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• W2170532016 abstract "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. 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. base excision repair polynucleotide kinase human polynucleotide kinase 3′-phosphatase open reading frame polymerase chain reaction non-homologous end joining A. californica nucleopolyhedrovirus group of overlapping clones glutathione S-transferase 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. 1Friedberg E.C. Walker G.C. Siede W. DNA Repair and Mutagenesis. American Society for Microbiology, Washington, D. C.1995Google Scholar). 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 3′ and 5′ 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 5′ 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 β (2Sobol R.W. Prasad R. Evenski A. Baker A. Yang X.P. Horton J.K. Wilson S.H. Nature. 2000; 405: 807-810Crossref PubMed Scopus (299) Google Scholar, 3Srivastava D.K. Berg B.J. Prasad R. Molina J.T. Beard W.A. Tomkinson A.E. Wilson S.H. J. Biol. Chem. 1998; 273: 21203-21209Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar) or certain glycosylases (4Girard P.M. Guibourt N. Boiteux S. Nucleic Acids Res. 1997; 25: 3204-3211Crossref PubMed Scopus (119) Google Scholar, 5Bhagwat M. Gerlt J.A. Biochemistry. 1996; 35: 659-665Crossref PubMed Scopus (123) Google Scholar) or in long-patch BER by flap excision and resynthesis (6Memisoglu A. Samson L. Mutat. Res. 2000; 451: 39-51Crossref PubMed Scopus (237) Google Scholar). The extensive 5′-resection that occurs in the first steps of recombination also likely removes blocking lesions at double-strand breaks (7Paques F. Haber J.E. Microbiol. Mol. Biol. Rev. 1999; 63: 349-404Crossref PubMed Google Scholar, 8Anderson D.G. Kowalczykowski S.C. Genes Dev. 1997; 11: 571-581Crossref PubMed Scopus (153) Google Scholar). Common blocking lesions on 3′ termini include phosphates, α,β-unsaturated aldehydes resulting from β-elimination reactions (9Piersen C.E. McCullough A.K. Lloyd R.S. Mutat. Res. 2000; 459: 43-53Crossref PubMed Scopus (95) Google Scholar), and phosphoglycolate moieties that are the primary product of bleomycin action (10Povirk L.F. Mutat. Res. 1996; 355: 71-89Crossref PubMed Scopus (335) Google Scholar). The most potent 3′-processing enzymes are the apurinic-apyrimidinic endonucleases, which, in addition to cleaving strands at abasic sites, possess 3′-diesterase activities capable of removing most nucleotide fragments (11Ramotar D. Biochem. Cell Biol. 1997; 75: 327-336Crossref PubMed Scopus (55) Google Scholar, 12Mol C.D. Hosfield D.J. Tainer J.A. Mutat. Res. 2000; 460: 211-229Crossref PubMed Scopus (216) Google Scholar, 13Johnson A.W. Demple B. J. Biol. Chem. 1988; 263: 18017-18022Abstract Full Text PDF PubMed Google Scholar). It is again possible that 3′-lesions might be resolved by a more extensive degradation during recombination, e.g. by RecBCD (8Anderson D.G. Kowalczykowski S.C. Genes Dev. 1997; 11: 571-581Crossref PubMed Scopus (153) Google Scholar). Polynucleotide kinase (PNK) is best known due to the utility of the T4 enzyme (14Midgley C.A. Murray N.E. EMBO J. 1985; 4: 2695-2703Crossref PubMed Scopus (49) Google Scholar) in molecular cloning, but it was demonstrated to exist in eukaryotes 30 years ago (15Ichimura M. Tsukada K. J. Biochem. ( Tokyo ). 