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Thymine DNA glycosylase (TDG) attracted attention because of its ability to remove thymine, i.e., the normal DNA base, from G:T irregular pairs. This indicated its function in DNA base excision repair in the repair of G:C base pairs after deamination of 5-methylcytosine. Because of its apparent catalytic inefficiency, TDG was considered by some to be a poor DNA repair enzyme without an important biological function. However, it has been demonstrated that this enzyme can act as a key player in transcriptional regulation through interactions with various nuclear receptors and transcription factors, indicating its function in gene regulation, which appears to be crucial in developmental processes. Others have shown that TDG is involved in the epigenetic regulation of gene expression by protecting CpG-rich promoters from de novo DNA methylation and can initiate aberrant repair. Recent animal studies have revealed an association between loss of TDG and the onset of tumorigenesis. Thus, the purpose of this review is to bring together the results of various experiments in which thymine DNA-glycosylase has been studied since its discovery and to assess their implications for determining the possible physiological roles of this enzyme. [ABSTRACT FROM AUTHOR]

Aerobic cellular respiration generates reactive oxygen species (ROS), which can damage macromolecules including lipids, proteins and DNA. It was proposed that aging is a consequence of accumulation naturally occurring unrepaired oxidative DNA damage. In human cells, on average approximately 2000 to 8000 DNA lesions occur per hour in each cell, or about 40000 to 200000 per cell per day. DNA repair systems are able to discriminate between regular and modified bases. For example, DNA glycosylases specifically recognize and excise damaged bases among vast majority of regular bases in the base excision repair (BER) pathway. However, mismatched pairs between two regular bases occur due to spontaneous conversion of 5-methylcytosine to thymine and DNA polymerase errors during replication. To counteract these mutagenic threats to genome stability, cells evolved special DNA repair systems that target the non-damaged DNA strand in a duplex to remove mismatched regular DNA bases. Mismatch-specific adenine- and thymine-DNA glycosylases (MutY/MUTYH and TDG/MBD4, respectively) initiated base excision repair (BER) and mismatch repair (MMR) pathways can recognize and remove normal DNA bases in mismatched DNA duplexes. Paradoxically, under certain circumstances in DNA repair deficient cells bacterial MutY and human TDG can act in the aberrant manner: MutY and TDG removes Adenine and Thymine opposite misincorporated 8-oxoguanine and damaged Adenine, respectively. These unusual activities lead either to mutations or futile DNA repair, thus indicating that the DNA repair pathways which target non-damaged DNA strand can act in aberrant manner and introduce genome instability in the presence of unrepaired DNA lesions. Evidences gathered showing that in addition to the accumulation of oxidative DNA damage in cells, the aberrant DNA repair can also contribute to cancer, brain disorders and premature senescence. This review summarises the present knowledge about the aberrant DNA repair pathways for oxidised base modifications and their possible role in aging. [ABSTRACT FROM AUTHOR]

Heterologous expression of Aspergillus niger endo-1,4-β-glucanase (ENG1) in Saccharomyces cerevisiae was tested both with an episomal plasmid vector (YEGAp/eng1) and a yeast vector capable of integration into the HO locus of the S. cerevisiae chromosome (pHO-GAPDH-eng1-KanMX4-HO). In both cases, eng1 gene expression in yeast, with its native signal sequence for secretion, was under the control of the strong glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter. We aimed to verify how each expression system affects protein expression, posttranslational modification, and biochemical properties. Expression of eng1 from the episomal plasmid vector YEGAp/eng1 significantly slowed the growth of a yeast cell culture. However, expression of eng1 from the vector integrated into the HO locus of the chromosome did not cause growth suppression, and the enzyme activity in a culture supernatant was maintained throughout the incubation time. ENG1 has optimum catalytic activity at pH 6.0, and is stable in the pH range 5.0-9.0. The enzyme's optimum temperature for catalytic activity at pH 6.0 is 70°C; importantly, more than 95% of the enzyme's initial activity remained after a 2-h incubation at 60°C. The biochemical characterization of ENG1 confirmed the correct expression of the protein and showed that ENG1 expressed by the pHO-GAPDH-eng1-KanMX4-HO vector, in addition to its N-linked sites, is overglycosylated at its O-glycosylation sites compared with ENG1 expressed by the YEGAp/eng1 vector. It is likely that the O-glycosylated form of the A. niger ENG1 retains more stable activity during continuous cultivation of recombinant yeasts than the form that is only N-glycosylated.

