THE JOURNAL OF BIOLOGICAL CHEMISTRY © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 277, No. 6, Issue of February 8, pp. 4050 –4055, 2002 Printed in U.S.A.
Pnk1, a DNA Kinase/Phosphatase Required for Normal Response to DNA Damage by ␥-Radiation or Camptothecin in Schizosaccharomyces pombe* Received for publication, September 28, 2001, and in revised form, November 2, 2001 Published, JBC Papers in Press, November 29, 2001, DOI 10.1074/jbc.M109383200
Maria Meijer‡§, Feridoun Karimi-Busheri§¶, Timothy Y. Huang‡, Michael Weinfeld¶, and Dallan Young‡储 From the ‡Departments of Biochemistry & Molecular Biology and Oncology, University of Calgary Health Sciences Center, Calgary, Alberta, T2N 4N1, Canada and the ¶Department of Experimental Oncology, Cross Cancer Institute, University of Alberta, Edmonton, Alberta, T6G 1Z2, Canada
We report the characterization of Pnk1, a 45-kDa homolog of the human polynucleotide kinase PNKP in Schizosaccharomyces pombe. Recombinant Pnk1 like human PNKP exhibits both 5ⴕ-DNA kinase and 3ⴕ-DNA phosphatase activities in vitro. Furthermore, we detected 3ⴕ-DNA phosphatase activity with a single-stranded substrate in extracts from wild-type yeast, but no activity was detected in pnk1⌬ strains. We have shown that GFP-tagged Pnk1 like mammalian PNKP localizes to the nucleus. Deletion of pnk1 does not affect cell growth under normal conditions but results in significant hypersensitivity to ␥-radiation or camptothecin, an inhibitor of topoisomerase I, suggesting that Pnk1 plays an important role in the repair of DNA strand breaks produced by these agents. The pnk1 deletion mutants were not hypersensitive to ethyl methanesulfonate, methyl methanesulfonate, or 4-nitroquinoline N-oxide. Expression of human PNKP in pnk1⌬ cells restores resistance to ␥-radiation or camptothecin, suggesting that the functions of yeast Pnk1 and human PNKP have been conserved.
DNA strand breaks arise during DNA base excision repair or can result from DNA damage by ionizing radiation and chemical agents, such as alkylating agents and topoisomerase inhibitors (1, 2). Such DNA damage often results in DNA strands with 5⬘-hydroxyl and 3⬘-phosphate termini. However, the repair of DNA damage by DNA polymerases and ligases requires 5⬘-phosphate and 3⬘-hydroxyl termini. Eucaryotic polynucleotide kinases (PNKs)1 were identified over 30 years ago (3, 4) and were subsequently shown to possess 3⬘-DNA phosphatase activity in addition to their 5⬘-DNA kinase activity (5, 6). The recent identification and characterization of human PNKP revealed that it too possesses both 5⬘-DNA kinase and 3⬘-DNA phosphatase activities (7, 8). Such dual DNA kinase/phosphatases could provide a mechanism for the repair of blocking lesions at the ends of DNA single strand breaks. Indeed, mammalian PNKP preferentially phosphorylates the 5⬘ termini associated with gaps and nicks, consistent with the idea that it is * This work was supported by grants from the Canadian Institutes of Health Research and the Alberta Cancer Board. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. § Both authors contributed equally to this work. 储 To whom correspondence should be addressed: 3330 Hospital Dr., N. W., Calgary, AB T2N 4N1, Canada. Tel.: 403-220-3030; Fax: 403283-8727; E-mail:
[email protected]. 1 The abbreviations used are: PNK, polynucleotide kinases; kbp, kilobase pair; HA, hemagglutinin; GFP, green fluorescent protein.
