Site-directed modification of DNA duplexes by chemical ligation.

The efficiency of chemical ligation method have been demonstrated by assembling a number of DNA duplexes with modified sugar phosphate backbone. Condensation on a tetradecanucleotide template of hexa(penta)- and undecanucleotides differing only in the terminal nucleoside residue have been performed using water-soluble carbodiimide as a condensing agent. As was shown by comparing the efficiency of chemical ligation of single-strand breaks in those duplexes, the reaction rate rises 70 or 45 times if the 3'-OH group is substituted with an amino or phosphate group (the yield of products with a phosphoramidate or pyrophosphate bond is 96-100% in 6 d). Changes in the conformation of reacting groups caused by mismatched base pairs (A.A, A.C) as well as the hybrid rU.dA pair or an unpaired base make the template-directed condensation less effective. The thermal stability of DNA duplexes was assayed before and after the chemical ligation. Among all of the modified duplexes, only the duplex containing 3'-rU in the nick was found to be a substrate of T4 DNA ligase.     

T4-DNA ligase: substrate properties of synthetic DNA-duplexes with structural anomalies

Structural defects, affecting T4 DNA ligase function, were revealed with the help of synthetic DNA duplexes, containing modifications at single nick. Changes of configuration at C2' and C3' atoms of furanose in the acceptor terminus lead to total blocking of the nick sealing activity of T4 DNA ligase. On the contrary, substitution of 3'-terminal deoxyribonucleotide for ribonucleotide doesn't affect the enzyme's action. The duplex looses all of it's substrate activity if the next from the nick G.C pair is substituted for the noncomplementary G.C pair. In DNA duplexes containing an unpaired base in the nick, elimination of the extrahelical nucleotide proceeds the ligation step. In these cases the duplex substrate activity decreases depending on the extent of extrahelical base stacking into the double stranded DNA.     

Nicks 3'' or 5'' to AP sites or to mispaired bases, and one-nucleotide gaps can be sealed by T4 DNA ligase.

Using synthetic oligodeoxynucleotides with 3'-OH ends and 32P-labelled 5'-phosphate ends and the technique of polyacrylamide gel electrophoresis, it is shown that, in the presence of the complementary polynucleotide, an AP (apurinic or apyrimidinic) site at the 3' or the 5' end of the labelled oligodeoxynucleotides does not prevent their ligation by T4 DNA ligase, although the reaction rate is decreased. This decrease is more severe when the AP site is at the 3' end; the activated intermediates accumulate showing that it is the efficiency of the adenyl-5'-phosphate attack by the 3'-OH of the base-free deoxyribose which is mostly perturbed. Using the same technique, it is shown that a mispaired base at the 3' or 5' end of oligodeoxynucleotides does not prevent their ligation. A one-nucleotide gap, limited by 3'-OH and 5'-phosphate, can also be closed by T4 DNA ligase although with difficulty; here again the activation of the 5'-phosphate end does not seem to be slowed down, but rather the 3'-OH attack of the adenyl-5'-phosphate. All these anomalous ligations take place with the nick or the gap in front of a continuous complementary strand. Blunt ends ligation of correct duplexes occurs readily; however an AP site or a mispaired base at the 3' or 5' end of one strand of the duplexes prevents ligation between these strands. But a missing nucleotide (responsible for one unpaired nucleotide protruding at the 3' or 5' end of the complementary strand) does not stop ligation of the shorter oligodeoxynucleotides between independent duplexes.     

Nuclear magnetic resonance spectroscopy and molecular modeling reveal that different hydrogen bonding patterns are possible for G.U pairs: one hydrogen bond for each G.U pair in r(GGCGUGCC)(2) and two for each G.U pair in r(GAGUGCUC)(2)

