Solution-state structure of the Dewar pyrimidinone photoproduct of thymidylyl-(3'----5')-thymidine

The preparation, spectroscopic investigation, structure determination, conformational analysis, and modeling of the Dewar pyrimidinone photoproduct of thymidylyl-(3'----5')-thymidine, previously referred to as TpT3 [Johns, H. E., Pearson, M. L., LeBlanc, J. C., & Heilleiner, C. W. (1964) J. Mol. Biol. 9, 503-524], is described. TpT3 was prepared in quantitative yield by photolysis of an aqueous solution of the (6-4) photoproduct of TpT with Pyrex-filtered medium-pressure mercury arc light. TpT3 was analyzed by FAB MS, IR, UV, and 1H, 13C, and 31P NMR spectroscopy. The spectroscopic data led to the conclusion that TpT3 results from the photoisomerization of the pyrimidinone ring of the (6-4) product of TpT to its Dewar valence isomer. Torsion angle and interproton distance information derived from coupling constants and NOE data was used to constrain ring conformation searches by utilizing the SYBYL molecular modeling program subroutine SEARCH. Sets of angles derived from the ring search procedure were then used to construct structures whose geometries were optimized by the energy-minimization subroutine MAXIMIN. A two-state model for the solution-state structure of the Dewar photoproduct was chosen which was energetically sound, fit the experimental coupling constants with an RMS deviation of 1.15 Hz, and was consistent with the NOE data. The model for the Dewar photoproduct was compared to a model for the (6-4) photoproduct and the TpT subunits of the Dickerson dodecamer structure by a least-squares fitting procedure. It was concluded that the Dewar photoproduct more closely resembles a B-form TpT unit than does the (6-4) photoproduct.     

The structure of d(TpA), the major photoproduct of thymidylyl-(3'5')-deoxyadenosine.

Irradiation of the dinucleotide TpdA and TA-containing oligonucleotides and DNA produces the TA* photoproduct which was proposed to be the [2+2] cyclo-addition adduct between the C5-C6 double bonds of the T and the A [Bose,S.N., Kumar,S., Davies,R.J.H., Sethi,S.K. and McCloskey,J.A. (1984) Nucleic Acids Res. 12, 7929-7947]. The proposed structure was based on a variety of spectroscopic and chemical degradation studies, and the assignment of a trans-syn-I stereochemistry was based on an extensive 1H-NMR and molecular modeling study of the dinucleotide adduct [Koning,T.M.G., Davies,R.J.H. and Kaptein,R. (1990) Nucleic Acids Res. 18, 277-284]. However, a number of properties of TA* are not in accord with the originally proposed structure, and prompted a re-evaluation of the structure. To assign the 13C spectrum and establish the bond connectivities of the TA* photoproduct of TpdA [d(TpA)*], 1H-13C heteronuclear multiple-quantum coherence (HMQC) and heteronuclear multiple bond correlation (HMBC) spectra were obtained. The 13C shifts and connectivities were found to be inconsistent with the originally proposed cyclobutane ring fusion between the thymine and adenine, but could be explained by a subsequent ring-expansion reaction to give an eight-membered ring valence isomer. The new structure for the d(TpA)* resolves the inconsistencies with the originally proposed structure, and could have a stereochemistry that arises from the anti, anti glycosyl conformation found in B form DNA.     

Mutation spectra of TA*, the major photoproduct of thymidylyl-(3'5')-deoxyadenosine, in Escherichia coli under SOS conditions.

The biological activity of TA*, the major photoproduct of thymidylyl-(3',5')-deoxyadenosine, has remained speculative since it was identified a decade ago. To determine the mutagenicity of TA* in Escherichia coli, we constructed the replicative form of an M13mp18-derived phage containing TA* in the (-)-strand by polymerase-catalyzed elongation of a TA*-containing 49mer opposite a uracil-containing (+)-strand of the phage. The in vitro synthesis mixture was transfected into an ung+, phr- E.coli host and the progeny were screened with a hybridization probe unique for the (-)-strand. TA* was found to block DNA replication substantially in the absence of SOS, but under SOS, TA* was bypassed more efficiently and was highly mutagenic. Among 56 analyzed (-)-strand progeny from two transfections, 46 (82%) were mutants, including six (11%) tandem mutants. The most abundant mutation was a 3'A-->T substitution (31/46, 56%). The possible biological consequences of TA* formation in the highly conserved TATA box consensus sequence on gene expression are discussed in light of the mutagenicity of TA*.     