involved in the repair of DNA single strand breaks (9). More recent studies have shown that human PNKP associates with a complex containing XRCC1 (10), a protein that has been implicated in the repair of DNA single strand breaks (11–13). Furthermore, XRCC1 stimulates the DNA kinase and phosphatase activities of PNKP at damaged DNA termini. Thus, the evidence strongly suggests that PNKP is involved in the repair of DNA single strand breaks induced by DNA-damaging agents such as ionizing radiation. A comparison of human PNKP with the DNA and protein sequence data bases revealed the existence of structurally homologous proteins among different eucaryotic organisms, including members of Animalia, Plantae, Fungi, and Protista (7, 8, 14, 15). However, several of these homologs lack either DNA kinase or phosphatase domains. For example, the budding yeast protein, Tpp1, has weak similarity to PNKP and has DNA phosphatase activity, but it lacks a DNA kinase domain and kinase activity (15). Here, we report the characterization of Pnk1, a homolog of human PNKP, in Schizosaccharomyces pombe. Our results demonstrate that Pnk1 has both 5⬘-DNA kinase and 3⬘-DNA phosphatase activities. Furthermore, we show that the deletion of the gene renders fission yeast hypersensitive to ionizing radiation and camptothecin, an inhibitor of topoisomerase I. Together, our results suggest that Pnk1 has a conserved role in DNA repair. EXPERIMENTAL PROCEDURES
DNA Manipulation and Analysis—Procedures used for DNA purification, restriction-site mapping, electrophoresis, and transformation have been described previously (16). Plasmids—The pnk1 gene including flanking regions (⫺726 to ⫹660 relative to the open reading frame) was amplified by PCR of S. pombe genomic DNA using the primers 5⬘-AGCTCTAGATCCTTAAAAAGAGCAACGC-3⬘ and 5⬘-AGCTCTCGAGTTAGCCTTGTATGCAGGTTT-3⬘. pBS-Pnk was constructed by cloning the PCR product into the XhoI and XbaI sites of pBluescript II SK (Stratagene). The pnk1 open reading frame was then removed by digestion with EcoRV and BclI and replaced with a 1.8-kbp ura4 fragment isolated from pBSUra4 by digestion with BamHI and HincII to form pBS-PnkUra4. The pREP41 and pREP41XHA expression vectors were described previously (17, 18). pREP41XHA-Pnk was constructed by the insertion of the pnk1-coding sequence derived from PCR of S. pombe genomic DNA using the primers 5⬘-GATCCTCGAGGATGTCGTCGAAAAAAAG-3⬘ and 5⬘-GATCGGATCCTTATTCGTACCAATAATTCC-3⬘ into the SalI and BamHI sites of the pREP41XHA polylinker. pAALNHA was constructed by cloning the HA epitope-coding sequence 5⬘-CTGCAGATCTCGAGATGTATCCTTATGACGTGCCTGACTATGCCAGCCTGGGAGGACCGTCGACAACTAGTAGCGGCCGCAGGATCC-3⬘ into PstI/BamHI sites in pAALN (19). pAALNGFPHA was constructed by inserting the enhanced green fluorescent protein (eGFP) tag amplified from peGFP (CLONTECH) using 5⬘-GTCAGCCTCGAGATGGTGAGCAAGGGCGAG-3⬘ and 5⬘-GATCTCCTCGAGCTTGTACAGCTCGTCCAT-3⬘ primers into the pAA-
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Pnk1, a Fission Yeast DNA Kinase/Phosphatase
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FIG. 1. Comparison of yeast Pnk1 and human PNKP protein sequences. Protein sequences were aligned using the ClustalW alignment algorithm (37). Regions of sequence identity are shaded. Motifs conserved among PNKP-related proteins in other organisms are indicated.
