Participation of glutamic acid 23 of T4 endonuclease V in the beta-elimination reaction of an abasic site in a synthetic duplex DNA.

T4 endonuclease V catalyzes the hydrolysis of the glycosyl bond of a thymine dimer in a DNA duplex and the cleavage of the 3'-phosphate by beta-elimination. We have previously identified a catalytic site for the first reaction (pyrimidine dimer-glycosylase activity) by systematic mutagenesis (Doi et al. Proc. Natl. Acad. Sci. USA 1992 in press) and by x-ray crystallography (Morikawa et al. Science, 256: 523-526, 1992). The results showed that replacement of Glu23 with either glutamine or aspartic acid completely abolished the glycosylase activity. We describe the investigation of the second reaction (apurinic/apyrimidinic endonuclease activity), using twenty two mutants of T4 endonuclease V plus a DNA mini duplex containing an abasic site. Replacement of Glu23 by glutamine abolished the second reaction, but replacement with aspartic acid did not. The pH optima of the mutant (23 Asp) and the wild type were found to be 5.0 and 5.5, respectively. We conclude that the carboxylate anion in position 23 may act as a general base in the beta-elimination reaction of the endonuclease.     

Sequence-dependent cleavage of DNA by alkylation with antisense oligodeoxyribonucleotides containing a 2-(N-iodoacetylaminoethyl)thio-adenine.

Antisense oligodeoxyribonucleotides (15mers), containing a 2-(N-iodoacetylaminoethyl)thio-adenine, were synthesized and tested for their ability to cleave complementary DNAs (21mers). Cleavage of the target DNAs was done by alkylation followed by treatment with piperidine, and the positions of the alkylated sites were estimated by identification of the cleaved products. By using several combinations of the modified strands and their target DNAs, it was determined that alkylation occurred at adenine or guanine, depending on the torsion angle of the modified nucleoside.     

NMR studies for identification of dI:dG mismatch base-pairing structure in DNA.

One- and two-dimensional NMR experiments have been undertaken to investigate deoxyinosine:deoxyguanosine (dI:dG) base pairing in a self-complementary dodecadeoxyribonucleotide, d(C1-G2-C3-I4-A5-A6-T7-T8-G9-G10-G11-G12) (designated IG-12), duplex. The NMR data indicate formation of a dI(syn):dG(anti) base pair in a B-DNA helix. This unusual base pairing results in altered NOE patterns between the base protons (H8 and H2) of the I4 residue and the sugar protons of its own and the 5'-flanking C3 residues. The dI(syn):dG(anti) base pair is accommodated in the B-DNA duplex with only a subtle distortion of the local conformation. Identification of the dI:dG base pairing in this study confirms that a hypoxanthine base can form hydrogen-bonded base pairs with all of the four normal bases, C, A, T, and G, in DNA.     

Characterization of interaction between Tb3+ and porcine intestinal brush-border membranes

Effects of ionic strength and temperature on the interaction between Tb3+ and porcine intestinal brush-border membrane vesicles were studied. When Tb3+ was added to the vesicle suspension, Tb3+ fluorescence increased with increasing concentration of Tb3+, showing a saturation. The apparent dissociation constant of one of at least two components of this binding reaction was estimated to be about 12.5 microM at 25 degrees C, pH 7.4. But the affinity of Tb3+ for the membrane vesicles was variable with changes of ionic strength and temperature. The affinity was lowered by addition of KCl to medium and by increase of temperature above 30 degrees C. In addition, temperature-induced change in the affinity of Tb3+ for the membranes was reversible over a temperature range from 13 to 46 degrees C. Temperature-dependence profiles of the excimer formation efficiency of pyrene-labeled membranes and of the harmonic mean of the rotational relaxation times of pyrene molecules in the membranes revealed that the phase transition of the membrane lipids occurs at about 30 degrees C. Based on these results, characteristics of Tb3+ binding to the membranes are discussed in relation to the nature of lipid phase and surface charges of the membranes.     

Modification of the amino acid acceptor stem of E. coli tRNAMetf by ligation of chemically synthesized ribooligonucleotides

The single-stranded region of the amino acid acceptor stem corresponding to the 3'-end of E. coli tRNAMetf was replaced by ligation of chemically synthesized ribooligonucleotides, in order to change the length of the single-stranded CCA terminus. The chemically synthesized ribooligomers, CCA, ACCA, AACCA and CAACCA, were ligated to nuclease-treated E. coli tRNAMetf, which lacked the ACCA sequence at the 3'-end. The methionine acceptor activities of these modified tRNAs were examined using E. coli methionyl-tRNA synthetase. Ligation of the chemically synthesized pentamer (AACCA) to the acceptor terminus restored the methionine acceptor activity, whereas ligation of the hexamer (CAACCA) or trimer (CCA) to the acceptor terminus did not Modification of the acceptor terminus had no effect on the formylation of accepted methionine.     

Production of monoclonal antibodies against Clostridium botulinum type E derivative toxin

Abstract Five monoclonal antibodies (MCA; E–8–2, 9–1, 11–2, 12–4, and 13–1) against Clostridium botulinum type E derivative toxin were prepared. Their ELISA titers were higher than or equivalent to that of conventional polyclonal antibody. Three of them (E-8–2, 12–4, and 13–1) possessed the neutralizing activity comparable to that of polyclonal antibody. The results of binding-competition experiments indicated that the monoclonal antibodies bound to different sites on the type E toxin molecule. Immunoblotting analyses demonstrated that E-8–2, 9–1, and 11–2 react to fragment I (heavy chain) of the toxin. By use of these monoclonal antibodies, it may be possible to scrutinize the structure-function relationship of botulinum toxins and cross reactions between type E and F toxins.     

Anti-O-phosphotyrosine antibodies in human sera

Antibodies reactive with O-phosphotyrosine (PTYR) were detected in 60 out of 621 inpatients, with high frequencies in hematologic and lung malignancies, hepatic diseases, cerebrovascular diseases, and autoimmune diseases. Affinity-purified antibodies proved capable of recognizing PTYR-containing proteins in a human carcinoma cell line, A431, both by immunofluorescent staining and by immunoaffinity chromatography, but had no detectable affinity for phosphorylated serine or threonine, or for the nucleotides tested. In these respects, the antibodies observed in human sera were indistinguishable from anti-PTYR antibodies raised experimentally in rabbits or mice.     

Raman diagnosis of nucleic acid structure: sugar-puckering and glycosidic conformation in the guanosine moiety.

Observations of Raman spectra of various nucleic acids indicate that the guanine ring breathing frequency is sensitive to the internal rotation angle around the glycosidic bond and to the conformation of the five-membered ring of the ribose residue that is directly connected with the guanine residue in question. It is found that 682 cm-1 for C2'-endo-anti, at 665 cm-1 for C3'-endo-anti, and at 625 cm-1 for C3'-endo-syn. A DNA octamer d(GpGpApApTpTpCpC) shows, in its aqueous solution, a broad Raman band at 680 cm-1 with a tail at 670 cm-1. This fact suggests that the guanosine residues in this oligomer take primarily C2'-endo-anti conformation but an appreciable amount of fluctuation of the ribose ring structure towards C3'-endo is involved.