1971; 69: 823-828Crossref PubMed Scopus (22) Google Scholar). Although not clearly indicated by their name, the PNK proteins studied to date bear two distinct catalytic activities, a 5′-kinase and a 3′-phosphatase (16Karimi-Busheri F. Daly G. Robins P. Canas B. Pappin D.J. Sgouros J. Miller G.G. Fakhrai H. Davis E.M. Le Beau M.M. Weinfeld M. J. Biol. Chem. 1999; 274: 24187-24194Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 17Jilani A. Ramotar D. Slack C. Ong C. Yang X.M. Scherer S.W. Lasko D.D. J. Biol. Chem. 1999; 274: 24176-24186Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar). Although the precise biological role of eukaryotic PNK remains to be determined, it is clearly suited to directly reverse the two reciprocal blocking lesions that would result from a strand break with a misplaced phosphate, i.e. a 3′-phosphate and 5′-hydroxyl. Indeed, the preferred substrate of mammalian PNK is a DNA nick (18Karimi-Busheri F. Weinfeld M. J. Cell. Biochem. 1997; 64: 258-272Crossref PubMed Scopus (34) Google Scholar), and its enzyme activities are stimulated by interaction with the XRCC1 repair protein (19Whitehouse C.J. Taylor R.M. Thistlewaite A. Zhang H. Karini-Busheri F. Lasko D.D. Weinfeld M. Caldecott K.W. Cell. 2001; 104: 107-117Abstract Full Text Full Text PDF PubMed Scopus (511) Google Scholar), strongly suggesting a role in BER/single-strand break repair. As a continuation of our interest in delineating the mechanisms by which terminal damage is resolved during DNA double-strand break repair, we have been attempting to identify and characterize PNK from the yeast Saccharomyces cerevisiae, whose existence we inferred from the successful repair of transformed dephosphorylated linear plasmids. The recent cloning of human PNK/3′-phosphatase (hPNKP) (16Karimi-Busheri F. Daly G. Robins P. Canas B. Pappin D.J. Sgouros J. Miller G.G. Fakhrai H. Davis E.M. Le Beau M.M. Weinfeld M. J. Biol. Chem. 1999; 274: 24187-24194Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 17Jilani A. Ramotar D. Slack C. Ong C. Yang X.M. Scherer S.W. Lasko D.D. J. Biol. Chem. 1999; 274: 24176-24186Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar) has greatly facilitated this by allowing homology searching against the yeast and other sequenced genomes. Surprisingly, we find that S. cerevisiae, at least one otherSaccharomyces yeast, and species as distantly related asArabidopsis thaliana contain a gene with homology to only the putative 3′-phosphatase portion of hPNKP. The S. cerevisiae protein, encoded by open reading frame (ORF) YMR156C, here named TPP1, shares many of the biochemical properties of the hPNKP 3′-phosphatase, but indeed is not a 5′-kinase. Despite the observed plasmid repair, structural comparisons and enzymatic assays failed to detect an unlinked 5′-kinase. Evolutionary models to explain these results are discussed in the context of alternative pathways for resolution of terminal damage during DNA repair. Wild-type Saccharomyces mikataestrain 1815, obtained from Dr. Mark Johnston, is described in Ref. 20Fischer G. James S.A. Roberts I.N. Oliver S.G. Louis E.J. Nature. 2000; 405: 451-454Crossref PubMed Scopus (258) Google Scholar.Schizosaccharomyces pombe strain FY254 (h −, leu1-32, ura4-048,ade6, can1-1) was obtained from Dr. Dennis Thiele. The S. cerevisiae strains used in the plasmid assay were the wild-type strain YW389 (MATα,ade2Δ0, his3Δ200,leu2, lys2-801, trp1Δ63,ura3Δ0) and its isogenic derivatives YW513 (dnl4-K282R) and YW514 (pol4-D367E). The chromosomal point mutations in these strains were constructed by thede novo mutation strategy described by Erdeniz et al. (21Erdeniz N. Mortensen U.H. Rothstein R. Genome Res. 1997; 7: 1174-1183Crossref PubMed Scopus (142) Google Scholar). Strains were verified by diagnostic PCR, allele sequencing, and documenting their deficiencies in previously describedDNL4- and POL4-dependent non-homologous end joining (NHEJ) assays (22Wilson T.E. Lieber M.R. J. Biol. Chem. 1999; 274: 23599-23609Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 23Wilson T.E. Grawunder U. Lieber M.R. Nature. 