LNHA XhoI site. pAALNGFPHA-Pnk1 was constructed by inserting the coding region of Pnk1 amplified using 5⬘-GATCACTAGTATGTCGTCGAAAAAAAGAAAGTCTC-3⬘ and 5⬘-GATCGGATCCTTATTCGTACCAATAATTCC-3⬘ primers into pAALNGFPHA SpeI and BamHI sites. pAALNGFPHA-Pnk1K4E,R6D was constructed by inserting a mutated Pnk1-coding sequence generated by amplification using 5⬘-GATCACTAGTATGTCGTCGGAAAAAGACAAGTCTCCC-3⬘ and 5⬘-GATCGGATCCTTATTCGTACCAATAATTCC-3⬘ primers into pAALNGFPHA SpeI and BamHI sites. pREP41-HPnk was constructed by cloning the human PNKP cDNA into the NdeI and BamHI sites of pREP41. pET33-Pnk was constructed by cloning the pnk1 cDNA derived from PCR of a S. pombe cDNA library using the primers 5⬘-GATCGAGCTCGATGTCGTCGAAAAAAAG-3⬘ and 5⬘-GATCCTCGAGTTATTCGTACCAATAATTCC-3⬘ into the SacI and XhoI sites of pET-33b (Novagen). Con- struction of the S. pombe cDNA library was described previously (20). Yeast Strains and Genetic Analysis—Yeast culture, transformation, iodine staining, tetrad analysis, and other genetic manipulations were performed as described previously (21). The S. pombe haploid strain AS7 (h⫹ leu1–32 ade6 –210) was described previously (19). SP826 (h⫹/h⫹ leu1–32/leu1–32 ade6 –210/ade6 –216 ura4-D18/ura4-D18) was obtained from Dr. D. Beach (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). pnk1 Deletion—S. pombe pnk1 was disrupted in the diploid strain, SP826, by gene replacement (22, 23). SP826 was transformed with the PCR-amplified insert from pBS-PnkUra4. Transformants were tested for stability of the Ura⫹ phenotype and analyzed by Southern blots to confirm the replacement of one copy of pnk1 with the selectable marker as follows. Genomic DNA was purified from SP826 and Ura⫹ transformants and digested with XhoI and EcoRV, and Southern blots were hybridized with a 32P-labeled DNA probe derived from PCR amplification of the 300-bp region located between ⫹684 and ⫹984 bp 3⬘ to the pnk1 stop codon. The replacement of one pnk1 allele with ura4 resulted in a 1.6-kbp XhoI/EcoRV DNA fragment in addition to the 1.0-kbp DNA fragment of the wild-type allele. h⫹/h90 revertants were detected by iodine vapor staining and subjected to tetrad analysis. pnk1⌬ (h⫹ leu1–32 ade6 –210 ura4-D18 pnk1::ura4⫹) and pnk1⫹ (h⫹ leu1–32 ade6 – 210 ura4-D18) haploid strains were isolated from spores of a single ascus.
Kinase and Phosphatase Assays—The assays for DNA kinase activity using either micrococcal nuclease-digested DNA or an oligonucleotide substrate and phosphatase activity using an oligonucleotide bearing a 3⬘-phosphate have been described previously (7, 9). Purification of Recombinant Pnk1 from Escherichia coli—pET33Pnk, which encodes His6-tagged Pnk1, was transfected into the E. coli strain BLR(DE3) (Novagen). The bacteria were grown at 37 °C to an A600 of 0.6 in 100 ml of Luria-Bertani medium containing 50 g/ml ampicillin and 12.5 g/ml tetracycline. His6-Pnk1 expression was induced at 30 °C for 3 h by the addition of 0.1 mM final concentration of isopropyl-1-thio--D-galactopyranoside (Sigma). The cells were then harvested by centrifugation at 5000 ⫻ g for 5 min at 4 °C and resuspended in 10 ml of extraction buffer (50 mM Tris-HCl, pH 7.5, 0.015 mM ZnCl2, 4 mM 2-mercaptoethanol). Lysozyme was added to a final concentration of 100 g/ml together with Triton X-100 (final concentration, 0.1%) and incubated at 30 °C for 15 min. The bacteria were then disrupted by sonication, and the soluble fraction was separated from the insoluble fraction by centrifugation at 12,000 ⫻ g for 15 min at 4 °C. The soluble fraction was subjected to a 65% ammonium sulfate precipitation followed by centrifugation at 10,000 ⫻ g for 15 min. The pellet was resuspended in a minimum volume of buffer A (10 mM sodium phosphate, pH 7.3, 0.2 M NaCl, 4 mM 2-mercaptoethanol, and protease inhibitor mixture (Sigma)) and dialyzed overnight in the same buffer. The solution was applied on a 25-ml SP-Sepharose fast flow cation exchange column (Amersham Biosciences, Inc.). The column was washed with 80 ml of buffer A, and the enzyme was eluted with a 300-ml linear gradient of buffer A containing 0.2–1.0 M NaCl. The active fractions were eluted between 0.4 – 0.6 M NaCl. The active fractions were pooled and concentrated by 65% ammonium sulfate. The precipitate was dissolved in buffer A and loaded onto a Superdex-75 PC 3.2/30 gel filtration column attached to a SMART micropurification system (Amersham Biosciences, Inc.). The active fractions coeluted with a purified protein at an estimated molecular mass of 40 –50 kDa. Mass spectral analysis of tryptic peptides confirmed that the purified protein was coded by the Pnk1 cDNA. Eight peptides were identified by their mass, HNNVFR (786 Da), YIHHNQK (939 Da), NSSSHPYHFK (1204 Da), QLPEIAFNSFK (1294 Da), SPPQESLTSYFEK (1513 Da), STLAESQIVTQGYER (1682 Da), DAADWTWWHPSVVPK (1795 Da), and YVGDAAGRPGDHNSTDLK (1873 Da).
