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PubMed1990

Localization in situ of specific mRNA using thymine-thymine dimerized DNA probes. Sensitive and reliable non-radioactive in situ hybridization.

In situ hybridization has been recognized as a powerful technique for localizing specific nucleic acids such as mRNA and viral DNA in individual cells. For in situ hybridization, the use of a non-radioactive probe is considered superior to that of a radioactive one from viewpoints of resolution, probe stability and personal safety. Although various non-radioactive labels are currently available, some interfere with the formation of hybrids and some increase steric hindrance and prevent penetration of the labeled nucleic acid probe into cells and tissues. Recently, we have developed a method using thymine-thymine (T-T) dimerized DNA as a non-radioactive probe. This is simple to make, since it dose not require separation of labeled DNA from unreacted labeling compounds, and T-T dimer will not alter the chemical and physical nature of the probe. In this paper, we describe the tissue processing procedures that are suited for the T-T dimerized DNA probes, and the recent new findings on methodological aspects, particularly the use of synthetic oligodeoxynucleotide probes.

Animals↗
PubMed1996

Thymine-thymine dimer bypass by yeast DNA polymerase zeta.

The REV3 and REV7 genes of the yeast Saccharomyces cerevisiae are required for DNA damage-induced mutagenesis. The Rev3 and Rev7 proteins were shown to form a complex with DNA polymerase activity. This polymerase replicated past a thymine-thymine cis-syn cyclobutane dimer, a lesion that normally severely inhibits replication, with an efficiency of approximately 10 percent. In contrast, bypass replication efficiency with yeast DNA polymerase alpha was no more than 1 percent. The Rev3-Rev7 complex is the sixth eukaryotic DNA polymerase to be described, and is therefore called DNA polymerase zeta.

Base Sequence↗
PubMed1992

Localization of hepatitis C virus RNA in human liver biopsies by in situ hybridization using thymine-thymine dimerized oligo DNA probes: improved method.

To establish the most proper method of in situ hybridization in detection of HCV-RNA in the liver, various detailed procedures were examined using frozen as well as paraffin-embedded sections of tissue derived from patients. In frozen sections of the liver from hepatitis C patients obtained at autopsy or surgery, HCV-RNA was detectable by in situ hybridization using thymine-thymine dimerized oligonucleotide DNA probes when the sections were treated with ethanol-acetic acid at first, then 0.2 N hydrochloric acid, proteinase K (0.02 u/ml) and DNase. When the paraffin-embedded liver sections were used, more intense proteinase K treatment (0.2-2 u/ml) was required to expose viral RNA and even after that, the positive HCV-RNA signals were less than those in frozen sections, because the cytoplasmic RNA in the routine paraffin-embedded sections was preserved unevenly and less than in frozen sections. These findings indicate that in situ hybridization of HCV-RNA is useful for diagnosing HCV infection and should be a potent tool for monitoring the state of virus activities during therapy. However, the liver biopsy method should be modified so that RNA is retained properly to utilize biopsies more effectively for the routine diagnosis of HCV infection.

Base Sequence↗
PubMed2005

Evidence for three thymine DNA glycosylases in human cell extracts: substrate specificities of thymine DNA glycosylase activities.

BACKGROUND: Purified human thymine DNA glycosylase (TDG) recognizes a G: T mispair in a CpG sequence context more strongly than in any other, in addition to its inactivity toward 2-aminopurine: T or 2,6 diaminopurine: T pairs. We investigated the multiplicity of TDG to establish a better relationship between in vitro G: T mismatch incision and in vivo repair of a G: T to a G: C pair. MATERIAL/METHODS: Cell-free extract was prepared from A1235-MR4 human glioma cells grown in tissue culture. Fractions containing TDG activities were separated on a strong anion-exchange column. 45-bp DNA containing a single G: T or altered G: T mispair was prepared for measuring mismatch-specific strand-incision. RESULTS: The extract yielded three fractions containing TDG activities. Each was further purified on a sizing column to exclude a relationship between a small fragment and TDG activity. While the substrate activity range of fraction III, eluting at the highest salt concentration, was the same as the known TDG, fractions eluted at medium and low concentrations were distinct: fractions I and II reacted with substrates of known TDG and DNA containing or 2-aminopurine: T (2,6-diaminopurine: T) base pairs. Modified m4T mispaired with G in DNA was acted on by fraction I and not II or III, suggesting fraction I activity is distinct. Each fraction showed strong activity on DNA with G: U and G: T mispairs in the CpG sequence context. CONCLUSIONS: The unique range of each TDG activity corresponding to the three fractions indicates that human cells possibly express three distinct TDGs.

