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'-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.
Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0602, USA.
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JR
Vance
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Wilson
D000595:Amino Acid Sequence; D001483:Base Sequence; D002474:Cell-Free System; D017931:DNA Primers; D019143:Evolution, Molecular; D008969:Molecular Sequence Data; D009708:Nucleotidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016366:Open Reading Frames; D011116:Polynucleotide 5'-Hydroxyl-Kinase / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D011116:Polynucleotide 5'-Hydroxyl-Kinase; D009708:Nucleotidases; C039170:deoxynucleotide 3'-phosphatase