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Originally published In Press as doi:10.1074/jbc.M011075200 on January 30, 2001

J. Biol. Chem., Vol. 276, Issue 18, 15073-15081, May 4, 2001
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Uncoupling of 3'-Phosphatase and 5'-Kinase Functions in Budding Yeast
CHARACTERIZATION OF SACCHAROMYCES CEREVISIAE DNA 3'-PHOSPHATASE (TPP1)*

John R. Vance and Thomas E. WilsonDagger

From the Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0602

Polynucleotide kinase is a bifunctional enzyme containing both DNA 3'-phosphatase and 5'-kinase activities seemingly suited to the coupled repair of single-strand nicks in which the phosphate has remained with the 3'-base. We show that the yeast Saccharomyces cerevisiae is able to repair transformed dephosphorylated linear plasmids by non-homologous end joining with considerable efficiency independently of the end-processing polymerase Pol4p. Homology searches and biochemical assays did not reveal a 5'-kinase that would account for this repair, however. Instead, open reading frame YMR156C (here named TPP1) is shown to encode only a polynucleotide kinase-type 3'-phosphatase. Tpp1p bears extensive similarity to the ancient L-2-halo-acid dehalogenase and DDDD phosphohydrolase superfamilies, but is specific for double-stranded DNA. It is present at high levels in cell extracts in a functional form and so does not represent a pseudogene. Moreover, the phosphatase-only nature of this gene is shared by Saccharomyces mikatae YMR156C and Arabidopsis thaliana K15M2.3. Repair of 3'-phosphate and 5'-hydroxyl lesions is thus uncoupled in budding yeast as compared with metazoans. Repair of transformed dephosphorylated plasmids, and 5'-hydroxyl blocking lesions more generally, likely proceeds by a cycle of base removal and resynthesis.

* This work was supported in part by the Pew Scholars Program in the Biomedical Sciences of the Pew Charitable Trusts (to T. E. W.) and by National Institutes of Health Postdoctoral Training Grant 5T32HL07157 (to J. R. V.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Pathology, University of Michigan Medical School, 1301 Catherine Rd., M4214 Medical Science I, P. O. Box 0602, Ann Arbor, MI 48109-0602. Tel.: 734-936-1887; Fax: 734-763-6476; E-mail: wilsonte@umich.edu.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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Copyright © 2001 by the American Society for Biochemistry and Molecular Biology.