Deoxyuridine triphosphate nucleotidohydrolase (dUTPase; EC 188.8.131.52) was purified from HeLa cells by immunoaffinity chromatography. Based on SDS- polyacrylamide gel electrophoresis, two distinct forms of dUTPase were evident in the purified preparation. These proteins were further characterized by a combination of NH2-terminal protein sequencing, mass spectrometry, and mass spectrometry-based protein sequencing. These analyses indicate that the two forms of dUTPase are largely identical, differing only in a short region of their amino-terminal sequences. Despite the structural difference, both forms of dUTPase exhibited identical binding characteristics for dUTP. Each form of dUTPase has a distinct cellular localization. Cellular fractionation and isopycnic density centrifugation indicate that the lower molecular weight form of dUTPase (DUT-N) is associated with the nucleus, while the higher molecular weight species (DUT-M) fractionates with the mitochondria. The DUT-N isoform is approximately 30-fold more abundant in HeLa cells than DUT-M as determined by densitometry. The NH2-terminal protein sequence of both DUT-N and DUT-M did not match previous reports of the predicted amino-terminal sequence for human dUTPase (McIntosh, E. M., Ager, D. D., Gadsden, M. H., and Haynes, R. H. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 8020-8024; Strahler, J. R., Zhu X., Hora, N., Wang, Y. K., Andrews, P. C., Roseman, N. A., Neel, J. V., Turka, L., and Hanash, S. M. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 4991-4995). A cDNA corresponding to the DUT-N isoform was isolated utilizing an oligonucleotide probe based on the determined NH2-terminal sequence. The cDNA contains a 164-amino acid open reading frame, encoding a protein of M(r) 17,748. The DUT-N cDNA sequence matches the previously cloned cDNAs with the exception of a few discrepancies in the 5' end. Our data indicate a 69-base pair addition to the 5' end of the previously reported open reading frame.
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Cell Biology