Isolation and characterization of the human uracil DNA glycosylase gene.

Vollberg, Thomas M.; Siegler, Katherine; Cool, Barbara L.; Sirover, Michael A.

doi:10.1073/pnas.86.22.8693

Cited by 31 publications

(14 citation statements)

References 32 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Indeed, three genes encoding uracil glycosylases have been identified to date in human cells (24)(25)(26). Although the major part of uracil is probably removed, as in the lower organisms, by the product of a single gene [as judged by the fact that its inactivation leads to a loss of >80o of the total uracil glycosylase activity (27) and by its sequence similarity with the bacterial gene (24)], it is likely that the other enzymes carry out more limited, but perhaps also more specialized, functions.…”

Section: Discussionmentioning

confidence: 99%

Efficient removal of uracil from G.U mispairs by themismatch-specific thymine DNA glycosylase from HeLa cells.

Neddermann

Jiricny

1994

Proc. Natl. Acad. Sci. U.S.A.

178

150

View full text Add to dashboard Cite

The uracil DNA glycosylases (EC 3.2.2.3) characterized to date remove uracil from DNA irrespective of whether it is base paired with adenine or mispaired with guanine in double-stranded substrates or whether it is found in single-stranded DNA. We report here the characterization of uracil glycosylase activity that can remove the base solely from a mispair with guanne. It does not recognize uracil either in A-U pairs or in single-stranded substrates. The enzyme, a 55-kDa polypeptide, was previously characterized as a mismatch-specific thymine DNA glycosylase and was thought to be responsible solely for the correction (to GaC) of G T mispais that arise as a result of spontaneous hydrolytic deaination of 5-methylcytosine to thymine. Given the broader substrate specificity of the enzyme (in addition to uracil and thymine, the protein can also remove 5-bromouracil from mispairs with guanine), we propose that its biological role in vivo may also incinde the correction of a subset of GNU mips inefficiently removed by the more abundant ubiquitous uraci glycosylases, such as those arising from cytosine deatnation in G+C-rich regions of the genome.Uracil residues arise in DNA either by cytosine deamination (1) or by incorporation into the nascent strand during replication as dUMP (2). The former process gives rise to mutagenic G-U mispairs, whereas the latter yields A-U "pairs." Uracil is removed from both these lesions and from singlestranded DNA by ubiquitous uracil DNA glycosylases (EC 3.2.2.3) (2, 3). A recent study addressing the removal ofuracil from a large number ofdifferent sequence contexts suggested that the major uracil glycosylases of both mammals and bacteria may require local strand separation for efficient removal of the base from DNA (4). Correspondingly, G-U mispairs in a G+C-rich sequence context were found to be poor substrates for these enzymes in vitro, although this may not be so for all uracil glycosylases (5). Interestingly, analysis of mutational spectra of several mammalian genes published to date revealed no evidence for an increased rate of C --T transition mutations that could be associated with G+C-rich regions (6, 7). This would imply either that the ubiquitous enzyme is so abundant that it manages to repair even the less accessible substrates or that a second enzyme may be able to compensate for the inefficiency ofthe ubiquitous activity. We describe here another glycosylase activity that, due to its different substrate recognition requirements, efficiently removes uracil from GNU mispairs in double-stranded DNA and may thus be a candidate for such a back-up role. MATERIALS AND METHODSAll the reagents and solvents used in this study were of analytical purity. The Preparation of Whole Cell Extracts. These extracts were prepared from 180 g offrozen HeLa cells in three 60-g batches as described (9).Fractionation of the Cell Extracts. The extract was diluted 1:4 with HE buffer [25 mM Hepes, pH 7.8/1 mM EDTA/1 mM dithiothreitol/10% (vol/vol) glycerol] and incubated batchwise for 1 h w...

show abstract

Section: Discussionmentioning

confidence: 99%

Efficient removal of uracil from G.U mispairs by themismatch-specific thymine DNA glycosylase from HeLa cells.

Neddermann

Jiricny

1994

Proc. Natl. Acad. Sci. U.S.A.

178

150

View full text Add to dashboard Cite

show abstract

“…Deoxyuridine levels in DNA isolated from a yeast strain deficient for uracil-DNA-glycosylase were only about 0.1% or less (3), but such low levels are still consistent with a role for uracil excision in targeting the mismatch repair system to the newly replicated strand. Second, uracil-DNA-glycosylase levels are increased in mammalian cells induced to undergo proliferation (30). Similarly, expression of the UNG1 gene in S. cerevisiae is regulated during the cell cycle in exactly the same fashion as are yeast replication genes, with steadystate mRNA levels rising manyfold in late G1 and early S and decreasing again late in the S phase of the cell cycle (13a).…”

mentioning

confidence: 90%

The spectrum of spontaneous mutations in a Saccharomyces cerevisiae uracil-DNA-glycosylase mutant limits the function of this enzyme to cytosine deamination repair

