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The mechanisms by which AP endonucleases recognize AP sites have not yet been determined. Based on our previous study with Escherichia coli exonuclease III (ExoIII), the ExoIII family AP endonucleases probably recognize the DNA-pocket formed at an AP site. The indole ring of a conserved tryptophan residue in the vicinity of the catalytic site presumably intercalates into this pocket. To test this hypothesis, we constructed a series of mutants of ExoIII and human APE1. Trp-212 of ExoIII and Trp-280 of APE1 were critical to the AP endonuclease activity and binding to DNA containing an AP site. To confirm the ability of the tryptophan residue to intercalate with the AP site, we examined the interaction between an oligopeptide containing a tryptophan residue and an oligonucleotide containing AP sites, using spectrofluorimetry and surface plasmon resonance (SPR) technology. The tryptophan residue of the oligopeptide specifically intercalated into an AP site of DNA. The tryptophan residue in the vicinity of the catalytic site of the ExoIII family AP endonucleases plays a key role in the recognition of AP sites.

Eight synthetic Holliday junction (HJ) oligonucleotides containing an immobile or a mobile junction were characterized by gel electrophoresis, ultraviolet absorption and circular dichroism (CD) spectroscopy. Four 24-mer deoxyribonucleotides formed stable immobile and mobile HJs in 0.1 M NaCl at 5 muM strand concentration at room temperature. However, the immobile HJ constructed from four 18-mers was less stable, and four 12-mers did not form the HJ structure under the conditions used. A comparison of the melting profiles of the HJs with those of the duplexes corresponding to the arms of four-way junctions indicated that the thermal stability of the HJ was similar to that of the individual arm and the cooperativity of the melting behavior of the HJ was relatively higher than that of the individual arm duplex. The Tms of the mobile HJs containing 4, 6, 8, and 10 base-pair homologous cores at junctions were essentially identical with that of the immobile HJ of the same size. There is a tendency that the HJ containing a larger homologous core region becomes more resistant to thermal denaturation. The addition of divalent metal cations, Mg2+ and Ca2+, to the solutions of the HJs raised their melting temperatures. The difference found for the CD spectra of the HJs which differ only in the arrangement of the HJ depended primarily upon the DNA sequence flanking the junction. The RuvC protein binds to the immobile and mobile HJs, regardless of the presence and the size of the homologous core at the junction.

The interaction between ethidium bromide and a parallel G4-DNA, which is a quadruplex composed from four oligonucleotides containing a dG cluster, has been investigated. [d(TTGGGGTT)]4 formed a complex with ethidium bromide, which was assumed to be intercalated between the adjacent guanine tetrads of the quadruplex. #This paper is dedicated to Prof. Yoshihisa Mizuno on the occasion of his 75th birthday.

Escherichia coli AP endonuclease (ExoIII) and its human homolog (APE1) have the sole tryptophan residue for AP site recognition (AP site recognizer) but these residues are at different positions near the catalytic sites. On the other hand, many bacterial AP endonucleases have two tryptophan residues at the same positions of both ExoIII and APE1. To elucidate whether these residues are involved in AP site recognition, the ExoIII homologs of Thermoplasma volcanium and Lactobacillus plantarum were characterized. These proteins showed AP endonuclease and 3'-5'exonculease activities. In each enzyme, the mutations of the tryptophan residues corresponding to Trp-280 of APE1 caused more significant reductions in activities and binding abilities to the oligonucleotide containing an AP site (AP-DNA) than those corresponding to Trp-212 of ExoIII. These results suggest that the tryptophan residue corresponding to Trp-280 of APE1 is the predominant AP site recognizer, and that corresponding to Trp-212 of ExoIII is the auxiliary recognizer.

