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A cell wall hydrolase homologue, Bacillus subtilis YddH (renamed CwlT), was determined to be a novel cell wall lytic enzyme. The cwlT gene is located in the region of an integrative and conjugative element (ICEBs1), and a cwlT-lacZ fusion experiment revealed the significant expression when mitomycin C was added to the culture. Judging from the Pfam data base, CwlT (cell wall lytic enzyme T (Two-catalytic domains)) has two hydrolase domains that exhibit high amino acid sequence similarity to dl-endopeptidases and relatively low similarity to lytic transglycosylases at the C and N termini, respectively. The purified C-terminal domain of CwlT (CwlT-C-His) could hydrolyze the linkage of d-γ-glutamyl-meso-diaminopimelic acid in B. subtilis peptidoglycan, suggesting that the C-terminal domain acts as a dl-endopeptidase. On the other hand, the purified N-terminal domain (CwlT-N-His) could also hydrolyze the peptidoglycan of B. subtilis. However, on reverse-phase HPLC and mass spectrometry (MS) and MS-MS analyses of the reaction products by CwlT-N-His, this domain was determined to act as an N-acetylmuramidase and not a lytic transglycosylase. Moreover, the site-directed mutagenesis analysis revealed that Glu-87 and Asp-94 are sites related with the cell wall lytic activity. Because the amino acid sequence of the N-terminal domain of CwlT exhibits low similarity compared with those of the soluble lytic transglycosylase and muramidase (goose lysozyme), this domain represents “a new category of cell wall hydrolases.”

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.

A hyper extracellular protein producer, Bacillus subtilis 327UH, produced large amounts of levan in a medium containing 20% sucrose, and the yield of levan after 10 hours was more than 60%, when based on the fructose amount of sucrose. After transformation of 327UH with a levanase-deficient 168SC (sacC::Cm(r)) chromosomal DNA, a Cm(r) transformant 327UHSC (sacC::Cm(r) degSU(Hy)) produced 3 times longer levan than that of the wild type.

The Escherichia coli RuvC protein endonucleolytically resolves Holliday junctions, which are formed as intermediates during genetic recombination and recombination repair. Previous studies using model Holliday junctions suggested that a certain size of central core of homology and a specific sequence in the junction were required for efficient cleavage by RuvC, although not for binding. To determine the minimum length of sequence homology required for RuvC cleavage, we made a series of synthetic Holliday junctions with various lengths of homologous sequence in the core region. It was demonstrated that a monomobile junction possessing only 2 base pairs of the homology core was efficiently cleaved by RuvC. To study the sequence specificity for cleavage, we made 16 bimobile junctions, which differed only in the homologous core sequence. Among them, 6 junctions were efficiently cleaved. Cleavage occurred by introduction of nicks symmetrically at the 3'-side of thymine in all cases. However, the nucleotide bases at the 3'-side of the thymines were not always identical between the two strands nicked. These results suggest that RuvC recognizes mainly topological symmetry of the Holliday junction but not the sequence symmetry per se, that the thymine residue at the cleavage site plays an important role for RuvC-mediated resolution, and that a long homologous core sequence is not essential for cleavage.

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.

Summary When a sperm and oocyte unite into one cell upon fertilization, membranous fusion between the sperm and oocyte occurs. In mice, Izumo1 and a tetraspanin molecule CD9 are required for sperm-oocyte fusion as one of the oocyte factors, and another tetraspanin molecule CD81 is also thought to involve in this process. Since these two tetraspanins often form a complex upon cell-cell interaction, it is probable that such a complex is also formed in sperm-oocyte interaction; however, this possibility is still under debate among researchers. Here we assessed this problem using mouse oocytes. Immunocytochemical analysis demonstrated that both CD9 and CD81 were widely distributed outside the oocyte cell membrane, but these molecules were separate, forming bilayers, confirmed by immunobiochemical analysis. Electron-microscopic analysis revealed the presence of CD9- or CD81-incorporated extracellular structures in those bilayers. Finally, microinjection of in vitro-synthesized RNA showed that CD9 reversed a fusion defect in CD81-deficient oocytes in addition to CD9-deficient oocytes, but CD81 failed in both oocytes. These results suggest that both CD9 and CD81 independently work upon sperm-oocyte fusion as extracellular components.