1997; 388: 495-498Crossref PubMed Scopus (345) Google Scholar). The S. cerevisiae strains used for biochemical assays were the wild-type strain YW465 and its isogenic derivatives YW573 (tpp1Δ::MET15), YW605 (apn1Δ::HIS3), and YW619 (tpp1Δ::MET15,apn1Δ::HIS3). The deletions in these strains were constructed by PCR-mediated gene replacement and verified by PCR as described (24Brachmann C.B. Davies A. Cost G.J. Caputo E. Li J. Hieter P. Boeke J.D. Yeast. 1998; 14: 115-132Crossref PubMed Scopus (2612) Google Scholar). Single mutants were constructed directly in YW465 and YW619 by disruption of APN1 in YW573. Plasmid pES26 has been previously described (23Wilson T.E. Grawunder U. Lieber M.R. Nature. 1997; 388: 495-498Crossref PubMed Scopus (345) Google Scholar). Methods of plasmid preparation and transformation were exactly as described (22Wilson T.E. Lieber M.R. J. Biol. Chem. 1999; 274: 23599-23609Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar), but with the following addition. After BglII digestion, but before extraction and precipitation, an equal volume of 2× calf intestinal alkaline phosphatase buffer (Roche Molecular Biochemicals) was added, followed by nothing (ligation-competent control plasmid) or 0.16 units of calf intestinal alkaline phosphatase/μg of plasmid (dephosphorylated plasmid) and further incubation at 37 °C for 30 min. BLASTP and Psi-BLAST homology searches were performed via the NCBI web server. Sequences included in the overall alignment were all hPNKP BLASTP matches from the non-redundant GenBankTM, expressed sequence tag, and sequence tagged sites data bases with E < 0.01, in addition to Trl1p, T4 PNK, and AcNPV-2. Expressed sequence tag and STS sequences were assembled into contigs and translated prior to inclusion in the alignment (details are available on request). Accession numbers for the sequences that are ostensibly complete but uncharacterized are as follows (see Table I): Mus musculus,AAF36487; Drosophila melanogaster (CG9601), AAF54229; At-1 (A. thaliana gene encoding a putative 3′-phosphatase),BAA97052; At-2 (A. thaliana gene encoding a putative 5′ kinase), CAB81914; S. pombe (C23C11.04C), CAB11157;Spodoptera exigua NPV (ORF54), AAF33584; AcNPV-1 (Ac-HisP),AAA66663; and AcNPV-2 (A. californica PNK/ligase),AAA66716. In the case of Caenorhabditis elegans (accession number T21197), we extended the putative ORF F21D5.5 by appending both amino- and carboxyl-terminal exon translations that were not originally included. Alignments were performed with MACAW (25Schuler G.D. Altschul S.F. Lipman D.J. Proteins Struct. Funct. Genet. 1991; 9: 180-190Crossref PubMed Scopus (893) Google Scholar).Table ISpecies distribution of 3′-phosphatase and 5′-kinase domains identified by homology and literature searchesSpeciesGene 1Gene 2EukaryotaeMetazoaHs, Human3′-Pase/5′-kinaseMm, Mouse3′-Pase/5′-kinaseGg, Chicken3′-Pase ESTXl, FrogESTDm, Fruitfly3′-Pase/5′-kinaseBm, Silkworm3′-Pase ESTOv, Roundworm3′-Pase ESTBma, Roundworm3′-Pase ESTCe, Roundworm3′-Pase/5′-kinaseViridiplantaeAt, Thale cress3′-Pase5′-KinaseGm, Soybean3′-Pase ESTZm, Corn3′-Pase ESTCr, Green algae3′-Pase/?5′-kinase? ESTFungiSp, Fission yeast3′-Pase/5′-kinaseSc, Bakers' yeast3′-PasetRNA ligase/5′-kinaseSm3′-PaseKl, Milk yeast3′-Pase STSEuglenozoaTc, Trypanosome3′-Pase ESTDictyosteliidaDd, Slime mold3′-Pase/5′-kinase EST5′-Kinase ESTViridaedsDNASeNPV, Baculovirus3′-Pase(No RNA)AcNPV, Baculovirus3′-PasetRNA ligase/5′-Kinase/3′-PaseT4, Bacteriophage5′-Kinase/3′-PaseSpecies are grouped by taxonomy. Abbreviations are as follows: Hs,Homo sapiens; Mm, M. musculus; Gg, Gallus gallus; Xl, Xenopus laevis; Dm, D. melanogaster; Bm, Bombyx mori, Ov, Onchocerca volvulus; Bma, Brugia malayi; Ce, C. elegans; At, A. thaliana; Gm, Glycine