The effects of unnatural base pairs and mispairs on DNA duplex stability and solvation

In an effort to develop unnatural DNA base pairs we examined six pyridine-based nucleotides, d3MPy, d4MPy, d5MPy, d34DMPy, d35DMPy and d45DMPy. Each bears a pyridyl nucleobase scaffold but they are differentiated by methyl substitution, and were designed to vary both inter- and intra-strand packing within duplex DNA. The effects of the unnatural base pairs on duplex stability demonstrate that the pyridine scaffold may be optimized for stable and selective pairing, and identify one self pair, the pair formed between two d34DMPy nucleotides, which is virtually as stable as a dA:dT base pair in the same sequence context. In addition, we found that the incorporation of either the d34DMPy self pair or a single d34DMPy paired opposite a natural dA significantly increases oligonucleotide hybridization fidelity at other positions within the duplex. Hypersensitization of the duplex to mispairing appears to result from global and interdependent solvation effects mediated by the unnatural nucleotide(s) and the mispair. The results have important implications for our efforts to develop unnatural base pairs and suggest that the unnatural nucleotides might be developed as novel biotechnological tools, diagnostics, or therapeutics for applications where hybridization stringency is important.     

Novel insertion mutation in Caenorhabditis elegans.

The mutation e1662 is an allele of the Caenorhabditis elegans unc-54 gene induced with the difunctional alkylating agent 1,2,7,8-diepoxyoctane. unc-54 encodes the major myosin heavy chain isozyme of body wall muscle cells. Filter-transfer hybridization and DNA sequence analysis show that e1662 is an insertion of 288 base pairs of DNA within unc-54. The inserted DNA is identical to a 288-base pair region of unc-54 located ca. 600 base pairs from the insertion site. Thus, e1662 is a displaced duplication. A 14-base pair sequence located at one end of the duplicated segment is found adjacent to the site of insertion. These homologous sequences are juxtaposed head-to-tail by the insertion event. e1662 thus contains a tandem direct repeat extending across one of its junctions.     

Equilibrium between a wobble and ionized base pair formed between fluorouracil and guanine in DNA as studied by proton and fluorine NMR

A synthetic oligonucleotide duplex containing the chemotherapeutic and mutagenic agent 5-fluorouracil paired with guanine has been studied in solution by proton and fluorine NMR. The 7-mer duplex containing a central FU.G base pair adopts a normal right-handed configuration. At low pH, the predominant base-paired structure is wobble, whereas at higher pH an ionized structure in Watson-Crick geometry is observed. The two structures are in a pH-dependent equilibrium with one another with an apparent pK of 8.3 at 23 degrees C. This is the first demonstration of an equilibrium between two distinct base pairing schemes and the first demonstration of a negatively charged base pair in DNA.     

The DNA sequence at echinomycin binding sites determines the structural changes induced by drug binding: NMR studies of echinomycin binding to [d(ACGTACGT)]2 and [d(TCGATCGA)]2

The complexes formed between the cyclic octadepsipeptide antibiotic echinomycin and the two DNA octamers [d(ACGTACGT)]2 and [d(TCGATCGA)]2 have been investigated by using one- and two-dimensional proton NMR spectroscopy techniques. The results obtained for the two complexes are compared to each other, to the crystal structures of related DNA-echinomycin complexes, and to enzymatic and chemical footprinting results. In the saturated complexes, two echinomycin molecules bind to each octamer by bisintercalation of the quinoxaline moieties on either side of each CpG step. Binding of echinomycin to the octamer [d(ACGTACGT)]2 is cooperative so that only the two-drug complex is observed at lower drug-DNA ratios, but binding to [d(TCGATCGA)]2 is not cooperative. At low temperatures, both the internal and terminal A.T base pairs adjacent to the binding site in the [d(ACGTACGT)]2-2 echinomycin complex are Hoogsteen base paired (Gilbert et al., 1989) as observed in related crystal structures. However, as the temperature is raised, the internal A.T Hoogsteen base pairs are destabilized and are observed to be exchanging between the Hoogsteen base-paired and an open (or Watson-Crick base-paired) state. In contrast, in the [d(TCGATCGA)]2-2 echinomycin complex, no A.T Hoogsteen base pairs are observed, the internal A.T base pairs appear to be stabilized by drug binding, and the structure of the complex does not change significantly from 0 to 45 degrees C. Thus, the structure and stability of the DNA in echinomycin-DNA complexes depends on the sequence at and adjacent to the binding site. While we conclude that no single structural change in the DNA can explain all of the footprinting results, unwinding of the DNA helix in the drug-DNA complexes appears to be an important factor while Hoogsteen base pair formation does not.     

