Uranyl photoprobing of nonbent A/T- and bent A-tracts. A difference of flexibility?

In this study, we have systematically compared the uranyl photocleavage of a range of bent A-tracts and nonbent TA-tracts as well as interrupted A-tracts. We demonstrate that uranyl photocleavage of A-tracts and TA-tracts is almost identical, indicating a very similar minor groove conformation. Furthermore, a 10 base pair A-tract is divided into two independent tracts by an intervening TA or GC step. Uranyl probing also clearly distinguishes the bent A4T4 and the nonbent T4A4 sequences as adopting different structures, and our interpretation of the data is consistent with a structure for the bent A4T4 sequence that resembles a continuous A-tract, whereas the nonbent T4A4 sequences are closer to two independent and opposite A-tracts that cancel each other in terms of macroscopic bending. Finally, we also note that even single TA and TAT steps are highly sensitive to uranyl photocleavage and propose that in addition to average minor groove width, uranyl also senses DNA helix flexibility/deformability. Thus, the structural difference of TA-tracts and A-tracts may to a large extent reflect a difference in flexibility, and DNA curvature may consequently require a rigid narrow minor groove conformation that creates distinct A-tract-B-DNA junctions as the predominant cause of the bending.     

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.     

Efficient, Mg(2+)-dependent photochemical DNA cleavage by the antitumor quinobenzoxazine (S)-A-62176

The quinobenzoxazines, a group of structural analogues of the antibacterial fluoroquinolones, are topoisomerase II inhibitors that have demonstrated promising anticancer activity in mice. It has been proposed that the quinobenzoxazines form a 2:2 drug-Mg(2+) self-assembly complex on DNA. The quinobenzoxazine (S)-A-62176 is photochemically unstable and undergoes a DNA-accelerated photochemical reaction to afford a highly fluorescent photoproduct. Here we report that the irradiation of both supercoiled DNA and DNA oligonucleotides in the presence of (S)-A-62176 results in photochemical cleavage of the DNA. The (S)-A-62176-mediated DNA photocleavage reaction requires Mg(2+). Photochemical cleavage of supercoiled DNA by (S)-A-62176 is much more efficient that the DNA photocleavage reactions of the fluoroquinolones norfloxacin, ciprofloxacin, and enoxacin. The photocleavage of supercoiled DNA by (S)-A-62176 is unaffected by the presence of SOD, catalase, or other reactive oxygen scavengers, but is inhibited by deoxygenation. The photochemical cleavage of supercoiled DNA is also inhibited by 1 mM KI. Photochemical cleavage of DNA oligonucleotides by (S)-A-62176 occurs most extensively at DNA sites bound by drug, as determined by DNase I footprinting, and especially at certain G and T residues. The nature of the DNA photoproducts, and inhibition studies, indicate that the photocleavage reaction occurs by a free radical mechanism initiated by abstraction of the 4'- and 1'-hydrogens from the DNA minor groove. These results lend further support for the proposed DNA binding model for the quinobenzoxazine 2:2 drug-Mg(2+) complex and serve to define the position of this complex on the minor groove of DNA.     

The use of diaminopurine to investigate structural properties of nucleic acids and molecular recognition between ligands and DNA.

2,6-Diaminopurine (DAP) is an analogue of adenine which can be converted to nucleotides that serve as substrates for incorporation into nucleic acids by polymerases in place of (d)AMP. It pairs with thymidine (or uracil), engaging in three hydrogen bonds of the Watson-Crick type. The result of DAP incorporation is to add considerable stability to the double helix and to impart other structural features, such as an altered groove width and disruption of the normal spine of hydration. DNA containing DAP may or may not be recognized by restriction endonucleases; RNA containing DAP may not engage in normal splicing. The DAP.T pair affects the local flexibility of DNA and impedes the interaction with helix bending proteins. By providing a non-canonical hydrogen bond donor in the minor groove and/or blocking access to the floor of that groove it strongly affects interactions with small molecules such as antibiotics and anticancer drugs. Examples which illustrate altered recognition of nucleotide sequences in DAP-containing DNA are presented: changed sites of cutting by bleomycin, photocleavage by uranyl nitrate and footprinting with mithramycin. Using DNA in which both A-->DAP and G-->Inosine substitutions have been made it is possible to assess precisely the role of the purine 2-amino group in ligand-DNA recognition.     

Uranyl photoprobing of a four-way DNA junction: evidence for specific metal ion binding.