max; Zm, Zea mays; Cr, Chlamydomonas reinhardtii; Sp,S. pombe; Sc, S. cerevisiae; Sm, S. mikatae; Kl, Kluyveromyces lactis; Tc,Trypanosoma cruzi; Dd, D. discoideum; SeNPV,S. exigua nucleopolyhedrovirus; AcNPV, A.californica nucleopolyhedrovirus. The domains present in gene 1 and, when relevant, gene 2 for a given species are indicated in amino- to carboxyl-terminal order. The H. sapiens gene is hPNKP;S. cerevisiae gene 1 is YMR156C (TPP1); S. cerevisiae gene 2 is TRL1; and the T4 gene is PNK. EST and STS designate partial sequences and therefore conclusions cannot be drawn regarding the domains that are not present. The C. reinhardtii gene has a sequence that can be aligned with the consensus Walker A motif, but it shows substantial divergence relative to all other proteins, and so it is unclear whether it represents a true 5′-kinase. 3′-Pase, 3′-phosphatase; dsDNA, double-stranded DNA. Open table in a new tab Species are grouped by taxonomy. Abbreviations are as follows: Hs,Homo sapiens; Mm, M. musculus; Gg, Gallus gallus; Xl, Xenopus laevis; Dm, D. melanogaster; Bm, Bombyx mori, Ov, Onchocerca volvulus; Bma, Brugia malayi; Ce, C. elegans; At, A. thaliana; Gm, Glycine max; Zm, Zea mays; Cr, Chlamydomonas reinhardtii; Sp,S. pombe; Sc, S. cerevisiae; Sm, S. mikatae; Kl, Kluyveromyces lactis; Tc,Trypanosoma cruzi; Dd, D. discoideum; SeNPV,S. exigua nucleopolyhedrovirus; AcNPV, A.californica nucleopolyhedrovirus. The domains present in gene 1 and, when relevant, gene 2 for a given species are indicated in amino- to carboxyl-terminal order. The H. sapiens gene is hPNKP;S. cerevisiae gene 1 is YMR156C (TPP1); S. cerevisiae gene 2 is TRL1; and the T4 gene is PNK. EST and STS designate partial sequences and therefore conclusions cannot be drawn regarding the domains that are not present. The C. reinhardtii gene has a sequence that can be aligned with the consensus Walker A motif, but it shows substantial divergence relative to all other proteins, and so it is unclear whether it represents a true 5′-kinase. 3′-Pase, 3′-phosphatase; dsDNA, double-stranded DNA. Primers were designed that corresponded to S. mikatae sequences (kindly provided by Dr. Mark Johnston) homologous to S. cerevisiae ORFs YMR154C and YMR157C (5′-TCCAGTTCAAAAGTAGGATTCC and 5′-TAGGTAAGGCCGACATCATC, respectively). These were used in a PCR with S. mikataegenomic DNA using the HF Advantage PCR kit (CLONTECH) according to the manufacturer's instructions. The resulting single ∼5-kilobase pair amplified fragment was sequenced directly by the University of Michigan DNA Sequencing Core by walking from YMR157C through YMR156C and into YMR155W. The entire S. mikatae YMR156C coding sequence (accession number AF326782) was read without ambiguities, including a stop codon read clearly from two independent runs. Yeast strains expressing GST fusion proteins were isolated from the ORF array described and kindly provided (via Dr. Dennis Thiele) by Dr. Eric Phizicky and co-workers (26Martzen M.R. McCraith S.M. Spinelli S.L. Torres F.M. Fields S. Grayhack E.J. Phizicky E.M. Science. 1999; 286: 1153-1155Crossref PubMed Scopus (354) Google Scholar). Yeast cells from the YMR156C well were streaked to single colonies, and anti-GST (Santa Cruz Biotechnology) Western blotting was used to identify isolates expressing proteins whose size corresponded to GST-Tpp1p and non-recombinant GST. Purification was by glass bead lysis and salt extraction, followed by batch chromatography on glutathione-agarose (Amersham Pharmacia Biotech) as described (26Martzen M.R. McCraith S.M. Spinelli S.L. Torres F.M. Fields S. Grayhack E.J. Phizicky E.M. Science. 