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• W2127698844 abstract "Human polydeoxyribonucleotide kinase is an enzyme that has the capacity to phosphorylate DNA at 5′-hydroxyl termini and dephosphorylate 3′-phosphate termini and, therefore, can be considered a putative DNA repair enzyme. The enzyme was purified from HeLa cells. Amino acid sequence was obtained for several tryptic fragments by mass spectrometry. The sequences were matched through the dbEST data base with an incomplete human cDNA clone, which was used as a probe to retrieve the 5′-end of the cDNA sequence from a separate cDNA library. The complete cDNA, which codes for a 521-amino acid protein (57.1 kDa), was expressed in Escherichia coli, and the recombinant protein was shown to possess the kinase and phosphatase activities. Comparison with other sequenced proteins identified a P-loop motif, indicative of an ATP-binding domain, and a second motif associated with several different phosphatases. There is reasonable sequence similarity to putative open reading frames in the genomes of Caenorhabditis elegans and Schizosaccharomyces pombe, but similarity to bacteriophage T4 polynucleotide kinase is limited to the kinase and phosphatase domains noted above. Northern hybridization revealed a major transcript of approximately 2.3 kilobases and a minor transcript of approximately 7 kilobases. Pancreas, heart, and kidney appear to have higher levels of mRNA than brain, lung, or liver. Confocal microscopy of human A549 cells indicated that the kinase resides predominantly in the nucleus. The gene encoding the enzyme was mapped to chromosome band 19q13.4. Human polydeoxyribonucleotide kinase is an enzyme that has the capacity to phosphorylate DNA at 5′-hydroxyl termini and dephosphorylate 3′-phosphate termini and, therefore, can be considered a putative DNA repair enzyme. The enzyme was purified from HeLa cells. Amino acid sequence was obtained for several tryptic fragments by mass spectrometry. The sequences were matched through the dbEST data base with an incomplete human cDNA clone, which was used as a probe to retrieve the 5′-end of the cDNA sequence from a separate cDNA library. The complete cDNA, which codes for a 521-amino acid protein (57.1 kDa), was expressed in Escherichia coli, and the recombinant protein was shown to possess the kinase and phosphatase activities. Comparison with other sequenced proteins identified a P-loop motif, indicative of an ATP-binding domain, and a second motif associated with several different phosphatases. There is reasonable sequence similarity to putative open reading frames in the genomes of Caenorhabditis elegans and Schizosaccharomyces pombe, but similarity to bacteriophage T4 polynucleotide kinase is limited to the kinase and phosphatase domains noted above. Northern hybridization revealed a major transcript of approximately 2.3 kilobases and a minor transcript of approximately 7 kilobases. Pancreas, heart, and kidney appear to have higher levels of mRNA than brain, lung, or liver. Confocal microscopy of human A549 cells indicated that the kinase resides predominantly in the nucleus. The gene encoding the enzyme was mapped to chromosome band 19q13.4. polynucleotide kinase or polydeoxyribonucleotide kinase a 21-mer oligonucleotide phosphorylated at the 3′- and 5′-termini phosphate-buffered saline polyacrylamide gel electrophoresis matrix-assisted laser desorption ionization N-succinimidyl-2-morpholine acetate base pair(s) untranslated region polymerase chain reaction Transient DNA strand breaks and short gaps are frequently observed in cellular DNA. Many arise during regular cellular activity such as DNA replication, recombination, or differentiation. Others occur as a consequence of exposure to endogenous or exogenous DNA damaging agents. Repair of these strand interruptions is usually mediated by DNA ligases and polymerases. Both of these classes of enzymes require 3′-hydroxyl DNA termini, and the DNA ligases also require 5′-phosphate termini. However, the termini generated by nucleases, such as DNase II, and many produced by ionizing radiation bear 3′-phosphate and 5′-hydroxyl groups (1Bernardi G. Adv. Enzymol. 