Metal ions are very important in mediating the folding of nucleic acids, as exemplified by the folding of the four-way DNA junction into the stacked X-conformation. Uranyl ion-mediated photocleavage provides a method for the localization of high-affinity ion binding sites in nucleic acids, and we have applied this to the four-way DNA junction. We have made the following observations. (i) Uranyl ions (UO2(2+)) suppressed the reactivity of junction thymine bases against attack by osmium tetroxide, indicating that the uranyl ion induces folding of the junction into a stacked conformation. (ii) DNA located immediately at the point of strand exchange on the two exchanging strands was hypersensitive to uranyl photocleavage. The relative hypersensitivity was considerably accentuated when the photocleavage was carried out in the presence of citrate ions. This suggests the presence of a tight binding site for the uranyl ion in the junction. (iii) The same positions were significantly protected from uranyl cleavage by the presence of hexamminecobalt (III) or spermidine. These ions are known to induce the folded conformation of the four-way junction with high efficiency, suggesting a direct competition between the ions. By contrast, magnesium ions failed to generate a similar protection against photocleavage. These results suggest that the uranyl, hexamminecobalt (III) and spermidine ions compete for the same high affinity binding site on the junction. This site is located at the centre of the junction, at the point where the exchanging strands pass between the stacked helices. We believe that we have observed the first known example of a metal ion 'footprint' on a folded nucleic acid structure.     

Control of DNA minor groove width and Fis protein binding by the purine 2-amino group

The width of the DNA minor groove varies with sequence and can be a major determinant of DNA shape recognition by proteins. For example, the minor groove within the center of the Fis–DNA complex narrows to about half the mean minor groove width of canonical B-form DNA to fit onto the protein surface. G/C base pairs within this segment, which is not contacted by the Fis protein, reduce binding affinities up to 2000-fold over A/T-rich sequences. We show here through multiple X-ray structures and binding properties of Fis–DNA complexes containing base analogs that the 2-amino group on guanine is the primary molecular determinant controlling minor groove widths. Molecular dynamics simulations of free-DNA targets with canonical and modified bases further demonstrate that sequence-dependent narrowing of minor groove widths is modulated almost entirely by the presence of purine 2-amino groups. We also provide evidence that protein-mediated phosphate neutralization facilitates minor groove compression and is particularly important for binding to non-optimally shaped DNA duplexes.     

Uranyl photofootprinting of triple helical DNA.

Two triple helix structures (15-mers containing only T.A-T triplets or containing mixed T.A-T and C.G-C triplets) have been studied by uranyl mediated DNA photocleavage to probe the accessibility of the phosphates of the DNA backbone. Whereas the phosphates of the pyrimidine strand are at least as accessible as in double stranded DNA, in the phosphates of the purine strand are partly shielded and more so at the 5'-end of the strand. With the homo A/T target increased cleavage is observed towards the 3'-end on the pyrimidine strand. These results show that the third strand is asymmetrically positioned along the groove with the tightest triple strand double strand interactions at the 5'-end of the third strand. The results also indicate that homo-A versus mixed A/G 'Hoogsteen-triple helices' have different structures.     

The influence of the 2-amino group of guanine on DNA conformation. Uranyl and DNase I probing of inosine/diaminopurine substituted DNA.

The conformation of the DNA helix is supposed to be a critical element in site-specific recognition by ligands both large and small. Groove width is one important measure of the conformation which varies with the local nucleotide composition, perhaps because of the presence of a purine 2-amino group on G.C base pairs. We have probed DNA with G-->inosine (I) and/or A-->diaminopurine (DAP) substitutions to see whether the location of the purine 2-amino group can indeed affect the minor groove width. At acid pH, the reactivity towards uranyl nitrate is modulated in substituted DNA quite differently from natural DNA, consistent with a marked narrowing of the minor groove at sites of G-->I substitution and widening at sites of A-->DAP replacement. The latter exerts the dominant effect. The expected changes in conformation are equally evident in the patterns of susceptibility to DNase I cleavage, but not to hydroxyl radical attack. Nuclease cleavage is maximal in normal and substituted DNA at regions of inferred moderate groove width which are generally little affected by the nucleotide substitutions. Consistent with models of sequence-dependent cutting by DNase I we find that the presence of a purine 2-amino group on the base pair three places upstream of the cutting site has a profound influence on the rate of reaction.     

Minor groove deformability of DNA: a molecular dynamics free energy simulation study

The conformational deformability of nucleic acids can influence their function and recognition by proteins. A class of DNA binding proteins including the TATA box binding protein binds to the DNA minor groove, resulting in an opening of the minor groove and DNA bending toward the major groove. Explicit solvent molecular dynamics simulations in combination with the umbrella sampling approach have been performed to investigate the molecular mechanism of DNA minor groove deformations and the indirect energetic contribution to protein binding. As a reaction coordinate, the distance between backbone segments on opposite strands was used. The resulting deformed structures showed close agreement with experimental DNA structures in complex with minor groove-binding proteins. The calculated free energy of minor groove deformation was approximately 4-6 kcal mol(-1) in the case of a central TATATA sequence. A smaller equilibrium minor groove width and more restricted minor groove mobility was found for the central AAATTT and also a significantly ( approximately 2 times) larger free energy change for opening the minor groove. The helical parameter analysis of trajectories indicates that an easier partial unstacking of a central TA versus AT basepair step is a likely reason for the larger groove flexibility of the central TATATA case.     

