Uncoupling of 3′-Phosphatase and 5′-Kinase Functions in Budding Yeast

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 yeastSaccharomyces 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.