A comparative conformational study of thymidylyl(3'----5')-thymidine, thymidylyl(3'----5')-5'-thio-5'- deoxythymidine and thymidinylacetamido-[3'(O)----5'(C)]-5'-deoxythymidine

A comparative 270 MHz NMR spectroscopic study on the solution structure of the dimer d(TpT) 1, and its two analogues, namely, d(TpST) 2, and NH2d(TcmT) 4 has been reported. Analysis of chemical shifts and coupling constants indicate that: (i) The sugar moieties of the constituent nucleotides are not affected by modification of the internucleotide linkages and adopt preferentially an S-type conformation. (ii) The C4'-C5' bond in the pT part of the modified dimers 2 and 4 shows a large conformational freedom (gamma+ = 32% and 35%, respectively) compared to 1 (gamma+ = 75%). (iii) The population of the trans conformer about C5'-O5' is less important in d(TpST) 2 compared to d(TpT) 1. (iv) The C3'-O3' bond in 2 adopts a trans conformation as in 1. (v) The glycosidic bonds in the modified dimers 2 and 4 showed preferential syn conformation. UV and CD data show that the modified dimers 2 and 4 have poor tendency to stack intramolecularly, they also base pair less efficiently with d(ApA) as compared to d(TpT) 1.     

Photooxidation of d(TpG) by riboflavin and methylene blue. Isolation and characterization of thymidylyl-(3',5')-2-amino-5-[(2-deoxy-beta-D- erythro-pentofuranosyl)amino]-4H-imidazol-4-one and its primary decomposition product thymidylyl-(3',5')-2,2-diamino-4-[(2-deoxy-beta-D- erythro-pentofuranosyl)amino]-5(2H)-oxazolone.

The major initial product of riboflavin- and methylene blue-mediated photosensitization of 2'-deoxyguanosine (dG) in oxygen-saturated aqueous solution has previously been identified as 2-amino-5-[(2-deoxy-beta-D-erythro-pentofuranosyl)amino] 4H-imidazol-4-one (dlz). At room temperature in aqueous solution dlz decomposes quantitatively to 2,2-diamino-4-[(2-deoxy-beta-D-erythro- pentofuranosyl)amino]-5(2H)-oxazolone (dZ). The data presented here show that the same guanine photooxidation products are generated following riboflavin- and methylene blue-mediated photosensitization of thymidylyl-(3',5')-2'-deoxyguanosine [d(TpG)]. As observed for the monomers, the initial product, thymidylyl-(3',5')-2-amino-5-[(2-deoxy- beta-D-erythro-pentofuranosyl)amino]-4H-imidazol-4-one [d(Tplz)], decomposes in aqueous solution at room temperature to thymidylyl-(3',5')-2,2-diamino-4- [(2-deoxy-beta-D-erythro-pentofuranosyl)amino]-5(2H)-oxazolone [d(TpZ)]. Both modified dinucleoside monophosphates have been isolated by HPLC and characterized by proton NMR spectrometry, fast atom bombardment mass spectrometry, chemical analyses and enzymatic digestions. Among the chemical and enzymatic properties of these modified dinucleoside monophosphates are: (i) d(Tplz) and d(TpZ) are alkali-labile; (ii) d(Tplz) reacts with methoxyamine, while d(TpZ) is unreactive; (iii) d(Tplz) is digested by snake venom phosphodiesterase, while d(TpZ) is unaffected; (iv) relative to d(TpG), d(TpZ) and d(Tplz) are slowly digested by spleen phosphodiesterase; (v) d(Tplz) and d(TpZ) can be 5'-phosphorylated by T4 polynucleotide kinase. The first observation suggests that dlz and dZ may be responsible for some of the strand breaks detected following hot piperidine treatment of DNA exposed to photosensitizers.     

Conformational multiplicity of the antibody combining site of a monoclonal antibody specific for a (6-4) photoproduct.

The antigen binding site of monoclonal antibody 64M5, which possesses a high degree of affinity for DNA containing pyrimidine (6-4) pyrimidone photoproducts, were investigated by use of stable-isotope-assisted NMR spectroscopy. A variety of 64M5 Fab fragments specifically labeled with 13C and 15N at backbone amide groups were prepared. Extensive assignments of amide resonances originating from the variable region of 64M5 were made by using 2D-HN(CO) measurements along with recombination of the heavy and light chains of 64M5. On the basis of chemical shift changes of the amide resonances caused upon addition of d(T[6-4]T) and d(GTAT[6-4]TATG), the binding sites of 64M5 Fab for the (6-4) photodimer and for the oligodeoxynucleotides flanking it were identified. It was revealed that the L1 and L3 segments, which are responsible for the binding to (6-4) photodimer, exhibit conformational multiplicities in the absence of antigens, and take different conformations between the d(T[6-4]T) and d(GTAT[6-4]TATG)-bound forms. On the basis of spectral comparison with another Fab fragment with a similarity in the amino acid sequence of the VL domain of 64M5, we suggest that the conformational multiplicities observed in the present study is caused by a substitution of an amino acid residue at the position of a key residue in L3 canonical structure, which leads to a preferable effect on the antigen binding, and by a specific combination of L1 and L3 canonical structures.     