1968; 31: 1-49PubMed Google Scholar, 2Coquerelle T. Bopp A. Kessler B. Hagen U. Int. J. Radiat. Biol. 1973; 24: 397-404Crossref Scopus (65) Google Scholar, 3Lennartz M. Coquerelle T. Hagen U. Int. J. Radiat. Biol. 1975; 27: 577-587Crossref Scopus (69) Google Scholar, 4Henner W.D. Rodriguez L.O. Hecht S.M. Haseltine W.A. J. Biol. Chem. 1983; 258: 711-713Abstract Full Text PDF PubMed Google Scholar), and therefore must be processed before they can be acted upon by DNA ligases or polymerases. One enzyme that possesses the capacity to both phosphorylate 5′-hydroxyl termini and dephosphorylate 3′-phosphate termini is polynucleotide kinase (PNK).1The PNK from T4 phage has found widespread application in molecular biology, especially for radiolabeling DNA and oligonucleotides (5Midgley C.A. Murray N.E. EMBO J. 1985; 4: 2695-2703Crossref PubMed Scopus (49) Google Scholar). It can act on DNA and RNA and even phosphorylate nucleoside 3′-monophosphates. However, the main cellular function of the T4 enzyme is not to repair DNA, but rather to counter the action of a phage endoribonuclease that cleaves tRNA (6Amitsur M. Levitz R. Kaufmann G. EMBO J. 1987; 6: 2499-2503Crossref PubMed Scopus (204) Google Scholar). Eukaryotic PNKs fall into two categories depending on whether their preferred substrate is DNA or RNA (7Kleppe K. Lillehaug J.R. Adv. Enzymol. 1979; 48: 245-275PubMed Google Scholar). While both can phosphorylate 5′-termini, only the former have an associated 3′-phosphatase activity (8Pheiffer B.H. Zimmerman S.B. Biochem. Biophys. Res. Commun. 1982; 109: 1297-1302Crossref PubMed Scopus (31) Google Scholar, 9Habraken Y. Verly W.G. FEBS Lett. 1983; 160: 46-50Crossref PubMed Scopus (23) Google Scholar, 10Habraken Y. Verly W.G. Eur. J. Biochem. 1988; 171: 59-66Crossref PubMed Scopus (32) Google Scholar, 11Karimi-Busheri F. Weinfeld M. J. Cell. Biochem. 1997; 64: 258-272Crossref PubMed Scopus (34) Google Scholar, 12Jilani A. Slack C. Matheos D. Zannis-Hadjopoulos M. Lasko D.D. J. Cell. Biochem. 1999; 73: 188-203Crossref PubMed Scopus (22) Google Scholar). Mammalian DNA kinases have been purified from a variety of sources including rat liver and testes and calf thymus (8Pheiffer B.H. Zimmerman S.B. Biochem. Biophys. Res. Commun. 1982; 109: 1297-1302Crossref PubMed Scopus (31) Google Scholar, 9Habraken Y. Verly W.G. FEBS Lett. 1983; 160: 46-50Crossref PubMed Scopus (23) Google Scholar, 10Habraken Y. Verly W.G. Eur. J. Biochem. 1988; 171: 59-66Crossref PubMed Scopus (32) Google Scholar, 11Karimi-Busheri F. Weinfeld M. J. Cell. Biochem. 1997; 64: 258-272Crossref PubMed Scopus (34) Google Scholar, 12Jilani A. Slack C. Matheos D. Zannis-Hadjopoulos M. Lasko D.D. J. Cell. Biochem. 1999; 73: 188-203Crossref PubMed Scopus (22) Google Scholar, 13Ichimura M. Tsukada K. J. Biochem. (Tokyo). 1971; 69: 823-828Crossref PubMed Scopus (22) Google Scholar, 14Teraoka H. Mizuta K. Sato F. Shimoyachi M. Tsukada K. Eur. J. Biochem. 1975; 58: 297-302Crossref PubMed Scopus (34) Google Scholar, 15Levin C. Zimmerman S.B. J. Biol. Chem. 1976; 251: 1767-1774Abstract Full Text PDF PubMed Google Scholar, 16Austin G.E. Sirakoff D. Roop B. Moyer G.H. Biochim. Biophys. Acta. 1978; 522: 412-422Crossref PubMed Scopus (14) Google Scholar, 17Tamura S. Teraoka H. Tsukada K. Eur. J. Biochem. 