G.U pairs occur frequently and have many important biological functions. The stability of symmetric tandem G.U motifs depends both on the adjacent Watson-Crick base pairs, e.g., 5'G > 5'C, and the sequence of the G.U pairs, i.e., 5'-UG-3' > 5'-GU-3', where an underline represents a nucleotide in a G.U pair [Wu, M., McDowell, J. A., and Turner, D. H. (1995) Biochemistry 34, 3204-3211]. In particular, at 37 degrees C, the motif 5'-CGUG-3' is less stable by approximately 3 kcal/mol compared with other symmetric tandem G.U motifs with G-C as adjacent pairs: 5'-GGUC-3', 5'-GUGC-3', and 5'-CUGG-3'. The solution structures of r(GAGUGCUC)(2) and r(GGCGUGCC)(2) duplexes have been determined by NMR and restrained simulated annealing. The global geometry of both duplexes is close to A-form, with some distortions localized in the tandem G.U pair region. The striking discovery is that in r(GGCGUGCC)(2) each G.U pair apparently has only one hydrogen bond instead of the two expected for a canonical wobble pair. In the one-hydrogen-bond model, the distance between GO6 and UH3 is too far to form a hydrogen bond. In addition, the temperature dependence of the imino proton resonances is also consistent with the different number of hydrogen bonds in the G.U pair. To test the NMR models, U or G in various G.U pairs were individually replaced by N3-methyluridine or isoguanosine, respectively, thus eliminating the possibility of hydrogen bonding between GO6 and UH3. The results of thermal melting studies on duplexes with these substitutions support the NMR models.     

Structural and kinetic aspects of chemical reactions in DNA duplexes. Information on DNA local structure obtained from chemical ligation data.

Chemical ligation of oligonucleotides in double-stranded helices has been considered in its structural-kinetic aspect. A study was made of (i) two series of DNA duplexes with various arrangements of reacting groups in the ligation junction induced by mispairing or by alteration of furanose structure (the replacement of dT unit with rU, aU, IU, xU, dxT ones) and of (ii) eight synthetic water-soluble carbodiimides with different substituents at N1 and N3 atoms. We assumed that some information on the local structure of modified sites in the duplex can be obtained from kinetic parameters of oligonucleotide coupling reaction. The ratio of kinetic constants k3/(k2 + k3) for productive and nonproductive decomposition of the activated phosphomonoester derivative apparently reflects the reaction site structure: for a given duplex this parameter is virtually independent of the condensing agent composition. Based on the analysis of the chemical ligation kinetics a suggestion has been made about the conformation of some modified units in the double helix.     

In vitro ligation of oligodeoxynucleotides containing C8-oxidized purine lesions using bacteriophage T4 DNA ligase

Ligases conduct the final stage of repair of DNA damage by sealing a single-stranded nick after excision of damaged nucleotides and reinsertion of correct nucleotides. Depending upon the circumstances and the success of the repair process, lesions may remain at the ligation site, either in the template or at the oligomer termini to be joined. Ligation experiments using bacteriophage T4 DNA ligase were carried out with purine lesions in four positions surrounding the nick site in a total of 96 different duplexes. The oxidized lesion 8-oxo-7,8-dihydroguanosine (OG) showed, as expected, that the enzyme is most sensitive to lesions on the 3' end of the nick compared to the 5' end and to lesions located in the intact template strand. In general, substrates containing the OG.A mismatch were more readily ligated than those with the OG.C mismatch. Ligations of duplexes containing the OA.T base pair (OA = 8-oxo-7,8-dihydroadenosine) that could adopt an anti-anti conformation proceeded with high efficiencies. An OI.A mismatch-containing duplex (OI = 8-oxo-7,8-dihydroinosine) behaved like OG.A. Due to its low reduction potential, OG is readily oxidized to secondary oxidation products, such as the guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp) nucleosides; these lesions also contain an oxo group at the original C8 position of the purine. Ligation of oligomers containing Gh and Sp occurred when opposite A and G, although the overall ligation efficiencies were much lower than those of most OG base pairs. Steady-state kinetic studies were carried out for representative examples of lesions in the template. Km increased by 90-100-fold for OG.C-, OI.C-, OI.A-, and OA.T-containing duplexes compared to that of a G.C-containing duplex. Substrates containing Gh.A, Gh.G, Sp.A, and Sp.G base pairs exhibited Km values 20-70-fold higher than that of the substrate containing a G.C base pair, while the Km value for OG.A was 5 times lower than that for G.C.     

