Muneo Sasaki

On the mechanism of preferential incorporation of dAMP at abasic sites in translesional DNA synthesis. Role of proofreading activity of DNA polymerase and thermodynamic characterization of model template-primers containing an abasic site.

DNA polymerase preferentially incorporate dAMP opposite abasic sites (A-rule). The mechanism of the A-rule can be studied by analyzing three dissected stages of the reaction including (i) initial nucleotide insertion, (ii) proofreading excision of the inserted nucleotide and (iii) extension of the nascent primer terminus. To assess the role of the stage (ii) in the A-rule, kinetic parameters of the proofreading excision of primer terminus nucleotides opposite abasic sites were determined using E.coli DNA polymerase I Klenow fragment. The relative efficiency of the excision (Vmax/Km) revealed that removal of A was the least favored of the four nucleotides, but the differences in the efficiencies between excision of A and the other nucleotides was less than 2-fold. In addition, in an attempt to reconcile kinetic data associated with the stage (i) or (ii), the differences in free energy changes (delta delta G degrees) for the formation of model template-primer termini containing XN pairs (X = abasic site, N = A, G, C or T) were determined by temperature dependent UV-melting measurements. The order of delta delta G degrees was XG > XA = XC > or = XT, with delta delta G degrees being 0.5 kcal/mol for the most stable XG and the least stable XT. Based on these data, the role of the stage (ii) and energetic aspects of the A-rule are discussed.

Application of the Thermodynamic Parameters of DNA Stability Prediction to Double-Helix Formation of Deoxyribooligonucleotides

Abstract The energetics of a double helix formation of deoxyribooligonucleotides has been studied by measuring optical melting curves and calculating thermodynamic values based on the nearest neighbor model and parameters. The results show that, though the model is valid, the values of the nearest-neighbor thermodynamic parameters reported previously should be improved for some base pairs in order to predict more precisely the stability of the double helix of the oligomers by the parameters.

RNA/DNA hybrid duplexes with identical nearest-neighbor base-pairs have identical stability

Energetic behaviors of eight pairs of RNA/DNA hybrid duplexes with identical nearest neighbors have been investigated by UV melting analysis. In the pairs with identical nearest‐neighbor pairs, the melting curve traces at the same strand concentration were very similar. The average difference in stabilization energy of these pairs was 4%, which was about expected within experimental error. These results indicate that the nearest‐neighbor model is valid for predicting the stability of RNA/DNA hybrid duplexes as well as RNA/RNA and DNA/DNA duplexes.

The Stability of DNA and RNA G-Quartets †

Abstract We have investigated the thermodynamic stability of DNA and RNA G-quartet by circular dichroism spectroscopy, gel electrophoresis, and melting analysis. The free energy (δG°37) for G-quartet formation of d(TTGGGG)4 and r(UUGGGG)4 at 100 mM NaCl and 37°C were 1.4 kcal mol-1 and −3.0 kcal mol-1, respectively. On the other hand, at 100 mh4 KCI, δG°37 of the DNA and RNA were −10.0 kcal mol-1 and −8.2 kcal mol-1. This result indicates that the dependence of DNA G-quartet stability on these ions is larger than that of RNA. †This paper is dedicated to Dr. Yoshihisa Mizuno on the occasion of his 75th birthday.

Substituent effects on proton tunnelling. Reaction between 2,4,6-trinitrotoluene and 1-substituted piperidines in acetonitrile

The proton/deuteron-transfer reaction of 2,4,6-trinitrotoluene (TNT) with 1-methylpiperidine, 1-ethylpiperidine or 1-phenethylpiperidine in acetonitrile is a simple reaction forming a 1:1 ion pair. The proton- and deuteron-transfer reactions have been followed at 5–35 °C by a stopped-flow method. The isotope rate ratio kH/kD at 25 °C is 14.7–16.2, the activation-energy difference Ea(D)–Ea(H) is 9.2–10.9 kJ mol–1 and the ratio of Arrhenius pre-exponential factors A(D)/A(H) is 2.5–4.0 for the forward reaction. Similar values of kH/kD, Ea(D)–Ea(H) and A(D)/A(H) are also found for the reverse reaction. All these values signify a tunnelling contribution to the proton-transfer reaction. An analysis based on an unsymmetrical parabolic potential barrier shows that as the bulkiness of the substituted group in the piperidines increases the top of the barrier becomes much rounder, and so tunnelling decreases.

Interactions of 5-(1-Pyrenyl)-10,15,20-tris(4-methylpyridinium)porphine with Double-Helix and Triple-Helix Nucleic Acids †

Abstract 5-(1 -Pyrenyl)-10,15,20-tris(4-methylpyridinium)porphine (H2PTMPP) having a porphyrin ring and a pyrenyl substituent was synthesized. The compound H2PTMPP bound to poly(dA)•poly(dT) double helix and poly(dA)•2poly(dT) triple helix in different styles. The results of H2PTMPP binding to oligonucleotides, dA14•dT14 and dA14•2dT14, was also shown. †This paper is dedicated to Dr. Yoshihisa Mizuno on the occasion of his 75th birthday.

Relationship between catalytic activity and secondary structure of a hammerhead ribozyme: A study using thermodynamic parameters for RNA structure prediction

Abstract The stabilization energy for the secondary structures of wild-type hammerhead and mutant ribozymes has been calculated at different salt conditions and temperatures by using the thermodynamic parameters for RNA structure prediction. The most stable structure at each condition has been searched and the obtained secondary structure is compared with the structure suggested phylogenetically or experimentally. The results indicate that the hammerhead-type secondary structure of the ribozyme and its reactivity correlate with each other. The multibranched loop containing the self-cleavage site of the ribozyme particularly should be a key structure in the hammerhead ribozyme reaction. The predicted secondary structures also suggest that the reactivity of the hammerhead ribozyme should be very much lower at 10°C than that at 37°C.

Application of improved free-energy parameters of RNA stability to codon-anticodon interactions

Free-energy parameters of the RNA duplex stability have been applied in order to investigate the complex between codon oligomers and anticodon loops in transfer RNA. The results suggested that the very large stability of the complex might be due to the stacking interactions of adjacent bases on the codon–anticodon helix and an additional hydrogen-bond formation of the core helix as well as a conformational change of the nucleic acid.Free-energy parameters of the RNA duplex stability have been applied in order to investigate the complex between codon oligomers and anticodon loops in transfer RNA. The results suggested that the very large stability of the complex might be due to the stacking interactions of adjacent bases on the codon–anticodon helix and an additional hydrogen-bond formation of the core helix as well as a conformational change of the nucleic acid.

Physico-chemical analysis on chemical bonding at adhesion interface between rubber and Pd alloy

Abstract Some spectroscopic analyses were carried out on the adhesive interface layers formed by cure-adhesion of natural rubber to electroless Pd and/or Pd–P alloys. The transmission electron microscopy revealed some clear images of the adhesion layer just at the interface between rubber and electroless-plating films of Pd and/or Pd–P alloys. The content ratio of Pd/S analyzed by energy dispersive X-ray spectroscopy changed gradually along the depth at the rubber–metal interface layer for Pd alloy, but was kept almost constant irrespective of depth for Pd–P alloy.

Melting Behavior and Stability of (dA)8·(dT)8 Double Helix

Abstract Melting behavior and stability of double helix of octadeoxyribonucleotides, (dA)8·(dT)8, have been studied by a UV measurement and a calculation of nearest-neighbor model. The helix of (dA)8·(dT)8 exhibited the thermodynamic parameters similar to those of B-form DNA.