Mismatch formation in solution and on DNA microarrays: how modified nucleosides can overcome shortcomings of imperfect hybridization caused by oligonucleotide composition and base pairing

The DNA microarray technology is a well-established and widely used technology although it has several drawbacks. The accurate molecular recognition of the canonical nucleobases of probe and target is the basis for reliable results obtained from microarray hybridization experiments. However, the great flexibility of base pairs within the DNA molecule allows the formation of various secondary structures incorporating Watson-Crick base pairs as well as non-canonical base pair motifs, thus becoming a source of inaccuracy and inconsistence. The first part of this report provides an overview of unusual base pair motifs formed during molecular DNA interaction in solution highlighting selected secondary structures employing non-Watson-Crick base pairs. The same mispairing phenomena obtained in solution are expected to occur for immobilized probe molecules as well as for target oligonucleotides employed in microarray hybridization experiments the effect of base pairing and oligonucleotide composition on hybridization is considered. The incorporation of nucleoside derivatives as close shape mimics of the four canonical nucleosides into the probe and target oligonucleotides is discussed as a chemical tool to resolve unwanted mispairing. The second part focuses non-Watson-Crick base pairing during hybridization performed on microarrays. This is exemplified for the unusual stable dG.dA base pair.     

Structural Basis for Promutagenicity of 8-Halogenated Guanine

8-Halogenated guanine (haloG), a major DNA adduct formed by reactive halogen species during inflammation, is a promutagenic lesion that promotes misincorporation of G opposite the lesion by various DNA polymerases. Currently, the structural basis for such misincorporation is unknown. To gain insights into the mechanism of misincorporation across haloG by polymerase, we determined seven x-ray structures of human DNA polymerase β (polβ) bound to DNA bearing 8-bromoguanine (BrG). We determined two pre-catalytic ternary complex structures of polβ with an incoming nonhydrolyzable dGTP or dCTP analog paired with templating BrG. We also determined five binary complex structures of polβ in complex with DNA containing BrG·C/T at post-insertion and post-extension sites. In the BrG·dGTP ternary structure, BrG adopts syn conformation and forms Hoogsteen base pairing with the incoming dGTP analog. In the BrG·dCTP ternary structure, BrG adopts anti conformation and forms Watson-Crick base pairing with the incoming dCTP analog. In addition, our polβ binary post-extension structures show Hoogsteen BrG·G base pair and Watson-Crick BrG·C base pair. Taken together, the first structures of haloG-containing DNA bound to a protein indicate that both BrG·G and BrG·C base pairs are accommodated in the active site of polβ. Our structures suggest that Hoogsteen-type base pairing between G and C8-modified G could be accommodated in the active site of a DNA polymerase, promoting G to C mutation.     