Dimerization of multicopy plasmids is widely assumed to be disadvantageous both for plasmid maintenance and for the host cell. It is known that dimerization causes plasmid instability; dimer-containing cells grow slower than their monomer-containing counterparts. However, as we demonstrate here, under conditions of selective stress, dimers provide an advantage for bacteria. Dimers facilitate segregation of mutants from numerous copies of the parental plasmid. Accelerated segregation greatly increases the rate of accumulation of plasmids carrying mutations that are adaptive for bacteria. In contrast, resolution of dimers by site-specific recombination decreases, 10(3)-10(5)-fold, the efficiency of selection of spontaneous reversions in the tet gene of pBR327.

The role of different DNA damages in the stimulation of homologous recombination was studied by using an in vivo plasmid recombination assay. Dimethyl sulphate (DMS) treatment of plasmid DNA induced a 20-50-fold increase in the frequency of recombinational events. DMS treatment also stimulated RecA protein binding to double-stranded DNA. In contrast, plasmid DNA containing uracil, which, like DMS, is also subject to repair, was less effective in stimulation of recombination. The ability of purified RecA protein to bind DMS-treated or uracil-containing DNA was tested by measuring its ATPase activity. The result indicates that DMS treatment, but not uracil incorporation, stimulates RecA protein binding to DNA. We conclude, that the main reason (or the first step) for stimulation of recombination by mutagens is activation of RecA binding to damaged DNA.

Methods for site-directed multiple modification of DNA have been developed and used for modification of either one or two strands of plasmid DNA. Plasmid DNAs modified in the region of the tet gene were transformed into Escherichia coli cells and Tet colonies were screened. It was shown that multiple lesions in one DNA strand performed using either N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or sodium bisulfite were effectively repaired in the cell by error-free mechanism. In contrast, modification of two DNA strands led to induction of mutations. The efficiency of mutagenesis in the case of modification of a local region of one DNA strand with sodium bisulfite and modification of the other strand with MNNG was 1.1-7.9%. Mutations were analysed by restriction mapping and sequencing. All of them were G----A transitions.

A new site-directed method for inserting long single-stranded DNA fragments into any region of a duplex vector is described. Its major advantage is independence of the location of the restriction sites. The method involves the assembly of single-stranded DNA fragments by ligation to both ends of the inserted fragments of two cohesive flanks that are complementary to the target region. Short oligonucleotide templates are used to direct the ligation. The resulting fragments, designated further as omega fragments with cohesive flanks, are hybridized with a gapped DNA vector. The heteroduplexes are transformed into Escherichia coli cells without enzymatic filling and sealing of gapped DNA. As a consequence of intracellular repair and heteroduplex resolution, insertion mutants are recovered. To demonstrate the method's efficiency, we inserted a 51-nucleotide synthetic DNA fragment containing a modified glucocorticoid receptor binding site into the region of pBR322, near the transcription starting point of the tet gene. The method we developed makes possible site-directed insertion of synthetic and genome-derived DNA fragments at least 200 nucleotides long.

A new method for obtaining the recombinant DNA based on heteroduplex-initiated site-directed insertion of alien nucleotide sequences is proposed. To generate a single-stranded region, plasmid DNA was nicked with restriction endonuclease in the presence of ethidium bromide with subsequent exonuclease III controlled digestion. The inserted DNA sequences flanked by nucleotide sequences complementary to single-stranded region were annealed with plasmid DNA and E. coli cells were transformed by the resulting heteroduplex molecules. The presented data show the possibility to insert as many as 200 nucleotides. The yield of recombinant DNA varied from 16 to 0.7% as the number of nucleotides inserted correspondingly varied from 15 to 200. The site of insertion does not depend crucially on the localization of the restriction site used.