Synthesis and antiviral activity assay of novel (E)-3',5'-diamino-5-(2-bromovinyl)-2',3',5'-trideoxyuridine

(E)-3',5'-diamino-5-(2-bromovinyl)-2',3',5'-trideoxyuridine (5), the diamino analogue of BVDU (1), was synthesized from BVDU. In contrast with BVDU, compound 5 did not show activity against herpes simplex virus or varicella-zoster virus.     

Chemical synthesis of oligodeoxyribonucleotides containing the Dewar valence isomer of the (6-4) photoproduct and their use in (6-4) photolyase studies

The pyrimidine(6–4)pyrimidone photoproduct, a major UV lesion formed between adjacent pyrimidine bases, is transformed to its Dewar valence isomer upon exposure to UVA/UVB light. We have synthesized a phosphoramidite building block of the Dewar photoproduct formed at the thymidylyl(3′–5′)thymidine site and incorporated it into oligodeoxyribonucleotides. The diastereoisomers of the partially protected dinucleoside monophosphate bearing the (6–4) photoproduct, which were caused by the chirality of the phosphorus atom, were separated by reversed-phase chromatography, and the (6–4) photoproduct was converted to the Dewar photoproduct by irradiation of each isomer with Pyrex-filtered light from a high-pressure mercury lamp. The Dewar photoproduct was stable under both acidic and alkaline conditions at room temperature. After characterization of the isomerized base moiety by NMR spectroscopy, a phosphoramidite building block was synthesized in three steps. Although the ordinary method could be used for the oligonucleotide synthesis, benzimidazolium triflate as an alternative activator yielded better results. The oligonucleotides were used for the analysis of the reaction and the binding of Xenopus (6–4) photolyase. Although the affinity of this enzyme for the Dewar photoproduct-containing duplex was reportedly similar to that for the (6–4) photoproduct-containing substrate, the results suggested a difference in the binding mode.     

Solution structure of a nucleic acid photoproduct of deoxyfluorouridylyl-(3'-5')-thymidine monophosphate (d-FpT) determined by NMR and restrained molecular dynamics: structural comparison of two sequence isomer photoadducts (d-U5p5T and d-T5p5U).

Acetone-sensitized irradiation using UV-B (sun lamp, lambda max = 313 nm) of deoxyfluorouridylyl-(3'-5')-thymidine monophosphate (d-FpT, F = fluorouracil), produces two major photoproducts, the cis-syn cyclobutane-type photodimer and a defluorinated (5-5) photoadduct, d-U5p5T. Product distribution is dependent on the pH of the irradiation solution, as was the case of irradiated d-TpF. At high pH (8-10) the (5-5) photoadduct is the major photoproduct. Irradiation of d-FpT shows a much faster photodegradation rate than the sequence isomer d-TpF. Multinuclear NMR experiments establish the formation of (5-5) covalent bonding between the C5 (d-U5p-, where the fluorine had been) and the C5 (-p5T) and the C6 (-p5T) acquires an OH group. NOE interproton distances and dihedral angles derived from J coupling analysis are constrained to refine model structures of d-U5p5T in restrained molecular dynamics calculations. The resultant structures obtained show 5S-6S as the most chiralities of the C5 and C6 atoms of the thymine, which is the opposite chirality to the corresponding atoms in the sequence isomer d-T5p5U. The orientation of the C5 substituents (-p5T fragment), the CH3 and the uracil are pseudo-axial and pseudo-equatorial respectively. Glycosidic angles are in the anti regions for both the d-U5p- and -p5T residues. Averaged backbone conformations of the two photoadducts, d-U5p5T and d-T5p5U, are similar, although the overall structure of d-U5p5T appears much more flexible than that of d-T5p5U. In particular, the sugar conformations of the 5'-end residues show a remarkable difference in flexibility.     