Unusual DNA duplex and hairpin motifs

Single-stranded DNA or double-stranded DNA has the potential to adopt a wide variety of unusual duplex and hairpin motifs in the presence (trans) or absence (cis) of ligands. Several principles for the formation of those unusual structures have been established through the observation of a number of recurring structural motifs associated with different sequences. These include: (i) internal loops of consecutive mismatches can occur in a B-DNA duplex when sheared base pairs are adjacent to each other to confer extensive cross- and intra-strand base stacking; (ii) interdigitated (zipper-like) duplex structures form instead when sheared G·A base pairs are separated by one or two pairs of purine·purine mismatches; (iii) stacking is not restricted to base, deoxyribose also exhibits the potential to do so; (iv) canonical G·C or A·T base pairs are flexible enough to exhibit considerable changes from the regular H-bonded conformation. The paired bases become stacked when bracketed by sheared G·A base pairs, or become extruded out and perpendicular to their neighboring bases in the presence of interacting drugs; (v) the purine-rich and pyrimidine-rich loop structures are notably different in nature. The purine-rich loops form compact triloop structures closed by a sheared G·A, A·A, A·C or sheared-like G_anti·C_syn base pair that is stacked by a single residue. On the other hand, the pyrimidine-rich loops with a thymidine in the first position exhibit no base pairing but are characterized by the folding of the thymidine residue into the minor groove to form a compact loop structure. Identification of such diverse duplex or hairpin motifs greatly enlarges the repertoire for unusual DNA structural formation.     

The RecA protein as a recombinational repair system

The Escherichia coli RecA protein plays a central role in homologous genetic recombination, recombinational repair, and several other processes in bacteria. In vitro, an extended filament involving thousands of RecA monomers promotes a reaction in which individual DNA strands switch pairing partners (DNA strand exchange). This reaction has been extensively studied as a paradigm for the central steps in recombination. Because the strand-exchange reaction is relatively simple and isoenergetic, the complexity of the RecA system that carries it out has led to controversy about the functional significance of many fundamental properties of RecA. Filamentous protein structures involving thousands of RecA monomers, which hydrolyse 100 ATPs per base pair of heteroduplex DNA formed, are hard to rationalize in the context of recombination between two homologous DNAs. The thermodynamic barriers to strand exchange are much too small. These molecular features of the system can be easily rationalized, however, by shifting the focus to DNA repair.     

Electron-Transfer-Induced Acidity/Basicity and Reactivity Changes of Purine and Pyrimidine Bases. Consequences of Redox Processes for Dna Base Pairs

Changes in the oxidation state of the DNA bases, induced by oxidation (ionization) or by reduction (electron capture), have drastic effects on the acidity or basicity, respectively, of the molecules. Since in DNA every base is connected to its complementary base in the other strand, any change of the electric charge status of a base in one DNA strand that accompanies its oxidation or reduction may affect also the other strand via proton transfer across the hydrogen bonds in the base pairs. The free energies for electron transfer to or from a base can be drastically altered by the proton transfer processes that accompany the electron transfer reactions. Electron-transfer (ET) induced proton transfer sensitizes the base opposite to the ET-damaged base to redox damage, i.e., damage produced by separation of charge (ionization) has an increased change of being trapped in a base pair. Of the two types of base pair in DNA, A-T and C-G, the latter is more sensitive to both oxidative and reductive processes than the former.

Proton transfer induced by ET does not only occur between the heteroatoms (o and N) of the base pairs (intra-pair proton transfer), but also to and from adjacent water molecules in the hydration shell of DNA (extra-pair proton transfer). These proton transfers can involve carbon and as such are likely to be irreversible. It is the A-T pair which appears to be particularly prone to such irreversible reactions.     

Molecular dynamics study of solvation effect on diffusivity changes of DNA fragments

DNA sequence analyzing and base pair separation techniques have attracted much attention, such as denaturing gradient gel electrophoresis, temperature gradient gel electrophoresis, and capillary electrophoresis. However, details of sequence separation mechanisms in electrophoresis are not clarified enough. Understanding and controlling flow characteristics of DNA are important not only for fundamental research but also for further developments of bio-nano technologies. In the present study, we theoretically discuss the relationship between diffusivity and hydrated structures of DNA fragments in water solvent using molecular dynamics methods. In particular, influence of base pair substitutions on the diffusivity is investigated, focusing on an adenine-thymine (AT) rich B–DNA decamer 5’-dCGTATATATA-3’. Consequently, it is found that water molecules that concentrate on dissociated base pairs form hydrated structures and change the diffusivity of DNA decamers. The diffusion coefficients are affected by the substitution of GC for AT because of the different manner of interactions between the base molecules and water solvent. This result predicts a possibility of base pair separation according to differences in the diffusivity.     

