Nuclear magnetic resonance (NMR) and model-building studies were carried out on the hairpin form of the octamer d(CGaCTAGCG) (aC = arabinofuranosylcytosine), referred to as the TA compound. The nonexchangeable protons of the TA compound were assigned by means of nuclear Overhauser effect spectroscopy (NOESY) and correlated spectroscopy (COSY). From a detailed analysis of the coupling data and of the NOESY spectra the following conclusions are reached: (i) The hairpin consists of a stem of three Watson-Crick type base pairs, and the two remaining residues, T(4) and dA(5), participate in a loop. (ii) All sugar rings show conformational flexibility although a strong preference for the S-type (C2'-endo) conformer is observed. (iii) The thymine does not stack upon the 3' side of the stem as expected, but swings into the minor groove. (This folding principle of the loop involves an unusual alpha t conformer in residue T(4).) (iv) At the 5'-3' loop-stem junction a stacking discontinuity occurs as a consequence of a sharp turn in that part of the backbone, caused by the unusual beta + and gamma t torsion angles in residue dG(6). (v) The A base slides over the 5' side of the stem to stack upon the aC(3) residue at the 3' side of the stem in an antiparallel fashion. On the basis of J couplings and a set of approximate proton-proton distances from NOE cross peaks, a model for the hairpin was constructed. This model was then refined by using an iterative relaxation matrix approach (IRMA) in combination with restrained molecular dynamics calculations. The resulting final model satisfactorily explains all the distance constraints.

NMR studies were carried out on various equimolar mixtures consisting of a combination of oligomers: d(ACGGCT) (I), d(pACGGCT) (Ia), d(TGCAGT) (II), d(AGCCGTACTGCA) (III), d(TGCAGTACGGCT) (IV). It is shown that I + II + III (MI) INTRODUCTION [1,2]The role of DNA ligases is well established in DNA replication, in DNA repair and in genetic recombination in prokaryotic and in eukaryotic cells [3]. These enzymes catalyse the restoration of an interruption of a single strand in double-helical DNA. For the joining activity the enzyme requires juxtaposed 3'-hydroxyl and 5'-phosphoryl ends aligned in a duplex structure [4]. The enzymes obtained from unaffected and from T4-infected E. coli have been investigated most thoroughly [4]. However, until now little information is available concerning structural details of the nicked duplex structure.The present work describes an NMR study, augmented with biochemical experiments, of a synthetic nicked duplex structure. The compound consists of a 12 base-paired duplex that features an interruption in the centre of one strand of the double helix. The conformational properties of the nicked duplex are compared with those of the intact doublehelical fragment. Furthermore, the influence of removal of the phosphate at the interruption is demonstrated. Thermodynamic analysis of duplex formation of the nicked as well as of the intact duplex structure is used to study the amount of cooperativity of the two hexamer strands in the melting behaviour of the nicked duplex. Finally, the T4 polynucleotide ligase activity upon this synthetic nicked duplex fragment is reported.

The two deoxyribotetranucleoside triphosphates d(T‐A‐T‐A) and d(A‐T‐A‐T) were investigated in aqueous solution by one‐ and two‐dimensional proton NMR at 300 and 500 MHz. It is demonstrated that both compounds occur predominantly in the single‐helical form. Accurate coupling constants are obtained by computer simulation of several 500‐MHz spectra. The data are interpreted in terms of N and S pseudorotational ranges. The geometry of the major S‐type conformers displays a clear sequence dependence, as expressed by variation of the endocyclic backbone angle δ (C5′‐C4′‐C3′‐O3′). A simple sum rule is proposed to predict δ variation in single‐helical DNA fragments. Comparisons are made with other sequence‐dependent geometries as observed in a double‐helical B‐DNA fragment in the crystalline state. Furthermore, one‐ and two‐dimensional nuclear Overhauser effect (NOE) spectroscopy was carried out on d(T‐A‐T‐A). An inventory is made of the observed intra‐ and inter‐residue NOEs. The NOE data confirm the presence of a highly stacked single‐helical conformation of d(T‐A‐T‐A) in solution. No indications are found for the formation of a bulge‐out structure as observed for analogous alternating purine‐pyrimidine oligoribonucleotides.