Histone specific proteinase and DNase activity of spleen lymphocyte nuclei during oncogenesis and under the effect of hydrobromide-5-(5',6' benzocoumaroyl-3')-methylaminouracil

The features of histone proteolysis, their carbonylation level and endonucleolysis intensity were studied in the spleen lymphocytes of rats with transplanted Guerin's carcinoma and under the conditions of hydrobromide-5-(5',6' benzocoumaroyl-3')-methylaminouracil (BCU) action. The intensification of oxidizing histone destruction and histone-specific protease activity during oncogenesis and under conditions of BCU action was shown. DNase I and DNase II enzymatic activities of spleen lymphocytes of rats with tumour were decreased on different stages of tumour development, however, they were increased under conditions of chemical compound administration.     

Detection of human autoantibodies specific for 5'-m7GMP and m7G(5')ppp(5')N

An enzyme-linked immunosorbent assay was utilized for the detection of spontaneously occurring antibodies with apparent specificities for m7G, 5'-m7GMP, and m7G(5')ppp(5')C. From the sera of 50 patients containing anti-nuclear antibodies, 48 (96%) possessed antibodies which bound to one or more immobilized nucleoside-BSA antigens (A-, G-, C-, U-, and T-BSA). Additionally, 8 (16%) of these sera contained immunoglobulins that reacted with m7G-BSA antigen. In these latter sera, soluble competitors such as m7G, 5'm7GMP, and m7G(5')ppp(5')C (but not 5'-AMP, -GMP, -CMP, -UMP, and -TMP or m1G and m22G) effectively inhibited antibody-binding to immobilized m7G-BSA. These results indicate the existence of spontaneously occurring anti-m7G antibodies in autoimmune diseases which are distinct from anti-G antibody populations.     

Catabolism of capped (3'-5')- and (2'-5')-adenylates in rat liver nuclei

We describe studies concerning the ability of a nuclear dinucleoside triphosphatase to act as a decapping enzyme in RNA catabolism. The enzymatic release of GMP from the Gp3A moiety was determined in the capped RNA model compounds Gp3A3'pA, Gp3A3'pA-isoprop and Gp3A2'pA in isolated rat liver nuclei; i.e., in the environment in which the dinucleoside triphosphatase operates in vivo. The Gp3A cap moiety is hydrolyzed in (3'-5') linked nucleotides only, whereas an extension of the Gp3A in the 2'-direction prevents the nuclear triphosphatase to operate.     

5-Bromo (or chloro)-6-azido-5,6-dihydro-2' -deoxyuridine and -thymidine derivatives with potent antiviral activity

Synthesis, antiviral, and cytotoxic activities of 5-bromo (or chloro)-6-azido-5,6-dihydro-2' -deoxyuridine (4,5) and -thymidine (6,7) are reported. Compounds 4 and 5 exhibited a broad spectrum of antiherpes activity against (HSV-1, HSV-2, HCMV, and VZV).     

Synthesis of 5'(3')-O-amino nucleosides

Synthetic methods leading to 5'(3')-O-amino nucleosides have been developed in an effort to prepare derivatives that may have antitumor or antiviral activities. They are based on ring opening of O2,5'-cyclonucleosides with the N-protected hydroxylamines and dehydrative coupling of 5'(3')-O-unprotected nucleosides with N-hydroxyphthalimide.     

Purification of a novel UV-damaged-DNA binding protein highly specific for (6-4) photoproduct.

UV damage-specific binding proteins are considered to play important roles in early responses of cells irradiated with UV, including damage recognition in the DNA repair process. We have surveyed nuclear and cytoplasmic proteins which bind selectively to UV-irradiated DNA using an electrophoretic mobility shift assay. We detected four distinct binding activities with different mobilities in fractions separated from HeLa cells by heparin chromatography. Three of them were found in nuclear extracts and one in cytoplasmic extracts. We purified one of the binding factors from nuclear extracts to homogeneity, which was designated NF-10 (the 10th fraction of nuclear extract on heparin chromatography). It migrated as a 40 kDa polypeptide in SDS-PAGE, and bound to UV-irradiated double- stranded DNA but not to unirradiated DNA. The binding pattern of the NF-10 protein to DNA irradiated with UV corresponded to the induction kinetics of (6-4) photoproduct. Removal of (6-4) photoproducts from UV- irradiated DNA by (6-4) photoproduct-specific photolyase diminished the binding of NF-10 protein. These results suggest that the NF-10 protein binds to UV-damaged DNA through (6-4) photoproduct. Immunoblot analysis using a monoclonal antibody revealed that the NF-10 protein was expressed in cell lines from all complementation groups of xeroderma pigmentosum, indicating that the NF-10 protein is a novel UV-damaged-DNA binding protein.