1981; 115: 449-453Crossref PubMed Scopus (11) Google Scholar, 18Bosdal T. Lillehaug J.R. Biochim. Biophys. Acta. 1985; 840: 280-286Crossref PubMed Scopus (9) Google Scholar, 19Prinos P. Slack C. Lasko D.D. J. Cell. Biochem. 1995; 58: 115-131Crossref PubMed Scopus (9) Google Scholar). The isolated enzymes share similar properties with regard to the kinase activity including an acidic pH (5.5–6.0) optimum (8Pheiffer B.H. Zimmerman S.B. Biochem. Biophys. Res. Commun. 1982; 109: 1297-1302Crossref PubMed Scopus (31) Google Scholar, 9Habraken Y. Verly W.G. FEBS Lett. 1983; 160: 46-50Crossref PubMed Scopus (23) Google Scholar, 10Habraken Y. Verly W.G. Eur. J. Biochem. 1988; 171: 59-66Crossref PubMed Scopus (32) Google Scholar, 11Karimi-Busheri F. Weinfeld M. J. Cell. Biochem. 1997; 64: 258-272Crossref PubMed Scopus (34) Google Scholar, 12Jilani A. Slack C. Matheos D. Zannis-Hadjopoulos M. Lasko D.D. J. Cell. Biochem. 1999; 73: 188-203Crossref PubMed Scopus (22) Google Scholar, 13Ichimura M. Tsukada K. J. Biochem. (Tokyo). 1971; 69: 823-828Crossref PubMed Scopus (22) Google Scholar, 14Teraoka H. Mizuta K. Sato F. Shimoyachi M. Tsukada K. Eur. J. Biochem. 1975; 58: 297-302Crossref PubMed Scopus (34) Google Scholar, 15Levin C. Zimmerman S.B. J. Biol. Chem. 1976; 251: 1767-1774Abstract Full Text PDF PubMed Google Scholar, 16Austin G.E. Sirakoff D. Roop B. Moyer G.H. Biochim. Biophys. Acta. 1978; 522: 412-422Crossref PubMed Scopus (14) Google Scholar, 17Tamura S. Teraoka H. Tsukada K. Eur. J. Biochem. 1981; 115: 449-453Crossref PubMed Scopus (11) Google Scholar, 18Bosdal T. Lillehaug J.R. Biochim. Biophys. Acta. 1985; 840: 280-286Crossref PubMed Scopus (9) Google Scholar), and the minimum size of oligonucleotide that can be phosphorylated is in the range of 8–12 nucleotides (11Karimi-Busheri F. Weinfeld M. J. Cell. Biochem. 1997; 64: 258-272Crossref PubMed Scopus (34) Google Scholar, 15Levin C. Zimmerman S.B. J. Biol. Chem. 1976; 251: 1767-1774Abstract Full Text PDF PubMed Google Scholar). The only significant discrepancy has been the molecular mass assigned to the polypeptides. Earlier reports regarding the PNK purified from rat organs indicated that the protein may be an 80-kDa homodimer composed of 40-kDa polypeptides (9Habraken Y. Verly W.G. FEBS Lett. 1983; 160: 46-50Crossref PubMed Scopus (23) Google Scholar, 10Habraken Y. Verly W.G. Eur. J. Biochem. 1988; 171: 59-66Crossref PubMed Scopus (32) Google Scholar, 18Bosdal T. Lillehaug J.R. Biochim. Biophys. Acta. 1985; 840: 280-286Crossref PubMed Scopus (9) Google Scholar), but PNK activity in tissue extracts detected on activity gels migrated as a 60-kDa polypeptide (20Ohmura Y. Uchida T. Teraoka H. Tsukada K. Eur. J. Biochem. 1987; 162: 15-18Crossref PubMed Scopus (7) Google Scholar). Estimates for the size of calf thymus PNK have ranged from 56 to 70 kDa (16Austin G.E. Sirakoff D. Roop B. Moyer G.H. Biochim. Biophys. Acta. 1978; 522: 412-422Crossref PubMed Scopus (14) Google Scholar, 17Tamura S. Teraoka H. Tsukada K. Eur. J. Biochem. 1981; 115: 449-453Crossref PubMed Scopus (11) Google Scholar). We and others have recently purified the DNA kinases from calf thymus and rat liver to near homogeneity, making use of a broad spectrum of proteolysis inhibitors (11Karimi-Busheri F. Weinfeld M. J. Cell. Biochem. 1997; 64: 258-272Crossref PubMed Scopus (34) Google Scholar, 12Jilani A. Slack C. Matheos D. Zannis-Hadjopoulos M. Lasko D.D. J. Cell. Biochem. 