A stable antiparallel cytosine-thymine base pair occurring only at the end of a duplex.

Ultraviolet hyperchromicity experiments indicate that in DNA duplex formation, a C-T mismatch is destabilizing in the center of a duplex, but behaves as a stable base pair at the terminus of a duplex. The C-T base pair is thought to contain two hydrogen bonds, but has thermodynamic parameters (delta Ho and delta Go of dissociation) that are similar to a G-C base pair. AMBER molecular mechanics calculations were performed to study the possible structural properties of DNA duplexes with central and terminal C-T combinations. These calculations also indicate that a central C-T pair destabilizes a duplex, while terminal C-T forms a stable base pair. Hydrogen bonding between cytosine and thymine occurs only in the energy-minimized structures when the helix diameter decreases and the propeller twist angle between the bases increases. These changes are found to occur only at the end of a duplex in the calculations, which may explain the experimental results.     

Divergent natural selection drives the evolution of reproductive isolation in an Australian wildflower

Ecological speciation occurs when reproductive isolation evolves between populations adapting to contrasting environments. A key prediction of this process is that the fitness of hybrids between divergent populations should be reduced in each parental environment as a function of the proportion of local genes they carry, a process resulting in ecologically dependent reproductive isolation (RI). To test this prediction, we use reciprocal transplant experiments between adjacent populations of an Australian wildflower, Senecio lautus, at two locations to distinguish between ecologically dependent and intrinsic genetic reproductive barriers. These barriers can be distinguished by observing the relative fitness of reciprocal backcross hybrids, as they differ in the contribution of genes from either parent while controlling for any intrinsic fitness effects of hybridization. We show ecologically dependent fitness effects in establishment and survival of backcrosses in one transplant experiment, and growth performance in the second transplant experiment. These results suggest natural selection can create strong reproductive barriers that maintain differentiation between populations with the potential to interbreed, and implies a significant role for ecology in the evolutionary divergence of S. lautus.     

Conformational changes during normal and error-prone incorporation of nucleotides by a Y-family DNA polymerase detected by 2-aminopurine fluorescence

Y-family polymerases are specialized to carry out DNA synthesis past sites of DNA damage. Their active sites make fewer contacts to their substrates, consistent with the remarkably low fidelity of these DNA polymerases when copying undamaged DNA. We have used DNA containing the fluorescent reporter 2-aminopurine (2-AP) to study the reaction pathway of the Y-family polymerase Dbh. We detected 3 rapid noncovalent steps between binding of a correctly paired dNTP and the rate-limiting step for dNTP incorporation. These early steps resemble those seen with high-fidelity DNA polymerases, such as Klenow fragment, and include a step that may be related to the unstacking of the 5' neighbor of the templating base that is seen in polymerase ternary complex crystal structures. A significant difference between Dbh and high-fidelity polymerases is that Dbh generates no fluorescence changes subsequent to dNTP binding if the primer lacks a 3'OH, suggesting that the looser active site of Y-family polymerases may enforce reliance on the correct substrate structure in order to assemble the catalytic center. Dbh, like other bypass polymerases of the DinB subgroup, generates single-base deletion errors at an extremely high frequency by skipping over a template base that is part of a repetitive sequence. Using 2-AP as a reporter to study the base-skipping process, we determined that Dbh uses a mechanism in which the templating base slips back to pair with the primer terminus while the base that was originally paired with the primer terminus becomes unpaired.     