Groove Width and Depth of B-DNA Structures Depend on Local Variation in Slide

Abstract

The groove widths of DNA helix, especially minor groove width, are generally believed to be important for recognition of DNA by various types of ligands. It has been postulated earlier that large negative propeller twist, in the AT rich regions compresses the minor groove of duplex DNA A systematic study has now been carried out by generating models with different values of local doublet and intra-basepair parameters and calculating their minor groove widths. It is found that several local doublet parameters affect the minor groove width but it depends most strongly on the local step parameters roll and slide when each parameter is considered individually. However, a detailed analysis of the various local parameters within the B-DNA family of crystal structures indicates that propeller twist and slide are most strongly correlated with the observed values of minor groove width. The groove depth is also strongly correlated with slide. Thus the local base sequence dependent variations in slide can modify both the groove width and depth and consequently determine the ligand binding properties of DNA.     

Groove width and depth of B-DNA structures depend on local variation in slide.

The groove widths of DNA helix, especially minor groove width, are generally believed to be important for recognition of DNA by various types of ligands. It has been postulated earlier that large negative propeller twist, in the AT rich regions compresses the minor groove of duplex DNA. A systematic study has now been carried out by generating models with different values of local doublet and intra-basepair parameters and calculating their minor groove widths. It is found that several local doublet parameters affect the minor groove width but it depends most strongly on the local step parameters roll and slide when each parameter is considered individually. However, a detailed analysis of the various local parameters within the B-DNA family of crystal structures indicates that propeller twist and slide are most strongly correlated with the observed values of minor groove width. The groove depth is also strongly correlated with slide. Thus the local base sequence dependent variations in slide can modify both the groove width and depth and consequently determine the ligand binding properties of DNA.     

Effect of a neutralized phosphate backbone on the minor groove of B-DNA: molecular dynamics simulation studies

Alternative models have been presented to provide explanations for the sequence-dependent variation of the DNA minor groove width. In a structural model groove narrowing in A-tracts results from direct, short-range interactions among DNA bases. In an electrostatic model, the narrow minor groove of A-tracts is proposed to respond to sequence-dependent localization of water and cations. Molecular dynamics simulations on partially methylphosphonate substituted helical chains of d(TATAGGCCTATA) and d(CGCGAATTCGCG) duplexes have been carried out to help evaluate the effects of neutralizing DNA phosphate groups on the minor groove width. The results show that the time-average minor groove width of the GGCC duplex becomes significantly more narrow on neutralizing the phosphate backbone with methylphosphonates. The minor groove of the AATT sequence is normally narrow and the methylphosphonate substitutions have a smaller but measurable affect on this sequence. These results and models provide a system that can be tested by experiment and they support the hypothesis that the electrostatic environment around the minor groove affects the groove width in a sequence-dependent dynamic and time-average manner.     

The mechanics of minor groove width variation in DNA, and its implications for the accommodation of ligands.

In duplex DNA, groove width and depth are salient structural features that may influence the binding of drugs and proteins. These features are affected by movement of the bases, which for example may enforce groove compression or expansion through a rolling action of the adjacent base-pairs. Moreover, the sugar-phosphate backbone can also undergo limited movement, independently of the bases, which will affect the groove shape. We have examined how the movement of the sugar-phosphate backbone may affect the minor groove width for a fixed base geometry. In agreement with earlier studies, the sugar-phosphate backbone is found to have a certain degree of conformational flexibility in A and B-like helices, and we note a comparable freedom even in the highly curved TATA element of the TATA-binding protein/DNA complex. Phosphate mobility is highly anisotropic in all cases with favoured directions that can significantly change the groove width, independent of any changes in base geometry. We describe how the movement of the sugar-phosphate backbone may affect the accommodation of drugs and proteins in the minor groove, and we present a co-ordinate scheme which emphasises the groove adjustments associated with ligand binding. The observations have implications for the related problem of how cognate molecules are accommodated in the major groove.     