To elucidate the conformational properties of a DNA duplex with a 2-deoxyribosylformamide residue (dF), oligomers containing this abasic residue and a propanediol residue in the center of the helix were synthesized and characterized by UV absorption and CD. By a comparison of the Tms of the duplexes containing a dF residue and a propanediol residue (P), the stability of the duplex containing the abasic damage was judged to depend on the kind of nucleotide residue on the opposite side of the abasic residue rather than the kind of abasic residue.

Mitochondrial DNA (mt DNA) from a patulin producer, Penicillium urticae (synonym P. griseofulvum), was 27.8 kb +/- 0.6 kb in size by electron microscopy and 27.2 kb by agarose gel electrophoresis. Restriction endonuclease maps for nine restriction enzymes were constructed, and eleven fragments which covered the total range of the mt DNA were cloned into the Escherichia coli plasmid vector pUC19. Southern analysis of the native genomes of P. urticae and P. chrysogenum with six of the cloned fragments as probes indicated similar genome arrangements as well as similar restriction maps. Both the large and small rRNA genes of P. urticae and P. chrysogenum were located on these restriction maps using Southern hybridization, and the result also supported the similar arrangement. Agarose/formaldehyde gel electrophoresis indicated that the small rRNA was 1.5 kb in size in both species; but, surprisingly, the large rRNA was 4.2 kb in size for P. urticae and 3.5 kb for P. chrysogenum. These sizes were, respectively, 1.1 kb and 0.4 kb larger than those from the very closely related Aspergillus nidulans.

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.

The Bacillus subtilis CwlC and theBacillus polymyxa var. colistinus CwlV are the cell wall lytic N-acetylmuramoyl-l-alanine amidases in the CwlB (LytC) family. Deletion in the CwlC amidase from the C terminus to residue 177 did not change the amidase activity. However, when the deletion was extended slightly toward the N terminus, the amidase activity was entirely lost. Further, the N-terminal deletion mutant without the first 19 amino acids did not have the amidase activity. These results indicate that the N-terminal half (residues 1–176) of the CwlC amidase, the region homologous to the truncated CwlV (CwlVt), is a catalytic domain. Site-directed mutagenesis was performed on 20 highly conserved amino acid residues within the catalytic domain of CwlC. The amidase activity was lost completely on single amino acid substitutions at two residues (Glu-24 and Glu-141). Similarly, the substitution of the two glutamic acid residues (E26Q and E142Q) of the truncated CwlV (CwlV1), which corresponded to Glu-24 and Glu-141 of CwlC, was critical to the amidase activity. The EDTA-treated CwlV1 did not have amidase activity. The amidase activity of the EDTA-treated CwlV1 was restored by the addition of Zn2+, Mn2+, and Co2+ but not by the addition of Mg2+ and Ca2+. These results suggest that the amidases in the CwlB family are zinc amidases containing two glutamic acids as catalytic residues.

N-acetylmuramoyl-L-alanine amidase CwlC of Bacillus subtilis was overproduced in Escherichia coli and purified 21-fold. The amidase hydrolyzed type A cell walls such as B. subtilis. The amidase bound slightly to the Microbacterium lacticum cell wall (type B), but did not entirely hydrolyze it. The presence of calcium or magnesium ion increased the resistance of the amidase to heat denaturation.

