed between the two carbonyl groups on opposite ends of the steroid and phosphate groups on adjacent strands of DNA. Molecular mechanics calculations revealed that insertion of the steroid resulted in a relatively stable complex i.e. both van der Waals and electrostatic energies were lowered due to favorable steric interactions and stereospecific hydrogen bonds, respectively. Three published X-ray crystal structures of progesterone exhibited similar complementarity. Ent-progesterone which does not occur naturally possessed very poor complementarity. These findings confirm that the structure of progesterone is directly reflected in the stereochemistry of DNA. While no mechanistic explanation for these results is proffered, we hypothesize that such complementarity must have played a decisive role in the evolution of steroid hormone structure and function.     

Recognition of DNA sequences by the repressor of bacteriophage 434

The structure of a complex between the DNA-binding domain of phage 434 repressor and a 14 base-pair synthetic DNA operator reveals the molecular interactions important for sequence-specific recognition. A set of contacts with DNA backbone, notably involving hydrogen bonds between peptide-NH groups and DNA phosphates, position the repressor and fix the DNA configuration. Direct interactions between amino acid side chains and DNA bases involve nonpolar van der Waals contacts as well as hydrogen bonds. The structures of the repressor domain and of the 434 cro protein are extremely similar. There appear to be no major conformational changes in the proteins when they bind to DNA.     

Protein-RNA interactions: a structural analysis

A detailed computational analysis of 32 protein–RNA complexes is presented. A number of physical and chemical properties of the intermolecular interfaces are calculated and compared with those observed in protein–double-stranded DNA and protein–single-stranded DNA complexes. The interface properties of the protein–RNA complexes reveal the diverse nature of the binding sites. van der Waals contacts played a more prevalent role than hydrogen bond contacts, and preferential binding to guanine and uracil was observed. The positively charged residue, arginine, and the single aromatic residues, phenylalanine and tyrosine, all played key roles in the RNA binding sites. A comparison between protein–RNA and protein–DNA complexes showed that whilst base and backbone contacts (both hydrogen bonding and van der Waals) were observed with equal frequency in the protein–RNA complexes, backbone contacts were more dominant in the protein–DNA complexes. Although similar modes of secondary structure interactions have been observed in RNA and DNA binding proteins, the current analysis emphasises the differences that exist between the two types of nucleic acid binding protein at the atomic contact level.     

Electrostatic deformation of DNA by a DNA-binding protein

Complementary electrostatic interactions between negatively charged B-DNA and a positively charged array on the lambda Cro repressor protein are shown to substantially contribute to the formation energy of sequence-specific and nonspecific Cro-DNA complexes. The electrostatic interactions favor Cro binding to a bent form of DNA, a geometry which optimizes hydrogen-bonding contacts between Cro and exposed base pair groups in the DNA major groove.     

Amino acid–base interactions: a three-dimensional analysis of protein–DNA interactions at an atomic level

To assess whether there are universal rules that govern amino acid–base recognition, we investigate hydrogen bonds, van der Waals contacts and water-mediated bonds in 129 protein–DNA complex structures. DNA–backbone interactions are the most numerous, providing stability rather than specificity. For base interactions, there are significant base–amino acid type correlations, which can be rationalised by considering the stereochemistry of protein side chains and the base edges exposed in the DNA structure. Nearly two-thirds of the direct read-out of DNA sequences involves complex networks of hydrogen bonds, which enhance specificity. Two-thirds of all protein–DNA interactions comprise van der Waals contacts, compared to about one-sixth each of hydrogen and water-mediated bonds. This highlights the central importance of these contacts for complex formation, which have previously been relegated to a secondary role. Although common, water-mediated bonds are usually non-specific, acting as space-fillers at the protein–DNA interface. In conclusion, the majority of amino acid–base interactions observed follow general principles that apply across all protein–DNA complexes, although there are individual exceptions. Therefore, we distinguish between interactions whose specificities are ‘universal’ and ‘context-dependent’. An interactive Web-based atlas of side chain–base contacts provides access to the collected data, including analyses and visualisation of the three-dimensional geometry of the interactions.     

Interactions of molecules with nucleic acids. XI. Generalization of techniques to generate nucleic acid structures with applications to intercalation sites and kinked structures

A generalized procedure to generate nucleic acid structures is presented. In this procedure, the bases of a base pair are oriented first for characterization of particular DNA receptor sites. The resultant sites are then used in the study of specific molecule–DNA interactions. For example, intercalation sites, kinked DNA, and twisted and tilted bases are envisioned. Alterations of structures via anti → syn orientations of bases, as well as crankshaft motion about collinear bonds, provide additional conformations without disrupting the overall backbone structure. These approaches to the generation of nucleic acid structures are envisioned as required in studies of the intercalation phenomenon, minor adjustments of DNA to accommodate denaturation, binding of carcinogens to DNA, complex formation of transition metals with DNA, and antitumor agents as ligands. For these base-pair and base orientations, backbone orientations are calculated by the AGNAS technique to yield physically meaningful conformations, namely, those conformations for which nonbonded contacts are favourable. A procedure is presented to generate dimer duplex units that are physically meaningful and to assemble these units into a polynucleotide duplex. Double helices that begin with B-DNA, undergo a transition to one of the above-mentioned receptor sites, and return to B-DNA can be assembled from a catalog of dimer duplexes. Stereographic projections of the various receptor sites already being used to model binding to DNA are presented.     