To elucidate whether the tryptophan residues in the vicinity of the catalytic site are involved in AP site recognition and are critical for AP endonuclease activity, the AP endonucleases of the four subtypes in the ExoIII AP endonuclease family were characterized and compared the positions of the tryptophan residues. The positions of the catalytic amino acid residues, corresponding to Glu-34, Asp-229, and His-259 of ExoIII, are strictly conserved. On the other hand, the positions of the tryptophan residues, which are critical to the incipient complex formation, do not exist at a fixed position. There are four patterns at the position of the essential tryptophan residue.

To discover the physiological role of the Bacillus subtilis ExoA protein, which is similar in amino acid sequence to Escherichia coli exonuclease III, an exoA::Cm disruption was constructed in the chromosomal DNA of B. subtilis. There was no clear difference in tolerance to hydrogen peroxide and alkylating agents between the disruptant and the wild type strain. An expression plasmid of the ExoA in E. coli was constructed by inserting the exoA gene into the expression vector pKP1500. The purified ExoA was used to clarify enzymatic characterizations using synthetic DNA oligomers as substrates. A DNA oligomer containing a 1', 2'-dideoxyribose residue as an AP site, a DNA-RNA chimera oligomer, and a 3' end 32P-labeled oligomer were synthesized. It has been shown that the ExoA has AP endonuclease, 3'-5' exonuclease, ribonuclease H, and 3'-phosphomonoesterase activities. Thus, it has been confirmed that ExoA is a multifunctional DNA-repair enzyme in B. subtilis that is very similar to E. coli exonuclease III except that ExoA has lower 3'-5' exonuclease activity than that of E. coli exonuclease III.

To elucidate the conformational properties of DNA with a 2-deoxyribosylformamide residue (dF), an oligodeoxyribonucleotide containing this abasic residue in a specific position of the nucleotide sequence was synthesized by the standard solid-phase phosphotriester method. Deprotection of the synthesized oligonucleotide was performed under routine alkaline and acidic conditions. The presence of a dF residue in the oligomer was confirmed by ion-spray mass spectrometry. A dF residue was found to affect considerably the stability of the DNA duplex, as determined from the melting behavior of the dF-containing duplex.

Oligonucleotides containing a dG cluster, d(TmGnTm), are models of single-stranded parts of telomeric deoxyribonucleic acid and substitutes for poly(dG). Electrophoretic and spectroscopic analyses of the oligomers indicate that the oligomers can form two alternative structures, single- and quadruple-stranded helices, in solution at room temperature. The transformation of the single-strandef form into the quadruple-stranded form or vice versa is undetectable in 0.1 M NaCl at 4.3 x 10(-5) M strand concentration at room temperature. However, at a 50-fold higher strand concentration, the single-stranded oligomer is gradually converted into the quadruplex. An increase in ionic strength stabilizes the single-stranded structure, so it seems to inhibit the formation of the quadruplex. The quadruplex, [d(TTGGGGTT)]4, is resistant to denaturation in 7 M urea, in which the Watson-Crick type d(TTGGGGTT).d(AACCCCAA) duplex dissociates. An increase in the number of T residues facilitates the dissociation of the quadruplex by heating. Thus the number of T residues surrounding the dG cluster might control the rigidity of the quadruplex structure.

To elucidate the mechanism of DNA strand scission by bleomycin, a d(C-G-C-G-C-G) duplex was treated with the bleomycin-iron ion complex in the presence of H2O2 and degradation products (1, 2, cytosine and deoxyguanosine 5'-phosphate) were identified. 1 and 2 contain a carboxymethyl group attached to the 3'-terminal phosphoryl group of d(C-Gp) and d(C-G-C-Gp), respectively. These compounds were identified by UV, 1H and 31P NMR spectroscopy and paper electrophoresis. 1 was synthesized from the protected dinucleotide and glycolic acid and the proton NMR spectrum was identical to that of 1 obtained as a degradation product. Thus the oligonucleotide fragments produced by the action of bleomycin on DNA were directly identified and cleavage of the C3'-C4' bond of the sugar residues was proved.