1999; 286: 1153-1155Crossref PubMed Scopus (354) Google Scholar), except that protein expression was induced by adding 0.1 mm CuSO4 for 3 h prior to harvest. Typical final GST-Tpp1p dialysates derived from 50 ml of yeast culture contained 50 μg/ml fusion protein in 600 μl of 20 mm Tris-HCl (pH 7.5), 2 mm EDTA, 4 mm MgCl2, 1 mm dithiothreitol, 50 mm NaCl, and 50% (v/v) glycerol. Crude whole-cell extracts of S. cerevisiae, S. mikatae, and S. pombe were all prepared by glass bead disruption of cells in ∼1 cell pellet volume of 50 mmTris-HCl (pH 7.5), 1 mm EDTA, 1 m NaCl, 10 mm MgCl2, 1 mm dithiothreitol, 10% glycerol, 2 μg/ml aprotinin, 1 μg/ml each leupeptin and pepstatin, and 1 mm phenylmethylsulfonyl fluoride, followed by centrifugation to remove cellular debris. Final extracts were diluted to 0.5 μg/μl protein. Oligonucleotides with and without 3′-phosphates were purchased from Operon Technologies, Inc. (see Figs.5 and 7 for sequences). Oligonucleotides were 5′-end-labeled with [γ-32P]ATP using 3′-phosphatase-free polynucleotide kinase (Roche Molecular Biochemicals). Final substrates were prepared by annealing labeled oligonucleotides to a 2-fold molar excess of the required unlabeled strands by heating to 90 °C, followed by slow cooling. Standard assays of 3′-phosphatase activity contained 50 fmol of DNA substrate and 10 fmol of GST-Tpp1p or 1 μg of crude cellular protein in a reaction volume of 10 μl such that the final buffer was 50 mm Tris-HCl (pH 7.5), 100 mm NaCl, 10 mm MgCl2, 1 mm dithiothreitol, and 50 μg/ml bovine serum albumin. Kinase assays were similar, except they used 1 pmol of GST-Tpp1p and 25 mm NaCl and also included 100 units of T4 DNA ligase (New England Biolabs Inc.), 1 mm ATP, and, where indicated, 5 units of T4 PNK (New England Biolabs Inc.) as an internal control. After incubation at 30 °C for 10 min, formamide/EDTA loading buffer was added, and samples were electrophoresed on 7 m urea and 12% polyacrylamide gels, followed by autoradiography.Figure 7Uncoupling of 3′-phosphatase and 5′-kinase activities in cell-free extracts of Saccharomycesyeast. A, glass bead extracts from wild-type (wt), tpp1, apn1, and tpp1 apn1 strains (lanes 3–6, respectively) were tested for 3′-phosphatase activity as described in the legend to Fig. 5. The extracts resulted variably in removal of the 3′-phosphate or 3′-terminal nucleotide, yielding 22- and 21-mer oligonucleotide products, respectively. B, GST-Tpp1p and glass bead extracts from S. cerevisiae (Sc), S. mikatae(Sm), and S. pombe (Sp) were used in 3′-phosphatase assays as described for A to provide an activity comparison with D. C, a nicked oligonucleotide substrate was used to detect 5′-kinase activity. Following phosphorylation of the 5′ terminus at the nick, ligation by exogenously added T4 DNA ligase led to an increase in size of the labeled strand (indicated by an asterisk) from 22 to 47 nucleotides (nt). D, T4 PNK (5 units) and the same protein samples used in B were incubated with the 5′-kinase substrate (50 fmol) in the presence of 100 units of T4 DNA ligase at 30 °C for 10 min and analyzed on a sequencing gel, followed by autoradiography. S. cerevisiae apn1mutant extracts were used to rule out inhibition by the Apn1p nuclease. Also, T4 PNK was included in a duplicate of all reactions to demonstrate that the extracts did not prevent detection of 5′-kinase activity (note that nicked DNA is a poor substrate for T4 PNK).View Large Image Figure ViewerDownload Hi-res image Download (PPT) To begin to determine the impact of 5′-hydroxyl blocking lesions on the repair of double-strand breaks, plasmid pES26 was digested in vitro with the restriction enzyme BglII and transformed in yeast cells, both with and without pretreatment with calf intestinal alkaline phosphatase. In this well established assay (22Wilson T.E. Lieber M.R. J. Biol. Chem. 1999; 274: 23599-23609Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 23Wilson T.E. Grawunder U. Lieber M.R. Nature. 