Structural and functional analysis of a bipolar replication terminus. Implications for the origin of polarity of fork arrest

We have delineated the amino acid to nucleotide contacts made by two interacting dimers of the replication terminator protein (RTP) of Bacillus subtilis with a novel naturally occurring bipolar replication terminus by converting RTP to a site-directed chemical nuclease and mapping its cleavage sites on the terminus. The data show a relatively symmetrical arrangement of the amino acid to base contacts, and a comparison of the bipolar contacts with that of a normal unipolar terminus suggests that the DNA-protein contacts play an important determinative role in generating polarity from structurally symmetrical RTP dimers. The amino acid to nucleotide contacts provided distance constraints that enabled us to build a three-dimensional model of the protein-DNA complex. The model is consistent with features of the bipolar Ter.RTP complex derived from mutational and cross-linking data. The bipolar terminus arrested Escherichia coli DNA replication and DnaB helicase and T7 RNA polymerase in vitro in both orientations. RTP arrested the unwinding of duplex DNA on the bipolar Ter DNA substrate regardless of the length of the duplex DNA. The latter result suggested further that the terminus arrested authentic DNA unwinding by the helicase rather than just translocation of helicase on DNA.     

Nucleotide mapping and a kinetic model of a heteroplasmic deletion of 4,666 base pairs from mitochondrial DNA in the Kearns-Sayre syndrome

We report the nucleotidic mapping of a 4,666 base pairs deletion of the human mitochondrial DNA localized at positions 8571 and 13237 in a Kearns-Sayre syndrome patient. The gene fusion between the 15 N terminal amino acid residues of ATP synthetase subunit 6 and the 303 C terminal aminoacids of NADH dehydrogenase yields a potential protein of 35,000 d MW called A6-ND5. Deletion boundaries show a short inverted repeat ATCXTA. The heteroplasmic deletion mechanism is discussed in view of these data.     

RNA base-amino acid interaction strengths derived from structures and sequences.

We investigate RNA base-amino acid interactions by counting their contacts in structures and their implicit contacts in various functional sequences where the structures can be assumed to be preserved. These frequencies are cast into equations to extract relative interaction energetics. Previously we used this approach in considering the major groove interactions of DNA, and here we apply it to the more diverse interactions observed in RNA. Structures considered are the three different tRNA synthetase complexes, the U1A spliceosomal protein with an RNA hairpin and the BIV TAR-Tat complex. We use binding data for the base frequencies for the seryl, aspartyl and glutaminyl tRNA-synthetase and U1 RNA-protein complexes. We compare with the previously reported DNA major groove peptide contacts the results for atoms of RNA bases, usually in the major groove. There are strong similarities between the rank orders of interacting bases in the DNA and the RNA cases. The apparent strongest RNA interaction observed is between arginine and guanine which was also one of the strongest DNA interactions. The similar data for base atomic interactions, whether base paired or not, support the importance of strong atomic interactions over local structure considerations, such as groove width and alpha-helicity.     

Mechanistic details of the DNA recognition by the Dnmt1 DNA methyltransferase

A recently solved Dnmt1-DNA crystal structure revealed several enzyme-DNA contacts and large structural rearrangements of the DNA at the target site, including the flipping of the non-target strand base of the base pair flanking the CpG site and formation of a non-canonical base pair between the non-target strand Gua and the flanking base pair. Here, we show that the contacts of the enzyme to the target base and the Gua:5mC base pair that are observed in the structure are very important for catalytic activity. The contacts to the non-target strand Gua are not important since its exchange by Ade stimulated activity. Except target base flipping, we could not find evidence that the DNA rearrangements have a functional role.     

Determinants of DNA mismatch recognition within the polymerase domain of the Klenow fragment.

The Klenow fragment of Escherichia coli DNA polymerase I catalyzes template-directed synthesis of DNA and uses a separate 3'-5' exonuclease activity to edit misincorporated bases. The polymerase and exonuclease activities are contained in separate structural domains. In this study, nine Klenow fragment derivatives containing mutations within the polymerase domain were examined for their interaction with model primer-template duplexes. The partitioning of the DNA primer terminus between the polymerase and 3'-5' exonuclease active sites of the mutant proteins was assessed by time-resolved fluorescence anisotropy, utilizing a dansyl fluorophore attached to the DNA. Mutation of N845 or R668 disrupted favorable interactions between the Klenow fragment and a duplex containing a matched terminal base pair but had little effect when the terminus was mismatched. Thus, N845 and R668 are required for recognition of correct terminal base pairs in the DNA substrate. Mutation of N675, R835, R836, or R841 resulted in tighter polymerase site binding of DNA, suggesting that the side chains of these residues induce strain in the DNA and/or protein backbone. A double mutant (N675A/R841A) showed an even greater polymerase site partitioning than was displayed by either single mutation, indicating that such strain is additive. In both groups of mutant proteins, the ability to discriminate between duplexes containing matched or mismatched base pairs was impaired. In contrast, mutation of K758 or Q849 had no effect on partitioning relative to wild type, regardless of DNA mismatch character. These results demonstrate that DNA mismatch recognition is dependent on specific amino acid residues within the polymerase domain and is not governed solely by thermodynamic differences between correct and mismatched base pairs. Moreover, this study suggests a mechanism whereby the Klenow fragment is able to recognize polymerase errors following a misincorporation event, leading to their eventual removal by the 3'-5' exonuclease activity.