The conserved U.G pair in the 5'' splice site duplex of a group I intron is required in the first but not the second step of self-splicing.

Group I self-splicing introns have a 5' splice site duplex (P1) that contains a single conserved base pair (U.G). The U is the last nucleotide of the 5' exon, and the G is part of the internal guide sequence within the intron. Using site-specific mutagenesis and analysis of the rate and accuracy of splicing of the Tetrahymena thermophila group I intron, we found that both the U and the G of the U.G pair are important for the first step of self-splicing (attack of GTP at the 5' splice site). Mutation of the U to a purine activated cryptic 5' splice sites in which a U.G pair was restored; this result emphasizes the preference for a U.G at the splice site. Nevertheless, some splicing persisted at the normal site after introduction of a purine, suggesting that position within the P1 helix is another determinant of 5' splice site choice. When the U was changed to a C, the accuracy of splicing was not affected, but the Km for GTP was increased by a factor of 15 and the catalytic rate constant was decreased by a factor of 7. Substitution of U.A, U.U, G.G, or A.G for the conserved U.G decreased the rate of splicing by an even greater amount. In contrast, mutation of the conserved G enhanced the second step of splicing, as evidenced by a trans-splicing assay. Furthermore, a free 5' exon ending in A or C instead of the conserved U underwent efficient ligation. Thus, unlike the remainder of the P1 helix, which functions in both the first and second steps of self-splicing, the conserved U.G appears to be important only for the first step.     

Synthesis and properties of DNA duplexes containing hydrocarbon bridges instead of a nucleoside residue

DNA duplexes 14 bp long containing an EcoRII and MvaI restriction site in which a nucleoside is substituted by 1,3-diaminopropane or 1,3-propanediol residue have been chemically synthesized. Diaminopropane bridge was introduced by the chemical ligation, whereas the oligonucleotide containing propanediol was prepared by automatic solid phase phosphoroamidite method on "Victoria-4M" synthesizer. As CD and UV spectra show, the modification destabilises the duplex by 18-20 degrees C without essential distortion of the double helix, except for increase of the conformational mobility in the modified site.     

Tri-partite assay for studying exon ligation by the ai5gamma group II intron

Group II introns self-splice via a two-step mechanism: cleavage at the 5' splice site followed by exon ligation at the 3' splice site. The second step has been difficult to study in vitro because it is generally faster than the first. Herein we describe development and partial kinetic characterization of a novel assay for studying the second step in isolation. In this system, a truncated linear intron (nucleotides 1-881) mediates exon ligation between two oligonucleotide substrates: a 19 nt 5' exon and a 3' substrate consisting of the last 6 nucleotides of the intron plus a 6 nucleotide 3' exon. We found that neither the exact structure of domain 6 nor the identity of nucleotides flanking the 3' splice site is critical for accurate 3' splice site choice by the ai5gamma group II intron. The multiple turnover k(cat) (0.14 min(-)(1)) is slower than the single turnover k(obs) (0.6-0.7 min(-)(1)), consistent with rate-limiting product release under steady-state conditions. Decreased single turnover rates at lower pHs were more consistent with loss of catalytic activity than with rate-limiting chemistry. Binding of the 3' substrate (K(m) = 2.6 microM) could be improved by changing a long-range A:U base pair involving the last intronic nucleotide (the gamma-gamma' interaction) to G:C (K(m(3)(')(substrate)) = 1 microM).     

Branch capture reactions: effect of recipient structure.