Chemical shifts of base and H1' protons of the single-stranded hexamers d(ATTACC) and d(GGTAAT), of the 1:1 mixtures of these complementary hexamers, and of the self-complementary dodecamer d(ATTACCGGTAAT) were measured at various temperatures in aqueous solution. Four different sample concentrations were used in the case of the dodecamer and of the mixture of the complementary hexamers; the individual hexamers were measured at two different DNA concentrations. Absorbance temperature profiles at five different NaCl concentrations were measured for the dodecamer in order to quantify the effect of the ionic strength on the duplex formation. Under suitable conditions of nucleotide concentration, temperature, and ionic strength, the dodecamer adopts either a B-DNA duplex or a hairpin-loop structure. Chemical shift vs temperature profiles, constructed for all samples, were used to obtain thermodynamic parameters either for the various stacking interactions in the single strands or for the duplex or the hairpin-loop formation. In the analysis of the duplex formation of the hexamers, a two-state approach appeared too simple, because systematic deviations were revealed. Therefore, a new three-state model (DUPSTAK) was developed. In order to investigate the magnitude of error arising from the use of the two-state approach in cases where the DUPSTAK model appears more appropriate, a series of test calculations was made. The magnitude of error in the enthalpy and in the entropy of duplex melting is found to depend linearly upon the actual melting temperature and not upon the individual delta Hd degrees and delta Sd degrees values. Thermodynamic analysis of the chemical shift vs temperature profiles in D2O solution (no added salt) yields an average Tmd value of 341 K (1M DNA) and delta Hd degrees of - 121 kJ.mol-1 for the dimer/random-coil transition of the hexamer duplex d(ATTACC).d(GGTAAT). For the duplex in equilibrium random-coil transition of the 12-mer d(ATTACCGGTAAT) an average Tmd value of 336 K (1M DNA) and delta Hd degrees of -372 kJ.mol-1 are found. The hairpin/random-coil transition of d(ATTACCGGTAAT) is characterized by a rather large delta Hh degrees value, -130 kJ.mol-1, and an average Tmh value of 304 K.

The self-complementary octamers d(CGCTAGCG) and d(CGaCTAGCG) (aC, arabinofuranosylcytidine) were studied by means of NMR spectroscopy. It is shown that d(CGaCTAGCG), under suitable conditions of oligonucleotide concentration, ionic strength and temperature, exclusively adopts a hairpin structure. However, under the same experimental conditions (5 mM DNA, no added salt, 295 K) d(CCCTAGCG) mainly adopts a B-DNA-type duplex. At lower temperatures (G290 K) the hairpin form of d(CGaCTACCG) occurs in slow exchange with an intact B-DNA-type duplex. When the DNA concentration of d(CGCTAGCG) is dramatically reduced (GO.5 mM) the hairpin form becomes highly favoured at the expense of the dimer. Moreover, protonchemical-shift considerations indicate that the structural features of the hairpin structure of d(CGCTAGCG) mimic, in part, those of the modified octamer d(CGaCTAGCG), i.e. a loop comprising only the two central residues with the thymine located into the minor groove (Pieters, J. M. L., de Vroom, E., van Recently, we carried out an NMR and model-building study on the modified octamer d(CGaCTAGCG) at low DNA concentration, ionic strength and temperature (Pieters et al., unpublished results). Under these experimental conditions the DNA compound exclusively prefers to adopt a monomeric hairpin structure. It was demonstrated that this hairpin structure consists of a double-helical stem formed by two WatsonCrick-type dC . dG base pairs and one aC . dG base pair, which closes the loop formed by the two remaining residues (Fig. 2). The vertical base stacking is not propagated into the loop region; instead, the thymine swings into the minor groove.In order to obtain detailed information regarding the effect of incorporation of aC into the DNA strand upon the Correspondence to C .

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