Immunological lesions in human uracil DNA glycosylase: association with Bloom syndrome.

Self Cite

A series of anti-human placental uracil DNA glycosylase monoclonal antibodies was used to screen a human placental cDNA library in phage lambda gt11. Twenty-seven immunopositive plaques were detected and purified. One clone containing a 1.2-kilobase (kb) human cDNA insert was chosen for further study by insertion into pUC8. The resultant recombinant plasmid selected by hybridization a human placental mRNA that encoded a 37-kDa polypeptide. This protein was immunoprecipitated specifically by an anti-human placental uracil DNA glycosylase monoclonal antibody. RNA blot-hybridization (Northern) analysis using placental poly(A)+ RNA or total RNA from four different human fibroblast cell strains revealed a single 1.6-kb transcript. Genomic blots using DNA from each cell strain digested with either EcoRI or Pst I revealed a complex pattern of cDNA-hybridizing restriction fragments. The genomic analysis for each enzyme was highly similar in all four human cell strains. In contrast, a single band was observed when genomic analysis was performed with the identical DNA digests with an actin gene probe. During cell proliferation there was an increase in the level of glycosylase mRNA that paralleled the increase in uracil DNA glycosylase enzyme activity. The isolation of the human uracil DNA glycosylase gene permits an examination of the structure, organization, and expression of a human DNA repair gene.

Section: Resultssupporting

confidence: 81%

Isolation and characterization of the human uracil DNA glycosylase gene.

Vollberg

Siegler

Cool

et al. 1989

Self Cite

“…Protein was electroblotted onto nitrocellulose, as described by Towbin et al (18). Immunoblot analysis was done as described (19,20) (Fig. 7).…”

Section: Resultsmentioning

confidence: 99%

“…These include several separate DNA metabolic defects that appear independent of each other (19,(36)(37)(38)(39)(40)(41)(42)(43). Although complementation analysis has suggested the presence of a single-genetic-locus for Bloom syndrome (20), attributing these diverse cellular abnormalities to only one gene has been difficult.…”

Section: Discussionmentioning

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

Self Cite

310

182

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.

“…The existence of a heat-resistant factor that is associated with DNA ligases and promotes ligase activity on linear DNA has been observed in human fibroblasts as well as Xenopus laevis ovaries (30,31). In addition, it has been proposed that the ligation of nonhomologous DNA ends is facilitated by proteins that align the ends (32) (26,33), and an incomplete cDNA encoding the 3' half of DNA ligase I is sufficient for complementation of cdc9 (23 (11)(12)(13)(14) have identified an alteration in the structure and expression of uracil DNA glycosylase and hypoxanthine DNA glycosylase in BS cells. These enzymes are unlikely to affect the levels of DNA ligase I activity directly.…”

Section: Discussionmentioning

confidence: 99%

“…In BS cells this induction is delayed, and maximal levels ofuracil and hypoxanthine DNA glycosylases are not attained until late in S phase, although the maximal activity of these enzymes is not reduced in BS cells. In addition, a monoclonal antibody that recognizes uracil DNA glycosylase from normal cells is unreactive with BS uracil DNA glycosylase, indicating that the enzyme is structurally altered or modified in BS cells (11)(12)(13)(14).…”

mentioning

confidence: 99%

A wild-type DNA ligase I gene is expressed in Bloom's syndrome cells.

Petrini

Huwiler

Weaver

1991

Alteration of DNA ligase I activity is a consistent biochemical feature of Bloom's syndrome (BS) cells.DNA ligase I activity in BS cells either is reduced and abnormally thermolabile or is present in an anamolously dimeric form. To assess the role of DNA ligase function in the etiology of BS, we have cloned the DNA ligase I cDNA from normal human cells by a PCR strategy using degenerate oligonucleotide primers based on conserved regions of the Saccharomyces cerevisiae and Schizosaccharomyces pombe DNA ligase genes.Human DNA ligase I cDNAs from normal and BS cells complemented a S. cerevisiae DNA ligase mutation, and protein extracts prepared from S. cerevisiae transformants expressing normal and BS cDNA contained comparable levels of DNA ligase I activity. DNA sequencing and Northern blot analysis of DNA ligase I expression in two BS human fibroblast lines representing each of the two aberrant DNA ligase I molecular phenotypes demonstrated that this gene was unchanged in BS cells. Thus, another factor may be responsible for the observed reduction in DNA ligase I activity associated with this chromosomal breakage syndrome.To assess the molecular basis of this disease, we have cloned the cDNA corresponding to the human DNA ligase I gene expressed in normal cells and from two BS fibroblast lines, GM8505 and GM5289. The DNA ligase I gene was isolated by a PCR strategy employing degenerate oligonucleotides based on conserved regions of the DNA ligase genes of Saccharomyces cerevisiae and Schizosaccharomyces pombe (15). A complete DNA sequence analysis from two BS cell lines revealed that each BS DNA ligase I gene was indistinguishable from the wild-type gene. A single 3.1-kilobase (kb) DNA ligase I mRNA was detected at normal steady-state levels in BS cells by Northern blot analysis. Also, the GM8505 DNA ligase I gene was able to complement the S. cerevisiae cdc9 DNA ligase mutation. Given the reduction in DNA ligase I activity in BS cells, these results suggest that a factor which modulates DNA ligase I activity in normal cells is defective in BS. Alternatively, a defect in a modifying activity that acts upon DNA ligase I and some DNA-repair enzymes may account for the altered repair phenotypes in BS cells.