Selective DNA Binding of (N-alkylamine)-Substituted Naphthalene Imides and Diimides to G+C-rich DNA

Abstract

Alkylamine-substituted naphthalene imides and diimides bind DNA by intercalation and have applications as anticancer agents. The unique structures of these imides in which two adjacent carbonyl groups lie coplanar to an extended aromatic ring system allow the possibility of sequence-selective interactions between the intercalated chromophore and guanine amino groups situated in the DNA minor groove. The binding affinities of N-[3- (dimethylamino)propyl amine]-1,8-naphthalenedicarboxylic imide (N-DMPrNI) and N, N′- bis[3,3′-(dimethylamino)propylamine]-naphthalene-1,4,5,8-tetracarboxylic diimide (N- BDMPrNDI) for natural DNAs of differing base composition were determined spectroscopically and by equilibrium dialysis. In agreement with the above proposition, binding studies indicated that both the naphthalene imide and diimide strongly prefer to intercalate into steps containing at least one G:C base pair. The dependencies of association constants on DNA base composition are consistent with a requirement for one G:C pair in the binding site of the monoimide, and two G:C pairs in binding sites of the diimide. These selectivities are comparable to or exceed that of actinomycin D, a classic G:C-selective drug. Protection footprinting with DNase I confirmed that the naphthalene monoimide (N-DMPrNI) prefers to bind adjacent to G:C base pairs, with a most consistent preference for “mixed” steps containing both a G:C and an A:T pair, excepting GA:TC. Several 5-CG-3′ steps were also good binding sites as indicated by nuclease protection, but few GC:GC or GG:CC steps were protected. The naphthalene diimide inhibited DNase I digestion, but did not yield a footprint. The base recognition ability and versatile chemistry make naphthalene imides and diimides attractive building blocks for design of highly sequence-specific, DNA-directed drug candidates including conjugated oligonucleotides or oligopeptides.     

Structural insights into the generation of single-base deletions by the Y family DNA polymerase dbh

Dbh is a Y family translesion DNA polymerase that accurately bypasses some damaged forms of deoxyguanosine, but also generates single-base deletion errors at frequencies of up to 50%, in specific hot spot sequences. We describe preinsertion binary, insertion ternary, and postinsertion binary crystal structures of Dbh synthesizing DNA after making a single-base deletion. The skipped template base adopts an extrahelical conformation stabilized by interactions with the C-terminal domain of the enzyme. DNA translocation and positioning of the next templating base at the active site, with space opposite to accommodate incoming nucleotide, occur independently of nucleotide binding, incorporation, and pyrophosphate release. We also show that Dbh creates single-base deletions more rapidly when the skipped base is located two or three bases upstream of the nascent base pair than when it is directly adjacent to the templating base, indicating that Dbh predominantly creates single-base deletions by template slippage rather than by dNTP-stabilized misalignment.     

Opposite base specificity in excision of pyrimidine ring-opened 1,N6-ethenoadenine by thymine glycol-DNA-glycosylases

A highly mutagenic DNA lesion, 1,N6-ethenoadenine ( epsilon A) is chemically unstable and either depurinates or converts to a pyrimidine ring-opened product upon water molecule addition to the C(2)z.sbnd;N(3) bond in epsilon dA (compound B). Compound B subsequently undergoes deformylation to yield compound C, which depurinates in the final step of the epsilon A rearrangement pathway. We have previously shown that epsilon A rearrangement products are not repaired by human N-methylpurine-DNA-glycosylase, which excises parental epsilon A. Compound B was shown to be eliminated from a B:T pair by Escherichia coli formamidopyrimidine-DNA-glycosylase (Fpg protein) and endonuclease III (Nth protein). Fpg protein excised B also from a B:C pair, and much less efficiently from B:A and B:G pairs [J. Biol. Chem. 276 (2001) 21821]. Here we show that efficiency of B excision by the Nth protein also depends on the opposite base in the pair. Most efficient repair is observed when this derivative is paired with dG (Km=18nM, kcat=12) and is less favourable when paired with dC (Km=40nM, kcat=13) and dT (Km=32nM, kcat=11). In physiological conditions, compound B is probably not excised by the Nth-glycosylase from a B:A pair, or from a single-stranded DNA, since kinetic constants in these conditions are an order or two orders of magnitude higher than when B is paired with T, C or G. A similar specificity for B excision was found for Saccharomyces cerevisiae Ntg2-glycosylase. Thus, when paired with A, an epsilon A derivative might be more persistent than when paired with other bases and give rise to AT-->TA transversions.     

