Hydrolytic deamination of 5-methylcytosine leads to the formation of G/T mismatches. We have shown previously that these G/T mispairs are corrected to G/C pairs by a mismatch-specific thymine-DNA glycosylase, TDG, which we subsequently purified from human cells. Here we describe the cloning of the human cDNA encoding TDG. We have identified two distinct cDNA species that differ by 100 nucleotides at the 3-untranslated region. These cDNAs contain a 410-amino acid open reading frame that encodes a 46-kDa polypeptide. The G/T glycosylase, expressed both in vitro and in Escherichia coli, migrated in denaturing polyacrylamide gels with an apparent size of 60 kDa. The substrate specificity of the recombinant protein corresponded to that of the cellular enzyme, and polyclonal antisera raised against the recombinant protein neutralized both activities. We therefore conclude that the cDNA described below encodes human TDG. Data base searches identified a serendipitously cloned mouse cDNA sequence that could be shown to encode the murine TDG homologue. No common amino acid sequence motifs between the G/T-specific enzyme and other DNA glycosylases could be found, suggesting that TDG belongs to a new class of baseexcision repair enzymes.

To avoid the mutagenic effect of spontaneous hydrolytic deamination of 5-methylcytosine, G-T inspirs, arising in DNA as a result of this process, should always be corrected to G-C pairs. We describe here the identification of a DNA glycosylase activity present in nuclear extracts from HeLa cells, which removes the mispaired thymine to generate an apyrimidinic (AP) site opposite the guanine. We further show, using a specific antibody and inhibitors, that the single nucleotide gap, created upon processing of the AP site, is filled in by DNA polymerase (3. This rmding substantiates the proposed role of this enzyme in short-patch DNA repair.Efficient correction of base-base mismatches, arising as errors of DNA polymerase or through recombination, requires that the cellular machinery be able to direct the repair to the strand carrying the original information. For this purpose, secondary signals such as adenine methylation or strand nicks are used (for a comprehensive review, see ref. 1). The GOT mispair is the only one that can arise also by an alternative pathway in "resting" (i.e., nonreplicating, nonrecombining) DNA-through the spontaneous hydrolytic deamination of 5-methylcytosine. In the correction of this latter type of G-T mismatch, no strand discrimination is required, as it should always be corrected to a G-C. Indeed, repair pathways thought to be dedicated to the correction of the deamination-associated GOT mispairs have been identified in Escherichia coli (2-4) as well as in mammalian cells (5, 6).In all organisms studied to date, correction of deamination damage is mediated by base-specific glycosylases, which remove the deaminated base from the sugar-phosphate backbone by cleaving the glycosylic bond. Uracdil DNA glycosylase and hypoxanthine DNA glycosylase remove the products of cytosine and adenine deamination, uracil (U) and hypoxanthine (H), respectively (for review, see refs. 7 and 8). Both enzymes are highly substrate specific and can remove their respective substrates from matched (A-U and C-H) as well as mismatched (G-U and T.H) duplexes. As neither U nor H is a natural DNA base, their excision can be mediated by the base-specific enzymes from both double-and singlestranded DNA.In our previous studies, we reported that G-T mispairs, incorporated in the simian virus 40 genome and transfected into monkey CV-1 (5, 6) or human (9) cells, were corrected with high efficiency and mostly to G-C pairs. Our in vitro experiments (10) demonstrated that in nuclear extracts from human (HeLa) cells, the mispaired thymidine was excised from DNA to generate a single nucleotide gap. Preliminary evidence also suggested that the initial step of this repair process was mediated by a DNA glycosylase. This was an unexpected finding. Thymine is a natural DNA base and any enzyme responsible for its removal from G-T mispairs would have to be, unlike the uracil and hypoxanthine glycosylases, fully inactive on single-stranded and matched doublestranded substrates. It therefore seemed likely that a thymine DNA glyco...

No abstract

In differentiated cells, only a specific subset of genes is expressed. Recently, several genes have been shown to be transcriptionally inactivated by methylation of cytosine residues, mainly within their promoter sequences. Spontaneous hydrolytic deamination of 5-methylcytosine to thymine, which has been estimated to generate up to 12 G.T mismatched base pairs in the human genome per day, could have a deleterious effect on the expression of such genes. We recently reported that mammalian cells possess a specific repair pathway, which counteracts the mutagenic effects of this deamination by correcting G.T mismatches almost exclusively to G.C pairs. We show here that, in nuclear extracts from HeLa cells, this repair is mediated by excision of the aberrant thymidine monophosphate residue, followed by gap-filling to generate a G.C pair. We also provide preliminary evidence that the initial step of this process involves a DNA glycosylase.

In a genetic screen aimed at the identification of trans-acting factors involved in mRNA 3-end processing of budding yeast, we have previously isolated two temperature-sensitive mutants with an apparent defect in the 3-end formation of a plasmid-derived pre-mRNA. Surprisingly, both mutants were rescued by the essential gene ESS1/PTF1 that encoded a putative peptidylprolyl-cis/trans-isomerase (PPIase) (Hani, J., Stumpf, G., and Domdey, H. (1995) FEBS Lett. 365, 198 -202). Such enzymes, which catalyze the cis/trans-interconversion of peptide bonds N-terminal of prolines, are suggested to play a role in protein folding or trafficking. Here we report that Ptf1p shows PPIase activity in vitro, displaying an unusual substrate specificity for peptides with phosphorylated serine and threonine residues preceding proline. Both mutations were found to result in amino acid substitutions of highly conserved residues within the PPIase domain, causing a marked decrease in PPIase activity of the mutant enzymes. Our results are suggestive of a so far unknown involvement of a PPIase in mRNA 3-end formation in Saccharomyces cerevisiae.Despite intensive efforts to unravel the complex process of mRNA 3Ј-end formation in Saccharomyces cerevisiae, the list of participating factors still awaits its completion.We have recently isolated a gene complementing the phenotype of two temperature-sensitive yeast mutants that were impaired in mRNA 3Ј-end formation. This gene, designated PTF1 (processing/termination factor 1; identical with the previously described ESS1 (1)), encodes a protein that, by virtue of sequence similarity, was identified as a peptidylprolyl-cis/ trans-isomerase (PPIase) 1 (2). PPIases are ubiquitous enzymes that catalyze the interconversion from cis to trans of peptide bonds preceding a proline and are thought to accelerate this often rate-limiting step in the folding of a number of proteins in vivo (3-6).PPIases are divided in three families, based on their sensitivities toward two clinically relevant immunosuppressants: the cyclosporin A-binding proteins (cyclophilins), the FK506-binding proteins, and a third family, named after the Escherichia coli protein parvulin, which is not inhibited by either of the two drugs (for review see Refs. 3-6). In addition, the members of each family are characterized by conserved but distinct amino acid domains. By this criterion, PTF1 was predicted to belong to the parvulin family of PPIases (2).Although disruption of PPIase genes did not generally impair cell growth (7-8), PTF1 was the first PPIase gene shown to be essential for cell viability (1). In fact, PTF1 is the only essential PPIase gene in S. cerevisiae as demonstrated more recently by the viability of a yeast mutant lacking the remaining 12 PPIases, members of the other two immunosuppressant binding families. (8). So far, the only other example of an essential PPIase is the recently discovered PIN1, a human protein, that is structurally and functionally related to Ptf1p (9 -10).In this paper we describe the genetic screen ...