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Pnk1, a Fission Yeast DNA Kinase/Phosphatase
FIG. 2. Pnk1 exhibits 5ⴕ-DNA kinase and 3ⴕ-DNA phosphatase activities. A. His6-Pnk1 was purified from E. coli expressing this recombinant protein as described under “Experimental Procedures.” Samples from the soluble fraction of E. coli extract (Ext), pooled active fractions eluted from a SP-Sepharose fast flow column (SSFF), and active His6-Pnk1 recovered from a Superdex-75 gel filtration column SD-75 were analyzed by 12% SDS-PAGE and Coomassie Blue staining. B, 5⬘-DNA kinase activity of fractions collected from the SP-Sepharose column was assayed by 5⬘-end labeling of micrococcal nuclease-digested calf thymus DNA. Micrococcal nuclease generates DNA fragments with 5⬘-OH termini. The samples were then examined by gel electrophoresis. Micrococcal nuclease-digested DNA labeled by T4 Pnk was used as a control. C, 3⬘-DNA phosphatase activity of recombinant His6-Pnk1 was assayed by the conversion of the 21-mer oligonucleotide p21p, bearing a 3⬘-phosphate to p21 lacking the 3⬘-phosphate. Partial Purification of Polynucleotide Kinase from S. pombe Strains—Fission yeast strains expressing either Pnk1 or PNKP were grown to a A600 of 1.0 and were washed in H2O. The cells were resuspended in 50 mM Tris-Cl, pH 8.0, 1 mM EDTA, 100 mM NaCl, and protease inhibitor mixture and disrupted by French press. To remove nucleic acid, the supernatant obtained from French press was mixed for 1 h with preswelled DE52 resin, which was then removed by low speed centrifugation. The supernatant was dialyzed against buffer A and loaded onto a 5-ml Hi-Trap SP cation exchange column (Amersham Biosciences, Inc.). The column was washed with 15 ml of buffer A, and the protein was eluted in twenty 3-ml fractions with a 60-ml linear gradient of buffer A containing 0.2–1.0 M NaCl. Fractions containing Pnk1 activity eluted between 0.3 and 0.5 M NaCl. The active fractions were pooled, concentrated by 65% ammonium sulfate, dialyzed against a minimal volume of buffer A, and used for kinase and phosphatase activities. Polyclonal Antiserum—The antigen used to raise the antibodies was gel-purified His6-Pnk1 recovered from the insoluble fraction from E. coli extracts as described previously (24). Approximately 5–10 mg of the inclusion body material was mixed with gel loading buffer (0.1 M TrisHCl, pH 6.8, 10% glycerol, 4% SDS, 100 mM dithiothreitol, 0.1% bromphenol blue), boiled for 5 min, and run on a 10% SDS-polyacrylamide gel. The His6-Pnk1 was electroeluted by excising the 48-kDa band from the gel, transferring the gel slices to dialysis tubing containing the SDS-PAGE-running buffer (0.025 M Tris, 0.192 M glycine, 0.1% SDS) immersed in a shallow layer of the same buffer in an electrophoresis