A recombinant lipase, CWB-LipB, localized on the Bacillus subtilis cell surface and retaining lipase activity was unstable and not accumulated in a high yield. To improve the accumulation, we examined cell wall binding protease (wprA)- and/or sigma D (sigD)-deficient mutants, and also a NprE and AprA protease-deficient mutant as host strains. The nprE aprA mutation did not lead to a significant increase in the CWB-LipB accumulation. The wprA mutant accumulated a greater amount than the wild-type only in the stationary phase, but the sigD mutant accumulated a greater amount in both the exponential and stationary phases. The double mutant exhibited great accumulation of CWB-LipB, the amount being 36% of the total proteins extracted from the cell surface.

cutL cDNA encoding an extracellular lipase, L1, from Aspergillus oryzae was fused to the cell wall-binding domain (CWB) region of a plasmid, pHCB3R. SDS-polyacrylamide gel electrophoresis (PAGE) and zymography of proteins extracted from the cell surface of Bacillus subtilis 168 harboring a fused lipase plasmid (pHCB3RCL) revealed that the fused gene product, CWB-CutL, was localized in the B. subtilis cell wall and retained lipase activity. B. subtilis WASD (wprA sigD), recently used for the accumulation of CWB-LipB (the CWB protein fused with B. subtilis lipase B), was also a suitable host for the accumulation of CWB-CutL, the amount being 10% of the total proteins extracted from the cell surface.

In order to elucidate the mechanism of AP site recognition by Echerichia coli exonuclease III (exoIII), site-directed mutagenesis of the Tyr109, the Trp212, and the Phe213 residues, which were conserved in the type II AP endonuclease from various organisms and located in the vicinity of the catalytic site, was performed. The exoIII-W212S mutant lacked any detectable AP endonuclease activity and binding ability to the duplex DNA containing an AP site, while the exoIII-Y109S and exoIII-F213W mutants retained a low level of activities (13% and 83%, respectively, compared with wild-type exoIII). This study suggests that the Trp212 is an important component for abasic site recognition by the E. coli exonuclease III.

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.

The Escherichia coli exonuclease III (AP endonuclease VI) is a DNA-repair enzyme that hydrolyzes the phosphodiester bond 5' to an abasic site in DNA. To study how the enzyme recognizes the abasic site, we used oligonucleotides containing a synthetic abasic site at any desired position in the sequence. We prepared oligonucleotides containing an abasic residue such as 2'-deoxyribosylformamide, 2'-deoxyribose, 1',2'-dideoxy ribofuranose or propanediol. Duplex oligonucleotides containing an abasic residue used in this study were cleaved on the 5' side of the abasic site by exonuclease III in spite of the varieties of the bases opposite and adjacent to the abasic site. In addition, we observed that the enzyme cleaved single-stranded oligonucleotides containing an abasic site on the 5' side of the abasic site. These findings suggest that the enzyme may principally recognize the DNA-pocket formed at an abasic site. The indole ring of the tryptophan 212 residue of the exonuclease III is probably intercalated to the abasic site. The tryptophan in the vicinity of the catalytic site is conserved in the type II AP endonuclease from various organisms.

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.

Three dinucleoside monophosphates containing 8, 2'-anhydro-8-thio-9-β-D-arabinofuranosyladenine (As) and its hypoxanthine derivative (Is), AspIs, IspAs and IspIs, were synthesized. Examination of their properties by ultraviolet absorption, circular dichroism and 1H nuclear magnetic resonance measurements and comparison with the properties of AspAs, which has been shown to take a left-handed stacked conformation, showed that all these dimers take a left-handed stacked conformation, and the order of extent of stacking is AspAsaIspAs>AspIsaIspIs. This sequence dependency of stability of stacking can be explained in terms of the mode of base-base overlap in a left-handed stack. A similar explanation may be applicable to the corresponding sequence dependency of natural dimers with a right-handed stack.

