CHARACTERIZATION OF DISTINCT NUCLEAR AND MITOCHONDRIAL FORMS OF HUMANDEOXYURIDINE TRIPHOSPHATE NUCLEOTIDOHYDROLASE

Deoxyuridine triphosphate nucleotidohydrolase (dUTPase; EC 3.6.1.23) w as 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, ma ss 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 ide ntical binding characteristics for dUTP. Each form of dUTPase has a di stinct cellular localization. Cellular fractionation and isopycnic den sity centrifugation indicate that the lower molecular weight form of d UTPase (DUT-N) is associated with the nucleus, while the higher molecu lar weight species (DUT-M) fractionates with the mitochondria. The DUT -N isoform is approximately 30-fold more abundant in HeLa cells than D UT-M as determined by densitometry. The NH2-terminal protein sequence of both DUT-N and DUT-M did not match previous reports of the predicte d 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 cor responding to the DUT-N isoform was isolated utilizing an oligonucleot ide probe based on the determined NH2-terminal sequence. The cDNA cont ains a 164-amino acid open reading frame, encoding a protein of M(r) 1 7,748. The DUT-N cDNA sequence matches the previously cloned cDNAs wit h the exception of a few discrepancies in the 5' end. Our data indicat e a 69-base pair addition to the 5' end of the previously reported ope n reading frame.