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

Abstract

Polynucleotide kinase is a bifunctional enzyme containing both DNA 3-phosphatase and 5-kinase activities seemingly suited to the coupled repair of singlestrand 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-haloacid 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.Oxidative damage to DNA can result from endogenously generated reactive oxygen species or from exposure to exogenous agents such as ionizing radiation or anticancer agents such as bleomycin and neocarzinostatin (reviewed in Ref. 1). Such damage and the enzymes involved in its repair frequently produce fragmentation of the deoxyribose sugar backbone, resulting in DNA strand breaks bearing abnormal structures at the 3Ј and 5Ј termini. These are termed "blocking lesions" because they prevent the reactions necessary to achieve final repair of the damaged strand, namely polymerization and ligation. Since both single-and double-strand lesions can occur with potential consequences that include replication failure and genomic rearrangement, the resolution of blocking lesions is of major importance in genome maintenance. Although many chemical forms are possible, important blocking lesions on 5Ј termini include hydroxyls and deoxyribose phosphates. As an example of the redundancies in end processing, deoxyribose phosphate moieties can be removed in short-patch base excision repair (BER) 1 by the lyase function of DNA polymerase ␤ (2, 3) or certain glycosylases (4, 5) or in long-patch BER by flap excision and resynthesis (6). The extensive 5Ј-resection that occurs in the first steps of recombination also likely removes blocking lesions at double-strand breaks (7,8). Common blocking lesions on 3Ј termini include phosphates, ␣,␤-unsaturated aldehydes resulting from ␤-elimination reactions (9), and phosphoglycolate moieties that are the primary product of bleomycin action (10). The most potent 3Ј-processing enzymes are the apurinic-apyrimidinic endonucleases, which, in addition to cleaving strands at abasic sites, po...

The Role of Yeast DNA 3′-Phosphatase Tpp1 and Rad1/Rad10 Endonuclease in Processing Spontaneous and Induced Base Lesions

“…Bacteria, in contrast, lack a related DNA 3Ј-phosphatase. Like the plant enzymes, Tpp1 is devoid of a polynucleotide kinase domain, and we have provided functional evidence that budding yeast in fact do not possess this activity (24). Thus, only the 3Ј-phosphatase function of this enzyme family is consistently conserved, and only in eukaryotes.…”

“…Duplex oligonucleotides bearing a uracil, or a 3Ј-phosphate at a nick, were labeled and annealed as previously described (24,25). The uracil was converted to a singlebase gap with a 3Ј-aldehyde by treatment with uracil-DNA glycosylase (Udg, 0.01 units/5 pmol oligonucleotide) and Nth (28 ng/pmol oligonucleotide) in Udg buffer (NEB) for 1 h at 37°C.…”

“…Enzyme Activity Assays-GST-Tpp1 and GST-Apn1 were purified from S. cerevisiae as previously described (24). Duplex oligonucleotides bearing a uracil, or a 3Ј-phosphate at a nick, were labeled and annealed as previously described (24,25).…”

“…For comparison, 10 fmol of GST-Tpp1 and 380 fmol of GST-Apn1 were shown in the same experiment to completely remove a 3Ј-phosphate. GST-Apn1 can additionally remove the 3Ј-nucleotide to form a 21-OH product as previously described (24). M indicates marker lanes with labeled synthetic product oligonucleotides.…”

“…We have identified and begun to characterize Tpp1, the S. cerevisiae phosphatase (24,25). Tpp1 is homologous to one portion of the bifunctional enzyme polynucleotide kinase/3Ј phosphatase (PNKP) found in Schizosaccharomyces pombe and metazoans (26 -29).…”