Uncoupling of 3 '-phosphatase and 5 '-kinase functions in budding yeast - Characterization of Saccharomyces cerevisiae DNA 3 '-phosphatase (TPP1)

Polynucleotide kinase is a bifunctional enzyme containing both DNA 3'-phosp hatase and 5'-kinase activities seemingly suited to the coupled repair of s ingle-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 consid erable efficiency independently of the end-processing. polymerase Pol4p. Ho mology searches and biochemical assays did not reveal a 5'-kinase that woul d account for this repair, however. Instead, open reading frame YMR156C (he re named TPP1) is shown to encode only a polynucleotide kinase-type 3'-phos phatase. Tpp1p bears extensive similarity to the ancient L-2-haloacid dehal ogenase and DDDD phosphohydrolase superfamilies, but is specific for double -stranded DNA. It is present at high levels in cell extracts in a functiona l form and so does not represent a pseudogene. Moreover, the phosphatase-on ly nature of this gene is shared by Saccharomyces mikatae YMR156C and Arabi dopsis thaliana K15M2.3. Repair of 3'-phosphate and 5'-hydroxyl lesions is thus uncoupled in budding yeast as compared with metazoans. Repair of trans formed dephosphorylated plasmids, and 5'-hydroxyl blocking lesions more gen erally, likely proceeds by a cycle of base removal and resynthesis.