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

Abstract

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.

Glyceraldehyde-3-phosphate dehydrogenase: Nuclear translocation participates in neuronal and nonneuronal cell death

“…Nuclear functions that might be influenced by GAPDH include its binding DNA (4,5) and its uracil glycosylase activity (2).…”

The NAD+ precursors, nicotinic acid and nicotinamide protect cells against apoptosis induced by a multiple stress inducer, deoxycholate

“…through RNA chaperone activity). 78 ± 83 Therefore, we tested the hypothesis that one mechanism by which NA and NAM could protect against apoptosis is through elevation of GAPDH protein levels. We used a monoclonal antibody to GAPDH, followed by fluorescent detection and confocal microscopy.…”

Flocculation of Saccharomyces cerevisiae is induced by transformation with the GAP1 gene from Kluyveromyces marxianus

“…The ubiquity of GAPDH has been the subject of several reports in the literature (Huitorel and Pantolini, 1985;Kawamoto and Caswell, 1986;Allen et al, 1987;Meyer-Siegler et al, 1991;Pancholi and Fischetti, 1992;Singh and Green, 1993;Robbins et al, 1995;GilNavarro et al, 1997) and the results presented in this paper clearly show that this GAPDH protein (p37) has other functions than its glycolytic enzymatic activity, as its oversynthesis in¯uences growth, biomass yield, cell wall stability, cell morphology and aggregation. There is now evidence that p37 can induce cell aggregation in Saccharomyces, although it is a Kluyveromyces protein.…”