Branch capture reactions (BCR) contain two DNA species: (i) a recipient restriction fragment terminating in an overhang and (ii) a displacer-linker duplex terminating in a displacer tail complementary to the overhang as well as contiguous nucleotides within the recipient duplex. Branched complexes containing both species are captured by ligation of the linker to the recipient overhang. Specificity depends upon branch migration and is increased by substitution of bromodeoxycytidine for deoxycytidine in the displacer. BCR rates and specificities were determined for recipient overhangs that were (i) 5' and 3', (ii) 3 and 4 nucleotides long, and (iii) 0-100% G+C. Model systems permitted independent determination of G+C and branching effects on ligation rates and verification of rapid equilibrium between the branched complex and its component species. With all 4-base overhangs, recipient duplexes permitting extensive branch migration became saturated with displacer-linker duplexes. With increasing G+C, increasing ligation at competing sites led to decreased BCR specificity. BCR may be used to label a DNA fragment prior to electrophoresis, mark a fragment for affinity chromatography, or introduce a new overhang sequence compatible with a restriction endonuclease site in a cloning vector. A protocol was confirmed for mapping restriction sites in cloned DNA.     

Chemical reactions in double-stranded nucleic acids. III. Synthesis of terminal inverted repeats of the IS1 element

Three 35 bp-DNA duplexes have been assembled from synthetic oligonucleotides by means of DNA ligase or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in two parallel series of experiments. The "top" strands of these duplexes correspond either to the imperfect (natural) or perfect terminal inverted repeats of the IS1. Tm of DNA duplexes composed of 2 to 6 different oligonucleotides were investigated by UV spectroscopy. It was shown that DNA ligase effectively joined oligonucleotides even under conditions of DNA duplex instability. However, there is a minimum duplex size (within the range of 9-15 bp) below which the enzymatic ligation is ineffective. Chemical assembly of duplexes took place only if the double helix was stable. The yield was 50% after two successive ligation cycles. Efficiency of the chemical ligation depends on the nature of the nucleotide units to be joined.     

Characterization of conformational features of DNA heteroduplexes containing aldehydic abasic sites

The DNA duplexes shown below, with D indicating deoxyribose aldehyde absic sites and numbering from 5' to 3', have been investigated by NMR. The 31P and 31P-1H correlation data indicate [formula: see text] that the backbones of these duplex DNAs are regular. One- and two-dimensional 1H NMR data indicate that the duplexes are right-handed and B-form. Conformational changes due to the presence of the abasic site extend to the two base pairs adjacent to the lesion site with the local conformation of the DNA being dependent on whether the abasic site is in the alpha or beta configuration. The aromatic base of residue A17 in the position opposite the abasic site is predominantly stacked in the helix as is G17 in the analogous sample. Imino lifetimes of the AT base pairs are much longer in samples with an abasic site than in those containing a Watson-Crick base pair. The conformational and dynamical properties of the duplex DNAs containing the naturally occurring aldehyde abasic site are different from those of duplex DNAs containing a variety of analogues of the abasic site.     

Interaction of EcoRII restriction and modification enzymes with synthetic DNA fragments. IX. Cleavage of substrates with point modifications in the recognition site and flanking sequences

Ability of the EcoRII restriction endonuclease to cleave 14-base-pair DNA duplexes with nucleotide substitutions in the recognition site CCA/TGG and in the adjacent base pair has been studied. Modifications leading to a local change in the substrate conformation (rU residue in and outside the recognition site, A.A- or A.C-pairs in the flanking sequence) reduce the rate of hydrolysis, the effect being maximal when the modified base pair is outside the recognition site. No digestion occurs when the internal dC-residue of the recognition site is 5-methylated in one or both strands. Replacement of dT residue in the EcoRII recognition site by dfl5U residue results in a dramatic inhibition of hydrolysis. Km and kcat for the cleavage of 14-base-pair DNA duplex have been determined. The cleavage rate of the dT-containing strand of the recognition site in 1.5 fold higher comparing with the dA-containing strand. The cleavage of both strands of the substrate by EcoRII endonuclease is confirmed to proceed in one enzyme-substrate complex.     

The use of BrCN for assembling modified DNA duplexes and DNA-RNA hybrids; comparison with water-soluble carbodiimide.