Three dinucleoside monophosphates containing 8-bromoguanosine (br8G), (2'-5')C-br8G, (3'-5')C-br8G, and dC-br8G, were synthesized and characterized by UV absorption, CD, and 1H NMR spectroscopy. The 1H NMR data show that all the br8G residues in these dimers take a syn glycosidic conformation. At low dimer strand concentration (5 X 10(-5) M), the UV hypochromicity data suggest that the degree of base stacking decreases in the following order, (2'-5')C-br8G greater than C-G approximately equal to dC-br8G greater than (3'-5')C-br8G. The CD data also suggest little stacking in (3'-5')C-br8G. At high dimer strand concentration (5 X 10(-3) M), only (3'-5')C-br8G shows duplex formation in 0.1 M NaCl. The duplex is assumed to take a left-handed helical structure similar to that of Z-DNA. The Tm of this duplex is surprisingly high for a dimer (about 35 and 45 degrees C at 5 X 10(-3) and 10(-2) M dimer strand concentration, respectively). The above results and the similarity between the CD spectra of (3'-5')C-br8G and poly(G-C) suggest the possible existence of Z-form structure in ribooligo- and ribopolynucleotides with alternating purine-pyrimidine sequences.

Two short DNA helices, d-(CpGpCpG)2 and d-(CpGpCpGpCpG)2, in dilute salt solutions have been studied thoroughly by 1H- and 31P-nmr spectroscopy as model systems for the calibration of nmr theory in its application to nucleic acids research. All the nonexchangeable proton resonances of these two short helices have been assigned by the “incremental method,” “sequential homodecoupling” of the sugar protons, thermal perturbation, and computer spectral simulation, as well as the NOE technique. The CD and uv data were also obtained at either identical or half concentrations of the oligomer used in the nmr studies in order to have comparable optical data for estimating helical content and for assigning conformation. The precise assignment of the sugar proton resonances and their corresponding coupling-constant values provide valuable information about the backbone conformation. The sugar conformation was shown to be predominantly in the 2E-form (>70%). The rotation of the C4′C5′ is in favor of the gg conformation (55–95%), the rotation of the C5′O5′ bond is highly in favor of g′g′ (83–91%), and the rotation of the C3′O3′ bond is restricted to the domain of ϕ′ ≃ 195°. The CD data and the sugar conformational analyses indicate that these CG helices are in the B-form in dilute salt solutions. The assignment of NHN hydrogen-bonded resonances of these two helices are based on their thermal stability. The through-space magnetic field effects on the base protons and the NHN protons in the helices were calculated and compared with the experimental values. This comparison yields two general conclusions: (1) For the base protons, application of both ring-current effects and local anisotropic effects in the computations are preferred; but for the NHN protons, the application of the ring-current effect alone is preferred over the application of both ring-current effects and local anisotropic effects; and (2) while the computed values in the best theoretical treatment cited above did support the conclusion that these CG helices are in the B-conformation, the differences among the computed values for the three conformations, A, A′, and B, are not very large, at least for the CG sequence. All the 31P resonances of these CG helices are unambiguously assigned through a specific heterodecoupling technique established previously [Cheng et al. (1982) Biopolymers21, 697–701]. The complete assignment of all proton resonances and 31P resonance of d-(CpGpCpG)2 and d-(CpGpCpGpCpG)2 helices in the B-form establishes these two deoxy CG helices as valuable model systems for the study of the nmr and polymorphism of nucleic acids.

The conformational properties of purine-pyrimidine and pyrimidine-purine dinucleoside monophosphates in which the glycosidic torsion is fixed to congruent to 120 degree by the formation of a covalent link between the base and the sugar ring are explored by 1H NMR spectroscopy in order to obtain information about the spatial configuration of high anti nucleic acids. The intramolecular stack of the high anti dimers were found to be left handed, in contrast to that (right handed) for natural oligomers, which are low anti. Even though both the high anti pyrimidine-purine and purine-pyrimidine dimers have similar backbone torsion angles, they display widely different relative geometry between the bases; thus in the former there is extensive base-base overlap in the stack, and in the latter there is negligible intramolecular base-base overlap. In addition it was found that purine-pyrimidine systems form miniature double helices in which there is substantial interstrand purine-purine interaction; on the other hand the pyridine-purine high anti dinucleosides have no proclivity to form such base-paired complexes in solution. Mathematical polymerization of the conformation of the high anti purine-pyrimidine dinucleoside monophosphates generates a left handed helix for high anti polynucleotides. This also means that the double helix for high anti-nucleic acids containing purine-pyrimidine repeated units may also be left handed, as had been suggested [Sundaralingam, M., & Yathindra, N. (1977) Int. J. Quantum Chem., Quantum Biol. Symp. 4, 285]. It is suggested that the plasticity in the structure of genomic DNA is such that, if under certain conditions of interactions the sugar-base torsion of certain domains assume high anti values, that domain will become left handed, and this in turn can be a mechanism for the control of expression by genomic DNA.

