Dihydrothymine lesion in X-irradiated DNA: characterization at the molecular level and detection in cells.

PURPOSE: To study the formation of the dihydrothymine lesion produced in DNA by ionizing radiation in an anaerobic environment. MATERIALS AND METHODS: The dihydrothymine lesion, along with other lesions, was isolated from an X-irradiated aqueous solution of the dinucleoside monophosphate d(TpA) and analysed by correlated two-dimensional nuclear magnetic resonance spectroscopy. The dihydrothymine lesion was obtained by enzymatic digestion of irradiated DNA in the form of modified dinucleoside monophosphates, d(T(d)A), where T(d) stands for dihydrothymidine. Liquid chromatography-tandem mass spectrometry was used to detect the lesion in the DNA of X-irradiated mouse fibroblast cells. RESULTS: The modified dinucleoside monophosphate, d(T(d)pA), fragments by two pathways so that altogether the lesion could be detected using two different sets of tandem mass spectrometry (precursor ion mass/daughter ion mass) values. CONCLUSION: The dihydrothymine lesion is a significant lesion in cells exposed to ionizing radiation in an anaerobic environment.

An NMR and conformational investigation of the trans-syn cyclobutane photodimers of dUpdT.

Both trans-syn cyclobutane-type photodimers of 2'-deoxyuridylyl (3'-5') thymidine (dUpdT) were formed by deamination of the corresponding trans-syn cyclobutane photodimers of 2'-deoxycytidylyl (3'-5') thymidine (dCpdT) and were examined by 1H-, 13C-, and 31P-nmr spectroscopy. One- and two-dimensional nmr experiments provided a nearly complete assignment of the 1H, 13C, and 31P resonances. Scalar and nuclear Overhauser effect contacts were used to determine the conformation of the deoxyribose rings, exocyclic bonds, cyclobutane rings, and glycosidic linkages. Isomer I (S-type class; CB-; SYN-ANTI) and isomer II (N-type class; CB+; ANTI-SYN) exhibit markedly different conformational features. 31P chemical shifts show that the relative flexibility is dUpdT > isomer II > isomer I. The conformations of these species are very similar to those of other previously examined trans-syn photodimers. Among bipyrimidine photodimers of a given diastereomeric form (i.e., trans-syn I or II), the nmr-derived conformational parameters are nearly invariant, regardless of base substitution pattern. This contrasts with the substituent-dependent variation of cyclobutane ring conformation observed by Kim et al. (Biopolymers, 1993, Vol. 33, pp. 713-721) for an analogous series of cis-syn photodimers. Steric crowding of cyclobutane ring substituents is offered as an explanation for the difference in substituent effects between the families of cis-syn and trans-syn photodimers.

Synthesis of oligosaccharides containing beta-D-Gal-(1-->3)-O-(6-O-sulfo-beta-D-GlcNAc) as a terminal unit.

The chemical synthesis of beta-D-Gal-(1-->3)-6-O-SO3Na-beta-D-GlcNAc-(1-->6)-alpha-D-Man-O-+ ++C6H4NO2 (1) and beta-D-Gal-(1-->3)-6-O-SO3Na-beta-D-GlcNAc-(1-->2)-alpha-D-Man-OMe (2) is reported using a key glycosyl donor, phenyl O-(2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl)-(1-->3)-4,6-di-O- chloroacetyl-2-deoxy-2-phthalimido-1-thio-beta-D-glucopyranoside (3).

An NMR and conformational investigation of the trans-syn cyclobutane photodimers of dTpdU.

Two trans-syn cyclobutane photodimers of thymidylyl (3'-5') deoxyuridine were formed by deamination of the corresponding trans-syn cyclobutane photodimers of thymidylyl (3'-5') deoxycytidine and were examined by 1H-, 13C-, and 31P-nmr spectroscopy. Correlation spectroscopy, nuclear Overhauser enhancement spectroscopy, and one-dimensional heterodecoupling experiments allowed a more complete assignment of the 1H spectra, compared with previous reports by Koning et al. [(1991) European Journal of Biochemistry, Vol. 195, pp. 29-40] and Liu and Yang [(1978) Biochemistry, Vol. 17, pp. 4865-4876]. Deoxyribose ring conformations were calculated from 1H coupling constants by pseudorotational analysis, and rotamer distributions of exocyclic bonds were calculated from the observed homonuclear and heteronuclear coupling constants. The cyclobutane ring configuration (CB) of each isomer was identified, using arguments based upon observed scalar and dipolar couplings. Glycosidic bond conformation was ascertained from nuclear Overhauser enhancements observed between base and deoxyribose protons. Isomer I (S-type class; CB-; SYN-ANTI) and isomer II (N-type class; CB+; ANTI-SYN) exhibit markedly different conformational features. 31P chemical shifts and exocyclic bond rotamer distributions indicate diminished backbone flexibility for both photoproducts relative to parent thymidylyl (3'-5') deoxyuridine. Isomer I (SYN-ANTI) is particularly rigid, while isomer II (ANTI-SYN) maintains some flexibility. Also, 13C spectra were acquired and assigned unequivocally with the aid of short- and long-range two-dimensional heteronuclear shift correlation experiments.

A 5-4 pyrimidine-pyrimidone photoproduct produced from mixtures of thymine and 4-thiouridine irradiated with 334 nm light.

The nucleoside 4-thiouridine, present in some bacterial tRNA species, is known to be a chromophore and a target for near-UV light-induced growth delay and also mediates both photoprotection and near-UV cell killing in various bacterial strains. To investigate the photoreaction of 4-thiouridine with DNA or its precursors, we irradiated aqueous mixtures of thymine and 4-thiouridine with 334 nm light and then separated photoproducts using two or more stages of reversed-phase high performance liquid chromatography. The two equally abundant major photoproducts were analyzed by UV absorbance spectrophotometry, fast-atom bombardment and electron-impact mass spectrometry, and 1H- and 13C-NMR spectroscopy, and have been identified as two diastereomers of 6-hydroxy-5-[1-(beta-D-erythro-pentofuranosyl)-4'-pyrimidin-2'- one]dihydrothymine (O6hThy[5-4]Pdo), of molecular weight = 370.32. These two diastereomers, although stable at room temperature or below, are interconvertible by heating (90 degrees C for 5 min) in aqueous solution. The possible biological significance of this photoproduct is discussed, and an application as a crosslinker for oligonucleotides to selectively block replication is suggested.