Both cyanogen bromide (BrCN) and 1-ethyl-3-(3'-dimethylaminopropyl) carbodiimide may be used as coupling reagents for the template-directed assembly of DNA duplexes containing the sugar-phosphate backbone modification. Both reagents show similar ligation site structure-specific trend. Practical recommendations are given for selection of the condensing reagent depending on the properties of the duplex. Based on 31P NMR spectroscopy data, a scheme is suggested for BrCN activation of the nucleotide phosphomonoester group. Using both condensing reagents, we studied the condensation of oligonucleotides containing ribo-segments (from mononucleotide residue to full sequence) on the DNA template. Efficiency of the chemical ligation of RNA oligomers was shown to be much lower than that of DNA analogues. The coupling yield depends on the position of the RNA segment in the hybrid duplexes and on the position of the phosphate group in the nick.     

Structure and function of the 3' terminal six nucleotides of the west nile virus genome in viral replication

Using a self-replicating reporting replicon of West Nile (WN) virus, we performed a mutagenesis analysis to define the structure and function of the 3'-terminal 6 nucleotides (nt) (5'-GGAUCU(OH)-3') of the WN virus genome in viral replication. We show that mutations of nucleotide sequence or base pair structure of any of the 3'-terminal 6 nt do not significantly affect viral translation, but exert discrete effects on RNA replication. (i). The flavivirus-conserved terminal 3' U is optimal for WN virus replication. Replacement of the wild-type 3' U with a purine A or G resulted in a substantial reduction in RNA replication, with a complete reversion to the wild-type sequence. In contrast, replacement with a pyrimidine C resulted in a replication level similar to that of the 3' A or G mutants, with only partial reversion. (ii). The flavivirus-conserved 3' penultimate C and two upstream nucleotides (positions 78 and 79), which potentially base pair with the 3'-terminal CU(OH), are absolutely essential for viral replication. (iii). The base pair structures, but not the nucleotide sequences at the 3rd (U) and the 4th (A) positions, are critical for RNA replication. (iv). The nucleotide sequences of the 5th (G) position and its base pair nucleotide (C) are essential for viral replication. (v). Neither the sequence nor the base pair structure of the 6th nucleotide (G) is critical for WN virus replication. These results provide strong functional evidence for the existence of the 3' flavivirus-conserved RNA structure, which may function as contact sites for specific assembly of the replication complex or for efficient initiation of minus-sense RNA synthesis.     

Structure and activity of Y-class DNA polymerase DPO4 from Sulfolobus solfataricus with templates containing the hydrophobic thymine analog 2,4-difluorotoluene

The 2,4-difluorotoluene (DFT) analog of thymine has been used extensively to probe the relative importance of shape and hydrogen bonding for correct nucleotide insertion by DNA polymerases. As far as high fidelity (A-class) polymerases are concerned, shape is considered by some as key to incorporation of A(T) opposite T(A) and G(C) opposite C(G). We have carried out a detailed kinetic analysis of in vitro primer extension opposite DFT-containing templates by the trans-lesion (Y-class) DNA polymerase Dpo4 from Sulfolobus solfataricus. Although full-length product formation was observed, steady-state kinetic data show that dATP insertion opposite DFT is greatly inhibited relative to insertion opposite T (approximately 5,000-fold). No products were observed in the pre-steady-state. Furthermore, it is noteworthy that Dpo4 strongly prefers dATP opposite DFT over dGTP (approximately 200-fold) and that the polymerase is able to extend an A:DFT but not a G:DFT pair. We present crystal structures of Dpo4 in complex with DNA duplexes containing the DFT analog, the first for any DNA polymerase. In the structures, template-DFT is either positioned opposite primer-A or -G at the -1 site or is unopposed by a primer base and followed by a dGTP:A mismatch pair at the active site, representative of a -1 frameshift. The three structures provide insight into the discrimination by Dpo4 between dATP and dGTP opposite DFT and its inability to extend beyond a G:DFT pair. Although hydrogen bonding is clearly important for error-free replication by this Y-class DNA polymerase, our work demonstrates that Dpo4 also relies on shape and electrostatics to distinguish between correct and incorrect incoming nucleotide.     