1991

View full text Add to dashboard Cite

Uracil-DNA-glycosylase has been proposed to function as the first enzyme in strand-directed mismatch repair in eukaryotic organisms, through removal of uracil from dUMP residues periodically inserted into the DNA during DNA replication (Aprelikova, 0. N., V. M. Golubovskaya, T. A. Kusmin, and N. V. Tomilin, Mutat. Res. 213:135-140, 1989). This hypothesis was investigated with Saccharomyces cerevisiae. Mutation frequencies and spectra were determined for an ungi deletion strain in the target SUP4-o tRNA gene by using a forward selection scheme. Mutation frequencies in the SUP4-o gene increased about 20-fold relative to an isogenic wild-type S. cerevisiae strain, and the mutator effect was completely suppressed in the ungl deletion strain carrying the wild-type UNGI gene on a multicopy plasmid. Sixty-nine independently derived mutations in the SUP4-o gene were sequenced. All but five of these were due to GC->AT transitions. From this analysis, we conclude that the mutator phenotype of the ungi deletion strain is the result of a failure to repair spontaneous cytosine deamination events occurring frequently in S. cerevisiae and that the UNGI gene is not required for strand-specific mismatch repair in S. cerevisiae.Strand-directed mismatch repair depends on a mechanism which allows the organism to distinguish between the parental and the newly replicated DNA strand during DNA replication. This form of repair is best understood for Escherichia coli (11,23). In methyl-directed DNA mismatch repair, the strand to be repaired is identified by its temporary lack of methylation at GATC sites during DNA replication.

show abstract

“…Recombinant plasmid pChug 20.1 was prepared and introduced into Escherichia coli DH1 as described (5). Cells transformed with pUC8 were used as a negative control.…”

Section: Methodsmentioning

confidence: 99%

“…In an examination of the molecular mechanisms involved in expression of human nuclear DNA-repair genes, we isolated a normal human placental cDNA that hybrid-selected the mRNA encoding the nuclear UDG (5). Northern (RNA) blot analysis revealed the presence of a 1.6-kilobase (kb) RNA transcript.…”

mentioning

confidence: 99%

A human nuclear uracil DNA glycosylase is the 37-kDa subunit of glyceraldehyde-3-phosphate dehydrogenase.

Meyer-Siegler

Mauro

Seal

et al. 1991

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

310

182

View full text Add to dashboard Cite

We have isolated and characterized a plasmid (pChug 20.1) that contains the cDNA of a nuclear uracil DNA glycosylase (UDG) gene Isolated from normal human placenta. This cDNA directed the synthesis of a fusion protein (Mr 66,000) that exhibited UDG activity. The enzymatic activity was specific for a uracil-containing polynucleotide substrate and was inhibited by a glycosylase antibody or a (3-galactosidase antibody. Sequence analysis demonstrated an open reading frame that encoded a protein of 335 amino acids of calculated Mr 36,050 and pI 8.7, corresponding to the Mr 37,000 and pI 8.1 of purified human placental UDG. No homology was seen between this cDNA and the UDG of herpes simplex virus, Escherichia coil, and yeast; nor was there homology with the putative human mitochondrial UDG cDNA or with a second human nuclear UDG cDNA. Surprisingly, a search of the GenBank data base revealed that the cDNA of UDG was completely homologous with the 37-kDa subunit of human glyceraldehyde-3-phosphate dehydrogenase. Human erythrocyte glyceraldehyde-3-phosphate dehydrogenase was obtained commercially in its tetrameric form. A 37-kDa subunit was isolated from it and shown to possess UDG activity equivalent to that seen for the purified human placental UDG.The multiple functions of this 37-kDa protein as here and previously reported indicate that it possesses a series of activities, depending on its oligomeric state. Accordingly, mutation(s) in the gene of this multifunctional protein may conceivably result in the diverse cellular phenotypes of Bloom syndrome.Human cells contain two major DNA excision-repair pathways to remove DNA lesions (1). Bulky DNA adducts are eliminated by the nucleotide-excision pathway, whereas most alkylated bases and alterations due to spontaneous damage are removed by the base-excision pathway. DNA glycosylases remove modified bases in the latter pathway by cleaving the base-sugar bond. Uracil present in DNA as a result of utilization of dUTP during DNA synthesis (2) or by deamination of existing cytosine residues (3, 4) is removed by the uracil DNA glycosylase (UDG).In an examination of the molecular mechanisms involved in expression of human nuclear DNA-repair genes, we isolated a normal human placental cDNA that hybrid-selected the mRNA encoding the nuclear UDG (5). Northern (RNA) blot analysis revealed the presence of a 1.6-kilobase (kb) RNA transcript. In this study we report that the nucleotide sequence of this human glycosylase cDNA* and the deduced amino acid sequence of the glycosylase are identical to those reported for the 37-kDa subunit of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (G3PD). This finding reveals an unusual and different biochemical function ofthe monomeric form ofG3PD in normal human cells as that of a UDG that functions in the base-excision repair of DNA.

show abstract

Isolation and characterization of the human uracil DNA glycosylase gene.

Cited by 31 publications

References 32 publications

Efficient removal of uracil from G.U mispairs by themismatch-specific thymine DNA glycosylase from HeLa cells.

Efficient removal of uracil from G.U mispairs by themismatch-specific thymine DNA glycosylase from HeLa cells.

The spectrum of spontaneous mutations in a Saccharomyces cerevisiae uracil-DNA-glycosylase mutant limits the function of this enzyme to cytosine deamination repair

A human nuclear uracil DNA glycosylase is the 37-kDa subunit of glyceraldehyde-3-phosphate dehydrogenase.

Contact Info

Product

Resources

About