To elucidate the conformational properties and their chain length dependency in solution of oligodeoxyribonucleotides containing an alternating C-G sequence, which have been shown to take a left-handed duplex structure in crystals, d (C-G), d (C-G-C-G) and d (C-G-C-G-C-G) were synthesized by a modified triester method and characterized by ultraviolet (UV) circular dichroism (CD) and 1H-nuclear magnetic resonance (NMR) spectroscopy. The effects of salt concentration (0.1 M and 4 M NaCl), oligomer strand concentration (10-5-10-2M) and temperature (0°-90°C) on the oligomer conformation were investigated. The dimer does not form a duplex under any conditions studied. The tetramer does form a right-handed duplex in 0.1M NaCl even at 3×10-5M strand concentration. In 4M NaCl, however, it does not form any duplex at the same strand concentration, and left-handed duplex formation is observed at 10-fold higher concentration. The hexamer forms a duplex in either salt concentration even at 2×10-5M strand concentration. The melting temperatures of these duplexes were measured by UV and CD methods. The CD-temperature profile in 4M NaCl, which is not sigmoidal when monitored at the λmin of the characteristic negative band, suggests that melting of the high salt duplex is a rather complex process that involves more than two species. The results of 1H-NMR-temperature studies support left-handed duplex formation in a high salt solution.

Two sequence isomers of dinucleoside monophosphates, 8, 2'-anhydro-8-thio-9-β-D-arabinofuranosyladenylyl-(3'-5')-6, 2'-anhydro-6-oxy-1-β-D-arabinofuranosyluracil (AspU°) (III) and 6, 2'-anhydro-6-oxy-1-β-D-arabinofuranosyluridylyl-(3'-5')-8, 2'-anhydro-8-thio-9-β-D-arabinofuranosyladenine (U°pAs) (IV), were synthesized by condensation of suitably protected nucleoside and nucleotide units using dicyclohexyl carbodiimide as a condensing reagent. Examination of the UV, CD and NMR spectra of these dimers led us to the conclusion that, whereas compound III did not take a stacked conformation, compound IV took a well-stacked conformation, in which the bases were stacked along a left-handed screw axis. The adoption of this conformation could be interpreted in terms of the high base torsion angles in both nucleoside units.

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.

To elucidate the conformational properties and their chain length dependency in solution of oligodeoxyribonucleotides containing an alternating C-G sequence, which have been shown to take a left-handed duplex structure in crystals, d (C-G), d (C-G-C-G) and d (C-G-C-G-C-G) were synthesized by a modified triester method and characterized by ultraviolet (UV) circular dichroism (CD) and 1H-nuclear magnetic resonance (NMR) spectroscopy. The effects of salt concentration (0.1 M and 4 M NaCl), oligomer strand concentration (10-5-10-2M) and temperature (0°-90°C) on the oligomer conformation were investigated. The dimer does not form a duplex under any conditions studied. The tetramer does form a right-handed duplex in 0.1M NaCl even at 3×10-5M strand concentration. In 4M NaCl, however, it does not form any duplex at the same strand concentration, and left-handed duplex formation is observed at 10-fold higher concentration. The hexamer forms a duplex in either salt concentration even at 2×10-5M strand concentration. The melting temperatures of these duplexes were measured by UV and CD methods. The CD-temperature profile in 4M NaCl, which is not sigmoidal when monitored at the λmin of the characteristic negative band, suggests that melting of the high salt duplex is a rather complex process that involves more than two species. The results of 1H-NMR-temperature studies support left-handed duplex formation in a high salt solution.