FIG. 3. pnk1⌬ strains lack 3ⴕ-DNA phosphatase activity. A, Western blot using anti-Pnk1 antibody of purified His6-Pnk1 from E. coli (first lane) and extracts from normal (WT) and pnk1⌬ cells containing control vectors or expressing Pnk1 or PNKP as indicated. B, 3⬘-DNA phosphatase activities of partially purified extracts from (i) WT, (ii) pnk1⌬, (iii) pnk1⌬-expressing Pnk1, and (iv) pnk1⌬ cells expressing human PNKP. The extracts were eluted from a Hi-Trap SP cation exchange column as described under “Experimental Procedures.” The number at the top of each lane denotes the elution fraction. Expression plasmids used were pREP41XHA (control vector), pREP41XHA-Pnk, and pREP41-HPnk. tank and electrophoresed overnight at 30 V. The protein concentration was determined by the Bradford method using a commercial protein assay system (Bio-Rad). For immunization, a New Zealand White female rabbit was given an initial intramuscular injection of 200 g of protein followed by three boosts with 100 g of protein injected at monthly intervals following a standard protocol (25). RESULTS
Comparison of Fission Yeast Pnk1 and Human PNKP Sequences—A standard BLAST search of the GenBankTM data base for proteins homologous to human PNKP revealed the presence of a homologous protein in S. pombe that we have named Pnk1 (7, 8). We amplified the Pnk1 cDNA by PCR and cloned it (see “Experimental Procedures”). The cDNA sequence revealed the presence of three introns in the pnk1 gene rather than two as reported in GenbankTM, and the exons encode a predicted protein of 408 residues (Fig. 1). Human PNKP (521 residues) and fission yeast Pnk1 are structurally homologous; they share 127 identical residues (Fig. 1). An alignment of Pnk1 with human PNKP and other related PNKs revealed a strong sequence similarity within five conserved regions of the kinase and phosphatase domains (7, 8, 15). In particular, Pnk1 contains a conserved ATP-binding Walker A motif (hGXPGXGKSTh) in the kinase domain and phosphotransferase
Pnk1, a Fission Yeast DNA Kinase/Phosphatase (DXDX(T/V)) and DDDD motifs in the phosphatase domain in addition to other conserved motifs that are found in human PNKP and other related eucaryotic PNKs (Fig. 1). Pnk1 Exhibits DNA Kinase and Phosphatase Activities—The high degree of sequence identity between the yeast Pnk1 and human PNKP suggests that they may be functionally conserved as well. Thus, we explored the possibility that Pnk1 possesses DNA kinase and phosphatase activities similar to human PNKP. First, we generated plasmids to express and purify recombinant His6-tagged Pnk1 from E. coli (see “Experimental Procedures”). An analysis of purified protein fractions by polyacrylamide gel electrophoresis indicated that they contained a homogeneous protein with a molecular mass of ⬃48 kDa corresponding to the predicted molecular mass of His6Pnk1. We found that purified His6-Pnk1 exhibited both 5⬘-DNA kinase and 3⬘-DNA phosphatase activities (Fig. 2). No activity was observed in corresponding proteins fractions from bacteria containing a control vector (data not shown). To further investigate the function of Pnk1, we generated
FIG. 4. Pnk1 localizes to the nucleus. Normal (AS7) fission yeast cells expressing GFP, GFP-Pnk1, or GFP-Pnk1K4E,R6D were briefly fixed before being mounted in medium containing 4⬘,6-diamidine-2⬘-phenylindole dihydrochloride as described previously (38). Cells were visualized by differential interference contrast (DIC) microscopy. DNA (DAPI) and GFP localization were visualized using fluorescence microscopy. Expression plasmids used were pAALNGFPHA, pAALNGFPHAPnk1, and pAALNGFPHA-Pnk1K4E,R6D.