Helix-coil transition of parallel-stranded DNA. Thermodynamics of hairpin and linear duplex oligonucleotides

The stabilities have been determined of different DNA double helices constructed with the two constituent strands in a parallel orientation. These molecules incorporate polarity-inverting loop structures (hairpins) or nucleotide sequences (duplexes) which impose the desired polarity on the two strands constituting the sugar-phosphate backbone. The hairpins consisted of d(A.T)n stems (n = 8 or 10) and either a 5'-p-5' linkage in a d(C)4 loop (ps-C8 and ps-C10) or a 3'-p-3' linkage in a d(G)4 loop (ps-G10). The linear duplexes had 21-nt (ps-C2.C3) and 25-nt (ps-D1.D2, ps-D3.D4) mixed A,T sequences and normal chemical linkages throughout. Reference molecules with normal antiparallel helical orientations (hairpins aps-C8, aps-C10, and aps-G10 and duplexes aps-C3.C7, aps-D1.D3, and aps-D2.D4) were also synthesized and studied. Hydrogen bonding in ps-DNA is via reverse Watson-Crick base pairs, and the various constructs display spectroscopic, chemical, biochemical, and electrophoretic properties distinct from those of their aps counterparts. For example, both the ps and aps molecules show a pronounced UV absorption hyperchromicity upon melting, but the spectral distribution is not the same. Thus, the difference spectra (ps-aps) in the native state are characterized by a positive peak at 252 nm, an isosbestic point at 267 nm, and a negative peak at 282 nm. Temperature-dependent absorbances were recorded at selected wavelengths and in the form of complete spectra to derive the thermodynamic parameters for the helix-coil transitions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Substrate recognition by Escherichia coli MutY using substrate analogs.

The Escherichia coli adenine glycosylase MutY is involved in the repair of 7,8-dihydro-8-oxo-2"-deoxyguanosine (OG):A and G:A mispairs in DNA. Our approach toward understanding recognition and processing of DNA damage by MutY has been to use substrate analogs that retain the recognition properties of the substrate mispair but are resistant to the glycosylase activity of MutY. This approach provides stable MutY-DNA complexes that are amenable to structural and biochemical characterization. In this work, the interaction of MutY with the 2"-deoxyadenosine analogs 2"-deoxy-2"-fluoroadenosine (FA), 2"-deoxyaristeromycin (R) and 2"-deoxyformycin A (F) was investigated. MutY binds to duplexes containing the FA, R or F analogs opposite G and OG within DNA with high affinity; however, no enzymatic processing of these duplexes is observed. The specific nature of the interaction of MutY with an OG:FA duplex was demonstrated by MPE-Fe(II) hydroxyl radical footprinting experiments which showed a nine base pair region of protection by MutY surrounding the mispair. DMS footprinting experiments with an OG:A duplex revealed that a specific G residue located on the OG-containing strand was protected from DMS in the presence of MutY. In contrast, a G residue flanking the substrate analogs R, F or FA was observed to be hypersensitive to DMS in the presence of MutY. These results suggest a major conformational change in the DNA helix upon binding of MutY that exposes the substrate analog-containing strand. This finding is consistent with a nucleotide flipping mechanism for damage recognition by MutY. This work demonstrates that duplex substrates for MutY containing FA, R or F instead of A are excellent substrate mimics that may be used to provide insight into the recognition by MutY of damaged and mismatched base pairs within DNA.     

Preferential recognition of I.T base-pairs in the initiation of excision-repair by hypoxanthine-DNA glycosylase.

Double-stranded synthetic oligonucleotides with a centrally located dIMP residue in a 5'-32P-labeled strand were employed as substrates for hypoxanthine-DNA glycosylase. The enzyme activity was monitored by the generation of a piperidine-sensitive site in the labeled oligonucleotide. The enzyme was purified approximately 5000-fold from calf thymus. The purified enzyme removed efficiently a hypoxanthine base residue from an I.T base pair, but 15-20 times more slowly from an I.C base pair. Similar results were obtained with oligonucleotides in which the deoxyinosine residue was placed in different surrounding nucleotide sequences. The enzyme had no detectable activity on mismatched G.T, A.G or A.C base pairs. The data indicate that hypoxanthine-DNA glycosylase participates in the repair of deaminated adenine residues in DNA.