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.

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Three monoclonal antibodies that react with uracil DNA glycosylase of normal human placenta were tested to determine whether one of the antibodies could be used as a negative marker for Bloom syndrome. As defined by enzyme-linked immunosorbent assay, monoclonal antibody 40.10.09, which reacts with normal human glycosylase, neither recognized nor inhibited native uracil DNA glycosylase from any of five separate Bloom syndrome cell strains. Immunoblot analyses demonstrated that the denatured glycosylase protein from all five Bloom syndrome cell strains was immunoreactive with the 40.10.09 antibody. Further, each native enzyme was immunoreactive with two other anti-human placental uracil DNA glycosylase monoclonal antibodies. In contrast, ELISA reactivity was observed with all three monoclonal antibodies in reactions of glycosylases from 5 normal human cell types and 13 abnormal human cell strains. These results experimentally verify the specificity of the aberrant reactivity of the Bloom syndrome uracil DNA glycosylase. The possibility arises that determination of the lack of immunoreactivity with antibody 40.10.09 may have value in the early diagnosis of Bloom syndrome.