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fission yeast strains lacking pnk1 by gene replacement with the ura4-selectable gene (see “Experimental Procedures”). Deletion of pnk1 did not have any apparent effect on cell growth, mating, or germination under normal conditions (data not shown). We generated an anti-Pnk1 polyclonal antibody, and we examined wild-type and pnk1⌬ yeast cell extracts by Western blot analysis to verify the presence or absence of Pnk1 expression. We detected a 45-kDa protein corresponding to the predicted size of Pnk1 in wild-type yeast, however, we did not detect a similar protein in pnk1⌬ cells (Fig. 3A). We also detected a similar band in pnk1⌬ cells overexpressing HA-Pnk1, but the antibody did not detect human PNKP in cells expressing this protein. Expression of human PNKP in S. pombe was verified by Western blotting with an antibody to PNKP (24). Next, we examined DNA phosphatase activities in partially purified extracts from pnk1⌬ and wild-type cells. To avoid any potential contribution to 3⬘-phosphatase activity arising from an apurinic/apyrimidinic endonuclease in the cell extract, we used a single-stranded oligonucleotide substrate, because all known apyrimidinic endonucleases appear to be double strandspecific (1, 26). Our results show that we detected significant 3⬘-DNA phosphatase activity in extracts from wild-type cells, but no detectable activity was observed in pnk1⌬ cell extracts (Fig. 3B). The phosphatase activity in the cell extracts coeluted from a cation-exchange column with recombinant Pnk1. We also detected 3⬘-DNA phosphatase activities in pnk1⌬ cells expressing either yeast Pnk1 or human PNKP (Fig. 3B). Attempts to recover 5⬘-DNA kinase activity from cell extract were unsuccessful. Our experience with the recombinant Pnk1 indicated that the kinase activity is less robust than that of human PNKP. Pnk1 Is Localized in the Nucleus—Human PNKP has been previously shown to localize in the nucleus of human A549 cells (7). To examine the localization of Pnk1 in fission yeast, we expressed GFP-tagged Pnk1 in wild-type cells. An examination of cells expressing GFP-Pnk1 by fluorescence microscopy revealed that it colocalized with 4⬘,6-diamidine-2⬘-phenylindole dihydrochloride staining, indicating that it was predominantly localized in the nucleus, whereas, GFP alone was distributed throughout the cell (Fig. 4). This observation is consistent with
FIG. 5. Deletion of pnk1 results in hypersensitivity to ␥-radiation and camptothecin, and expression of human PNKP rescues pnk1 null phenotypes. A, to assay for radiosensitivity, overnight cultures of normal (WT) and pnk1⌬ strains were serially diluted, plated onto 60-mm minimal medium plates, and irradiated with 1000 Gy using a Gammacell 1000 137Cs x-ray source (Nordion) at 2.5 Gy/min. Colony number was counted after 3 days of incubation at 30 °C. The graph indicates the percentage survival (y axis) of WT or pnk1⌬ cells harboring a control vector or pnk1⌬ cells expressing either Pnk1 or human PNKP. B, to test for sensitivity to camptothecin, cells were grown at 30 °C for 4 days on PMA plates containing 0.1% Me2SO, 25 mM HEPES, pH 7.2, in the presence or absence of 5 g/ml camptothecin (Sigma). Expression plasmids used were pREP41XHA (control vector), pREP41XHA-Pnk, and pREP41-HPnk.