Increased temperature and 2-methyl-2,4-pentanediol change the DNA structure of both curved and uncurved adenine/thymine-rich sequences

DNA curvature is affected by elevated temperature and dehydrating agents such as 2-methyl-2,4-pentanediol (MPD) (used in crystallization). This effect of MPD has been ascribed to a specific distortion of the structure of adenine tracts (A-tracts), probably through a deformation of the characteristic narrow minor groove. Uranyl photoprobing indicates that a narrowed minor groove is present in all A/T regions containing four or more A/T base pairs. Consequently, this technique may be employed to study conformational changes in other A/T-rich sequences than pure A-tracts. In this study we use uranyl photoprobing to demonstrate that the effect of elevated temperature and MPD is analogous on both "normal" and curve-inducing A/T-rich sequences. The results therefore indicate that under these conditions the minor groove is widened in all A/T sequences and not only in pure A-tracts as previously suggested. Thus, the rather subtle structural difference of AT regions and A-tracts in nonbent DNA versus A-tracts in bent DNA may be quantitative rather than qualitative; i.e., the structure is more persistent and/or rigid in bent DNA.     

A 1H NMR study of the oligonucleotide binding of [(en)Pt(mu-dpzm)2Pt(en)]Cl4

The non-covalent binding of [(en)Pt(mu-dpzm)2Pt(en)]4+ to the dodecanucleotides d(CGCGAATTCGCG)2 and d(CAATCCGGATTG)2 has been studied by 1H NMR spectroscopy in order to gain a greater understanding of the pre-covalent binding association of cationic dinuclear platinum(II) anti-cancer drugs. NOESY experiments showed that the metal complex bound in the minor groove at the A/T rich regions of both dodecanucleotides. The metal complex did not induce any major DNA conformational changes. However, given the relative dimensions of the DNA minor groove and the metal complex, it is reasonable to expect that the metal complex binding significantly widens the minor groove at the A/T rich binding sites. The results of this study suggest that although dinuclear platinum(II) anti-cancer drugs covalently bind at GC sequences in the DNA major groove, they will preferentially associate with AT sequences in the minor groove before the covalent binding.     

Evaluation of uranyl photocleavage as a probe to monitor ion binding and flexibility in RNAs

In order to evaluate uranyl photocleavage as a tool to identify and characterize structural and dynamic properties in RNA, we compared uranyl cleavage sites in five RNA molecules with known X-ray structures, namely the hammerhead and hepatitis delta virus ribozymes, the P4-P6 domain of the Tetrahymena group I intron, as well as tRNA(Phe) and tRNA(Asp) from yeast. Uranyl photocleavage was observed at specific positions in all molecules investigated. In order to characterize the sites, photocleavage was performed in the absence and in increasing amounts of MgCl(2). Uranyl photocleavage correlates well with sites of low calculated accessibility, suggesting that uranyl ions bind in tight RNA pockets formed by close approach of phosphate groups. RNA foldings require ion binding, usually magnesium ions. Thus, upon the adoption of the native structure, uranyl ions can no longer bind well except in flexible and open to the solvent regions that can undergo induced-fit without disrupting the native fold. Uranyl photocleavage was compared to N-ethyl-N-nitrosourea and lead-induced cleavages in the context of the three-dimensional X-ray structures. Overall, the regions protected from ENU attack are sites of uranyl cleavage, indicating sites of low accessibility which can form ion binding sites. On the contrary, lead cleavages occur at flexible and accessible sites and correlate with the unspecific cleavages prevalent in dynamic and open regions. Applied in a magnesium-dependent manner, and only in combination with other backbone probing agents such as N-ethyl-N-nitrosourea, lead and Fenton cleavage, uranyl probing has the potential to reveal high-affinity metal ion environments, as well as regions involved in conformational transitions.     

Induced topological changes in DNA complexes: influence of DNA sequences and small molecule structures

Heterocyclic diamidines are compounds with antiparasitic properties that target the minor groove of kinetoplast DNA. The mechanism of action of these compounds is unknown, but topological changes to DNA structures are likely to be involved. In this study, we have developed a polyacrylamide gel electrophoresis-based screening method to determine topological effects of heterocyclic diamidines on four minor groove target sequences: AAAAA, TTTAA, AAATT and ATATA. The AAAAA and AAATT sequences have the largest intrinsic bend, whereas the TTTAA and ATATA sequences are relatively straight. The changes caused by binding of the compounds are sequence dependent, but generally the topological effects on AAAAA and AAATT are similar as are the effects on TTTAA and ATATA. A total of 13 compounds with a variety of structural differences were evaluated for topological changes to DNA. All compounds decrease the mobility of the ATATA sequence that is consistent with decreased minor groove width and bending of the relatively straight DNA into the minor groove. Similar, but generally smaller, effects are seen with TTTAA. The intrinsically bent AAAAA and AAATT sequences, which have more narrow minor grooves, have smaller mobility changes on binding that are consistent with increased or decreased bending depending on compound structure.