Base Pairing↗
PubMed2001

Enhanced loop DNA folding induced by thymine-CH3 group contact and perpendicular guanine-thymine interaction.

A remarkable stabilizing effect induced by T-CH3 group and perpendicular guanine-thymine interactions in the DNA loop conformation has been demonstrated for the d(TTTG) loop structure using UV melting, high resolution NMR, distance geometry, and molecular dynamics studies. Contrary to the previously published d(TTCG) sequence that exhibits no specific inter-nucleotide interaction, we have found that d(TTTG), which differs only by one nucleotide with the d(TTCG) sequence (C7 --> T7), forms a rather stable and well-defined loop structure. Two characteristic structural features account for the stabilization of an otherwise flexible loop structure; the second loop T (T6) residue folds into the minor groove and engages in perpendicular interaction with the G8-NH2, while the third loop T (T7) residue stacks well upon the closing T5 x G8 wobble base pair and exhibits good contacts with many of the loop T5 and T6 sugar protons, which may form a hydrophobic core in the loop region. The importance of the bulky T7-CH3 was also proved by the UV melting study; while d(TTCG) hairpin exhibits a lower melting point (74.5 degrees C) than d(TTTG) hairpin (80.5 degrees C), d(TT(5-methyl)CG) hairpin resumes the same higher melting point (80 degrees C). Similarly, the fact that the melting temperature (74 degrees C) of d(TTTI) is lower than that of d(TTTG) indicates the critical role played by the G8-NH2 group. Our structural studies of the d(TTTG) loop indicate that DNA and RNA use a different strategy to establish stable tertiary folds. Comparison with several other pyrimidine-rich loop hairpins suggests that different minor-groove folding modes exist for the folding thymidine residue.

Base Pairing↗
PubMed2007

A structural rationale for stalling of a replicative DNA polymerase at the most common oxidative thymine lesion, thymine glycol.

Thymine glycol (Tg) is a common product of oxidation and ionizing radiation, including that used for cancer treatment. Although Tg is a poor mutagenic lesion, it has been shown to present a strong block to both repair and replicative DNA polymerases. The 2.65-A crystal structure of a binary complex of the replicative RB69 DNA polymerase with DNA shows that the templating Tg is intrahelical and forms a regular Watson-Crick base pair with the incorporated A. The C5 methyl group protrudes axially from the ring of the damaged pyrimidine and hinders stacking of the adjacent 5' template guanine. The position of the displaced 5' template guanine is such that the next incoming nucleotide cannot be incorporated into the growing primer strand, and it explains why primer extension past the lesion is prohibited even though DNA polymerases can readily incorporate an A across from the Tg lesion.

Crystallography, X-Ray↗
PubMed1990

ESR study of the thymine anion radical in a single crystal of thymine monohydrate.

ESR spectroscopy was used to study free radicals in an irradiated single crystal of thymine monohydrate at 77 K. At low microwave power (20 microW), a broad doublet is found to be super-imposed on the well known resonance patterns of the 5-yl and 7-yl radicals. The doublet spectrum has been analysed as a difference spectrum. Its spectroscopic properties and the observed transformation into the 5-yl, H-addition radical on warming the crystal are consistent with its anion nature, T(-).

Anions↗
PubMed2002

Global genome removal of thymine glycol in Escherichia coli requires endonuclease III but the persistence of processed repair intermediates rather than thymine glycol correlates with cellular sensitivity to high doses of hydrogen peroxide.

Using a monoclonal antibody that specifically recognizes thymine glycol (Tg) in DNA, we measured the kinetics of the removal of Tg from the genomes of wild-type and repair gene mutant strains of Escherichia coli treated with hydrogen peroxide. Tg is rapidly and efficiently removed from the total genomes of repair-proficient cells in vivo and the removal of Tg is completely dependent on the nth gene that encodes the endonuclease III glycosylase. Hence, it appears that little redundancy in the repair of Tg occurs in vivo, at least under the conditions used here. Moreover, previous studies have found that nth mutants are not sensitive to killing by hydrogen peroxide but xth mutant strains (deficient in the major AP endonuclease, exonuclease III) are sensitive. We find that cell death correlates with the persistence of single-strand breaks rather than the persistence of Tg. We attempted to measure transcription-coupled removal of Tg in the lactose operon using the Tg-specific monoclonal antibody in an immunoprecipitation approach but were not successful in achieving reproducible results. Furthermore, the analysis of transcription-coupled repair in the lactose operon is complicated by potent inhibition of beta-galactosidase expression by hydrogen peroxide.

Cell Death↗