DNA sequencing by hybridization to microchip octa-and decanucleotides extended by stacked pentanucleotides.

The efficiency of sequencing by hybridization to an oligonucleotide microchip grows with an increase in the number and in the length of the oligonucleotides; however, such increases raise enormously the complexity of the microchip and decrease the accuracy of hybridization. We have been developing the technique of contiguous stacking hybridization (CSH) to circumvent these shortcomings. Stacking interactions between adjacent bases of two oligonucleotides stabilize their contiguous duplex with DNA. The use of such stacking increases the effective length of microchip oligonucleotides, enhances sequencing accuracy and allows the sequencing of longer DNA. The effects of mismatches, base composition, length and other factors on the stacking are evaluated. Contiguous stacking hybridization of DNA with immobilized 8mers and one or two 5mers labeled with two different fluorescent dyes increases the effective length of sequencing oligonucleotides from 8 to 13 and 18 bases, respectively. The incorporation of all four bases or 5-nitroindole as a universal base into different positions of the 5mers permitted a decrease in the number of additional rounds of hybridization. Contiguous stacking hybridization appears to be a promising approach to significantly increasing the efficiency of sequencing by hybridization.     

Solution structures of a DNA dodecamer duplex with and without a cisplatin 1,2-d(GG) intrastrand cross-link: comparison with the same DNA duplex containing an oxaliplatin 1,2-d(GG) intrastrand cross-link

Proteins that discriminate between cisplatin-DNA adducts and oxaliplatin-DNA adducts are thought to be responsible for the differences in tumor range, toxicity, and mutagenicity of these two important chemotherapeutic agents. However, the structural basis for differential protein recognition of these adducts has not been determined and could be important for the design of more effective platinum anticancer agents. We have determined high-resolution NMR structures for cisplatin-GG and undamaged DNA dodecamers in the AGGC sequence context and have compared these structures with the oxaliplatin-GG structure in the same sequence context determined previously in our laboratory. This structural study allows the first direct comparison of cisplatin-GG DNA and oxaliplatin-GG DNA solution structures referenced to undamaged DNA in the same sequence context. Non-hydrogen atom rmsds of 0.81 and 1.21 were determined for the 15 lowest-energy structures for cisplatin-GG DNA and undamaged DNA, respectively, indicating good structural convergence. The theoretical NOESY spectra obtained by back-calculation from the final average structures showed excellent agreement with the experimental data, indicating that the final structures are consistent with the NMR data. Several significant conformational differences were observed between the cisplatin-GG adduct and the oxaliplatin-GG adduct, including buckle at the 5' G6.C19 base pair, opening at the 3' G7.C18 base pair, twist at the A5G6.T20C19 base pair step, slide, twist, and roll at the G6G7.C19C18 base pair step, slide at the G7C8.C18G17 base pair step, G6G7 dihedral angle, and overall bend angle. We hypothesize that these conformational differences may be related to the ability of various DNA repair proteins, DNA binding proteins, and DNA polymerases to discriminate between cisplatin-GG and oxaliplatin-GG adducts.     

Measuring the bleomycin-induced paired damage of two DNA chains by a method of psoralen photocross-linking

DNA of bacteriophage PM2 was allowed to react with bleomycin in the presence of Fe(II) and oxygen and the "paired" DNA lesions of two types were measured: (1) double-strand breaks, (2) lesions converted to double-strand breaks after introducing into the DNA a large number of psoralen cross-links (about 10(-2) per base pair) and alkali treatment. The mean numbers of each lesion type per DNA molecule are found to be proportional to the square of bleomycin concentration over the range of 3 X 10(-7) to 3 X 10(-6) M. These findings indicate that paired lesions are formed as a result of action of two bleomycin molecules at the same DNA site.