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conserved nuclear roles for human PNKP and fission yeast Pnk1. We also expressed mutant GFP-Pnk1K4E,R6D in which residues in the putative N-terminal nuclear localization signal have been replaced. As predicted, this mutant protein failed to localize to the nucleus (Fig. 4). Deletion of pnk1 Results in Hypersensitivity to DNA-damaging Agents—To further investigate the roles of Pnk1, we examined pnk1⌬ cells for sensitivity to DNA damage by ionizing radiation. We found that pnk1⌬ cells were hypersensitive to ␥-radiation compared with wild-type cells (Fig. 5A), suggesting that Pnk1 is important for the normal cellular response to DNA damage, and may be directly involved in DNA repair. We also found that pnk1⌬ cells were hypersensitive to camptothecin (5 g/ml), an inhibitor of topoisomerase I (Fig. 5B). However, we did not observe a significant difference in the sensitivity to other DNA-damaging agents including ethyl methanesulfonate, methyl methanesulfonate, and 4-nitroquinoline N-oxide. Expression of Pnk1 restored normal resistance to ␥-radiation or camptothecin in pnk1⌬ cells. Furthermore, we found that the expression of human PNKP in pnk1⌬ cells restored normal resistance to either ␥-radiation or camptothecin, suggesting that the functions of these proteins have been conserved (Fig. 5). DISCUSSION
DNA damage can lead to serious cellular consequences including mutation, gene rearrangement, cell cycle arrest, and cell death. Thus, mechanisms to repair DNA lesions are important for the selective fitness and survival of all species. Consequently, several DNA repair mechanisms have evolved and are highly conserved among eucaryotes. A role for polynucleotide kinases in DNA repair has been previously suggested, and recent evidence has implicated mammalian PNKP in single strand break repair, although the details remain to be determined. The high degree of conservation of PNKP with related proteins in other eucaryotic organisms suggests that a role for these proteins in DNA repair may be widely conserved. Fission yeast Pnk1 exhibits strong homology with human PNKP. Our results from in vitro and in vivo assays demonstrate that Pnk1 exhibits both DNA kinase and phosphatase activities, indicating that its enzymatic properties are conserved with human PNKP. Also, our complementation assays indicate that the human PNKP can functionally substitute for Pnk1, further suggesting that these proteins have been functionally as well as structurally conserved. In contrast, budding yeast Tpp1 shares some sequence similarity with PNKP but lacks DNA kinase activity. Thus, fission yeast may provide a better model system for exploring the role of the conserved dual polynucleotide kinase/phosphatase-related proteins in DNA repair. It has been shown previously that the expression of human PNKP into HeLa cells or the XRCC1 mutant Chinese hamster ovary cell line, EM9, affords a degree of resistance to the alkylating agent ethyl methanesulfonate (10). Here, we provide clear evidence for the involvement of Pnk1 in the normal response of S. pombe to ionizing radiation and the antineoplastic agent, camptothecin. It has long been known that ionizing radiation can generate strand breaks with 3⬘-phosphate and or 5⬘-hydroxyl termini (27, 28). Thus, there is an important role for a polynucleotide 5⬘-kinase/3⬘-phosphatase in the restoration of these termini to a configuration suitable for strand rejoining by a DNA polymerase and ligase. In many organisms, 3⬘-phosphate groups can be removed by an apyrimidinic endonuclease (29). However, little progress has been made in identifying an active apyrimidinic endonuclease in S. pombe extracts (30, 31), and it remains to be seen whether Pnk1 is the major 3⬘-DNA phosphatase in this organism. Camptothecin inhibits topoisomerase I after the enzyme has
