The nature of enzymes involved in uracil-DNA repair: isoform characteristics of proteins responsible for nuclear and mitochondrial genomic integrity.
The absence of uracil from DNA genomes is a consequence of enzyme functions that eliminate intracellular dUTP pools and that purposefully recognize and remove uracil moieties from DNA. These enzymatic functions are dUTP nucleotidohydrolase (dUTPase) and uracil-DNA glycosylase (UDG), respectively. There are distinct nuclear and mitochondrial isoforms of each of these enzymes in human cells. The mitochondrial isoform of dUTPase (DUT-M) begins as a 31 kilodalton precursor protein containing an arginine-rich, amino-terminal presequence required for targeting to the mitochondria. This precursor is processed into a 23 kilodalton protein that resides, in mature form, in the mitochondria. The nuclear isoform of dUTPase (DUT-N) is an 18 kilodalton protein. Both species of dUTPase are nearly identical except for their amino-termini. Analysis of protein expression reveals that DUT-M is constitutive and independent of cell cycle phase or proliferation status of the cell. In contrast, DUT-N protein and mRNA levels are tightly regulated to coincide with nuclear DNA replication. The common sequence for both nuclear and mitochondrial isoforms includes a cyclin-dependent kinase consensus site. However, only the nuclear form appears to be phosphorylated at this site in vivo. Studies on dUTPase genomic organization reveal that both isoforms are encoded by the same gene. Isoform specific transcripts arise through the use of alternate 5' exons. Uracil-DNA glycosylase (UDG1) is but one of a growing family of enzymes that repairs potentially mutagenic events caused by uracil in DNA. Human cells contain two isoforms of UDG1 which are also nearly identical except for their amino termini. One isoform (UDG1-M), which is constitutively expressed, is targeted to the mitochondria. This form originates as a 35,000 dalton precursor and is N-terminally processed to a mature 29,000 dalton protein as it transits into the mitochondria. The other isoform is targeted to the nucleus and its expression is a function of cellular proliferation status. As with dUTPase, UDG1 isoform specific transcripts arise through the use of alternate 5prie; exons. Both of these enzymatic functions are a unique illustration, in humans, of the use of alternate exons to generate differentially expressed proteins targeted to different organelles. There are questions as to whether the nuclear isoform of UDG (UDG1-N) is also processed (at the N-terminus) to a lower molecular weight form. Polyclonal antisera generated to the unique N-terminal region of this isoform, reveals that UDG1-N exists as a 36,000 dalton protein in human cell nuclei. Since the epitope for this antibody resides in the first 24 amino acids of UDG1-N, it is apparent that the majority of this isoform is not processed and retains its amino terminus. Evidence also indicates that UDG1-N exists as a serine/threonine phosphoprotein and that phosphorylation occurs in the unique N-terminal region. This was initially deduced from the observation that nuclear UDG1-N migrates as multiple bands on SDS-PAGE and as a single band subsequent to phosphatase treatment. Cdc2 kinase is at least one of the enzymes that can phosphorylate UDG1-N. This review will summarize the current information on isoform characteristics of both dUTPase and uracil-DNA glycosylase. It will also focus on evidence for phosphorylation and speculate as to the purpose of these post-translational events.
D000595:Amino Acid Sequence; D015179:Colorectal Neoplasms / Q000201:enzymology; D045647:DNA Glycosylases; D004260:DNA Repair; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D009699:N-Glycosyl Hydrolases / Q000378:metabolism*; D010641:Phenotype; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D011755:Pyrophosphatases / Q000378:metabolism*; D014498:Uracil / Q000378:metabolism*; D051981:Uracil-DNA Glycosidase
D007527:Isoenzymes; D024101:Mitochondrial Proteins; D014498:Uracil; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase; D011755:Pyrophosphatases; C020020:dUTP pyrophosphatase