1997; 388: 495-498Crossref PubMed Scopus (345) Google Scholar), recircularization by NHEJ (7Paques F. Haber J.E. Microbiol. Mol. Biol. Rev. 1999; 63: 349-404Crossref PubMed Google Scholar, 23Wilson T.E. Grawunder U. Lieber M.R. Nature. 1997; 388: 495-498Crossref PubMed Scopus (345) Google Scholar, 27Featherstone C. Jackson S.P. Curr. Biol. 1999; 9: R759-R761Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar) is required for plasmid stability and thus for expression of the plasmid URA3 marker gene. Because the BglII site resides in an essential region of theADE2 marker gene also on this plasmid, transformation to Ade+ further requires that repair be precise (imprecise repair yields ade2 colonies that appear red instead of white). Dephosphorylated plasmid transformed into wild-type yeast showed only a 2–3-fold decrease in Ura+ colony recovery as compared with ligation-competent plasmid (Fig.1). Moreover, there was no increase in red colony recovery, indicating that joining remained precise. Transformation by dephosphorylated plasmids was decreased 54-fold in yeast deficient in the ligase required for NHEJ (dnl4-K282R) (23Wilson T.E. Grawunder U. Lieber M.R. Nature. 1997; 388: 495-498Crossref PubMed Scopus (345) Google Scholar), verifying that the damaged ends are not routed into another pathway. As an additional control, it was verified that calf intestinal alkaline phosphatase-treated plasmids could not be religated in vitro by T4 DNA ligase unless further treated with T4 PNK, as indicated by gel electrophoresis and a >700-fold decrease in colony recovery following bacterial transformation (data not shown). As shown in Fig. 1 A, there are at least two ways that NHEJ of 5′-hydroxyl lesions might be achieved. In the first, the damaged 5′-nucleotide is nucleolytically removed and subsequently resynthesized on the opposite side of the break. We have previously observed that the yeast PolX family polymerase Pol4p can catalyze such base addition during NHEJ (22Wilson T.E. Lieber M.R. J. Biol. Chem. 1999; 274: 23599-23609Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). pol4-D367E mutant yeast, which expresses catalytically inactive Pol4p (22Wilson T.E. Lieber M.R. J. Biol. Chem. 1999; 274: 23599-23609Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar), showed the same pattern as the wild type, however. Importantly, some polymerization-dependent NHEJ events show only a 2-fold defect in pol4 mutants (22Wilson T.E. Lieber M.R. J. Biol. Chem. 1999; 274: 23599-23609Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar), so it possible that another polymerase can substitute for Pol4p at dephosphorylated lesions as well. Nonetheless, these observations were consistent with the alternative model in which the 5′-hydroxyl lesion is directly reversed by a PNK 5′-kinase. We sought to identify this in the experiments described below. BLAST searches against the non-redundant GenBankTM, expressed sequence tag, and sequence tagged sites data bases using hPNKP as the query revealed a set of 22 distinct PNK-like genes from 20 different species and viruses (TableI). Among these was the S. cerevisiae ORF YMR156C, but this weaker matche (E= 0.006) corresponded to only a portion of the human protein and surprisingly lacked an apparent Walker A (i.e. P-loop) motif for ATP binding (28Walker J.E. Saraste M. Runswick M.J. Gay N.J. EMBO J. 1982; 1: 945-951Crossref PubMed Scopus (4248) Google Scholar), which would be expected for a 5′-kinase. Absent as matches were three genes identified from literature searching that are known or believed to encode 5′-kinases: TRL1 tRNA ligase from S. cerevisiae (29Koonin E.V. Gorbalenya A.E. FEBS Lett. 1990; 268: 231-234Crossref PubMed Scopus (20) Google Scholar, 30Westaway S.K. Phizicky E.M. Abelson J. J. Biol. Chem. 1988; 263: 3171-3176Abstract Full Text PDF PubMed Google Scholar), bacteriophage T4 PNK (14Midgley C.A. Murray N.E. EMBO J. 1985; 4: 2695-2703Crossref PubMed Scopus (49) Google Scholar), and a putative PNK from AcNPV (31Ayres M.D. Howard S.C. Kuzio J. Lopez-Ferber M. Possee R.D. Virology. 