incised the DNA, thus trapping the enzyme while it is covalently attached to the DNA terminus via a 3⬘-phosphate linkage (32). The 5⬘ terminus of the DNA at such a break bears a hydroxyl group. Several investigators have proposed that the repair of such strand breaks would require a polynucleotide kinase to phosphorylate the 5⬘ terminus and remove the 3⬘phosphate if such a group remains at the 3⬘ terminus after hydrolysis of the DNA-topoisomerase linkage (33–35). Support for this supposition was enhanced with the discovery and cloning of a gene in S. cerevisiae encoding a tyrosine-DNA phosphodiesterase that can hydrolyze the topoisomerase I-DNA complex (36). Our data showing that the deletion mutants of Pnk1 are hypersensitive to camptothecin provide substantial support for a DNA repair mechanism involving a 5⬘-kinase/3⬘phosphatase. That human PNKP can efficiently substitute for the fission yeast enzyme suggests that a similar mechanism for repair of camptothecin-blocked topoisomerase-DNA complexes may be operative in human cells, and this could have implications for the resistance of tumor cells to camptothecin and similar topoisomerase I inhibitors. Acknowledgments—We thank Dr. Hiroyuki Yamano (Japan Science and Technology Corporation) for excellent advice and assistance, Dr. Aghdass Rasouli-Nia and Mary Packer (Department of Oncology, University of Alberta) for technical support, and Dr. Liang Li and Jing Zheng (Department of Chemistry, University of Alberta) for providing the mass spectral analysis of His6-Pnk1. REFERENCES 1. Friedberg, E. C., Walker, G. C., and Siede, W. (1995) DNA repair and mutagenesis, ASM Press, Washington, D. C. 2. Li, T. K., and Liu, L. F. (2001) Annu. Rev. Pharmacol. Toxicol. 41, 53–77 3. Novogrodsky, A., Tal, M., Traub, A., and Hurwitz, J. (1966) J. Biol. Chem. 241, 2933–2943 4. Ichimura, M., and Tsukada, K. (1971) J. Biochem. (Tokyo) 69, 823– 828 5. Pheiffer, B. H., and Zimmerman, S. B. (1982) Biochem. Biophys. Res. Commun. 109, 1297–1302 6. Habraken, Y., and Verly, W. G. (1983) FEBS Lett. 160, 46 –50 7. Karimi-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., and Weinfeld, M. (1999) J. Biol. Chem. 274, 24187–24194 8. Jilani, A., Ramotar, D., Slack, C., Ong, C., Yang, X. M., Scherer, S. W., and Lasko, D. D. (1999) J. Biol. Chem. 274, 24176 –24186 9. Karimi-Busheri, F., and Weinfeld, M. (1997) J. Cell. Biochem. 64, 258 –272 10. Whitehouse, C. J., Taylor, R. M., Thistlethwaite, A., Zhang, H., Karimi-Busheri, F., Lasko, D. D., Weinfeld, M., and Caldecott, K. W. (2001) Cell 104, 107–117 11. Tebbs, R. S., Flannery, M. L., Meneses, J. J., Hartmann, A., Tucker, J. D., Thompson, L. H., Cleaver, J. E., and Pedersen, R. A. (1999) Dev. Biol. 208, 513–529 12. Thompson, L. H., and West, M. G. (2000) Mutat. Res. 459, 1–18 13. Thompson, L. H., Brookman, K. W., Jones, N. J., Allen, S. A., and Carrano, A. V. (1990) Mol. Cell. Biol. 10, 6160 – 6171 14. Betti, M., Petrucco, S., Bolchi, A., Dieci, G., and Ottonello, S. (2001) J. Biol. Chem. 276, 18038 –18045 15. Vance, J. R., and Wilson, T. E. (2001) J. Biol. Chem. 276, 15073–15081 16. Sambrook, J., Maniatis, T., and Fritsch, E. F. (1989) Molecular cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 17. Basi, G., Schmid, E., and Maundrell, K. (1993) Gene (Amst.) 123, 131–136 18. Loewith, R., Hubberstey, A., and Young, D. (2000) J. Cell Sci. 113, 153–160 19. Sengar, A. S., Markley, N. A., Marini, N. J., and Young, D. (1997) Mol. Cell. Biol. 17, 3508 –3519 20. Kawamukai, M., Gerst, J., Field, J., Riggs, M., Rodgers, L., Wigler, M., and Young, D. (1992) Mol. Biol. Cell 3, 167–180 21. Moreno, S., Klar, A., and Nurse, P. (1991) Methods Enzymol. 194, 795– 823 22. Rothstein, R. J. (1983) Methods Enzymol. 101, 202–211 23. Russell, P., and Nurse, P. (1986) Cell 45, 145–153 24. Fanta, M., Zhang, H., Bernstein, N., Glover, M., Karimi-Busheri, F., and Weinfeld, M. (2001) Hybridoma 20, 237–242 25. Harlow, E., and Lane, D. (1988) Antibodies: A Laboratory Manual, pp. 101–120, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 26. Suh, D., Wilson, D. M., III, and Povirk, L. F. (1997) Nucleic Acids Res. 25, 2495–2500 27. Henner, W. D., Rodriguez, L. O., Hecht, S. M., and Haseltine, W. A. (1983) J. Biol. Chem. 258, 711–713 28. Coquerelle, T., Bopp, A., Kessler, B., and Hagen, U. (1973) Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med. 24, 397– 404 29. Demple, B., and Harrison, L. (1994) Annu. Rev. Biochem. 63, 915–948 30. Ramotar, D., Vadnais, J., Masson, J. Y., and Tremblay, S. (1998) Biochim. Biophys. Acta 1396, 15–20 31. Kanno, S., Iwai, S., Takao, M., and Yasui, A. (1999) Nucleic Acids Res. 27,
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