1994; 202: 586-605Crossref PubMed Scopus (848) Google Scholar). To examine the relationship between YMR156C and the other PNK-related genes in more detail, we performed an extensive multiple sequence alignment as shown in Figs. Figure 2, Figure 3, Figure 4. Two sequence motifs, the Walker A box and the phosphotransferase motif DXDX(T/V) (32Collet J.F. Stroobant V. Pirard M. Delpierre G. Van Schaftingen E. J. Biol. Chem. 1998; 273: 14107-14112Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar), were used as a means of unambiguously identifying the 5′-kinase and 3′-phosphatase catalytic cores, respectively, as suggested by previous alignments of smaller numbers of bifunctional PNK sequences (16Karimi-Busheri F. Daly G. Robins P. Canas B. Pappin D.J. Sgouros J. Miller G.G. Fakhrai H. Davis E.M. Le Beau M.M. Weinfeld M. J. Biol. Chem. 1999; 274: 24187-24194Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 17Jilani A. Ramotar D. Slack C. Ong C. Yang X.M. Scherer S.W. Lasko D.D. J. Biol. Chem. 1999; 274: 24176-24186Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar). It was first apparent that genes may contain just the 3′-phosphatase domain, just the 5′-kinase domain, or both. This was not limited to S. cerevisiae. For example, A. thaliana contains two hypothetical genes, one encoding a 3′-phosphatase (designated for simplicity as At-1; see “Experimental Procedures” for accession numbers) and one encoding a 5′-kinase (At-2). These genes are on different chromosomes, and examination of the genomic sequence surrounding At-1 did not reveal any cryptic 5′-kinase domain exons. In addition, in some species, there is an apparent redundancy of function. For example, Dictyostelium discoideum has two distinct putative 5′-kinase genes. We note, however, that these genes correspond to two structurally evident 5′-kinase groupings (see below), so it is possible, if not likely, that 5′-kinases perform distinct tasks in the cell. Finally, in genes containing both domains, each of the two possible orders are observed.Figure 3Structural motifs of the PNK 3′-phosphatase domain. Selected sequence regions from the complete alignment are shown that correspond to the conserved motifs of the 3′-phosphatase (3′ Pase) domain. Positions identical in all species from human to bacteriophage T4 are highlighted asyellow-on-red. Positions identical or similar to a consensus of 50% of the sequences are highlighted as yellow-on-blueand white-on-green, respectively. Two PNK phosphatase subtypes evident in the alignment are separated by a horizontal line. Motifs discussed under “Results” are indicated, with the corresponding E. coli HisB sequence shown above the alignment where relevant. Asterisks mark amino acid positions in motifs 1–4 that, in addition to motifs A and B, provide strong discrimination between the histidinol and DNA 3′-phosphatase families. Gene/species designations are abbreviated as in Table I, with a hyphenated number corresponding to the gene number.View Large Image Figure ViewerDownload Hi-res image" @default.
• W2170532016 created "2016-06-24" @default.
• W2170532016 creator A5029634644 @default.
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