1999; 73: 188-203Crossref PubMed Scopus (22) Google Scholar). The major protein band migrated as a 60-kDa peptide on polyacrylamide gels, but a minor band was observed at 40 kDa in the rat liver preparation. At present, the cellular function of mammalian DNA kinases has not been elucidated. Clearly, one possibility is participation in the repair of strand breaks induced by DNA damaging agents, such as ionizing radiation or topoisomerase inhibitors (21Karimi-Busheri F. Lee J. Tomkinson A.E. Weinfeld M. Nucleic Acids Res. 1998; 26: 4395-4400Crossref PubMed Scopus (101) Google Scholar, 22Yang S.-W. Burgin Jr A.B. Huizenga B.N. Robertson C.A. Yao K.C. Nash H.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11534-11539Crossref PubMed Scopus (327) Google Scholar). We have shown that, unlike T4 phage PNK, calf thymus PNK is able to efficiently phosphorylate the 5′-OH terminus at a nick and a one-nucleotide gap in a double-stranded DNA substrate (11Karimi-Busheri F. Weinfeld M. J. Cell. Biochem. 1997; 64: 258-272Crossref PubMed Scopus (34) Google Scholar). Furthermore, an in vitro system consisting of purified mammalian PNK, DNA polymerase β, and DNA ligase I was able to effect the complete repair of nicks and short gaps bounded by 3′-phosphate and 5′-OH termini (21Karimi-Busheri F. Lee J. Tomkinson A.E. Weinfeld M. Nucleic Acids Res. 1998; 26: 4395-4400Crossref PubMed Scopus (101) Google Scholar). Alternatively, PNK could participate in a more regular function. For example, it has been observed that a proportion of Okazaki fragments have 5′-OH termini (23Pohjanpelto P. Hölttä E. EMBO J. 1996; 15: 1193-1200Crossref PubMed Scopus (41) Google Scholar), which would have to be phosphorylated prior to ligation. As part of our ongoing study to address the question of the role of eukaryotic PNKs, this paper describes the molecular cloning, sequencing, cellular localization, and chromosomal mapping of human PNK. The DNA substrate containing 5′-OH termini was prepared by digestion of calf thymus DNA with micrococcal nuclease as described by Richardson (24Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1965; 54: 158-165Crossref PubMed Scopus (493) Google Scholar). Each 5′-phosphorylation reaction mixture (20 μl ), containing 10 μg of DNA substrate, 3 μCi of [γ-32P]ATP (3000 Ci/mmol, Amersham Pharmacia Biotech), 500 nm unlabeled ATP, 80 mm succinic acid, pH 5.5, 10 mmMgCl2, 1 mm dithiothreitol, 1 mmEGTA, 2 μg of bovine serum albumin, and protein fraction (typically 4 μl), was incubated for 20 min at 37 °C. The reaction was stopped and the DNA precipitated by addition of 200 μl of 20% trichloroacetic acid and 100 μl of 250 μm sodium pyrophosphate containing 50 μg of bovine serum albumin. Following centrifugation at 10,000 × g for 10 min, the pellets were resuspended in 80 μl of 0.1 m NaOH and reprecipitated by addition of 400 μl of 10% trichloroacetic acid. This wash step was repeated once more before the radioactivity of the pellet was determined. As a control for kinase specificity (i.e. DNA versus protein), parallel reactions were carried out in the absence of the DNA substrate. The 3′-dephosphorylation of a 21-mer oligonucleotide (p21p) catalyzed by recombinant human PNK in Escherichia coli cell extracts was assayed by gel electrophoresis as described previously (21Karimi-Busheri F. Lee J. Tomkinson A.E. Weinfeld M. Nucleic Acids Res. 1998; 26: 4395-4400Crossref PubMed Scopus (101) Google Scholar). A pellet of frozen HeLa S3 cells (3 × 1010, approximately 50 ml packed cell volumes) was thawed in 200 ml of hypotonic buffer (10 mm Tris-HCl, pH 7.5, 2 mm MgCl2, 5 mm dithiothreitol, and 0.5 mm EDTA) containing a mixture of protease inhibitors (25 μg/ml N ε-p-tosyl-l-lysine chloromethyl ketone, 5 μg/ml chymotrypsin, 1 μg/ml aprotinin, 0.5 μg/ml leupeptin, 0.5 μg/ml pepstatin, and 1 mmα-toluenesulfonyl fluoride) and held for 20 min at 0 °C before disruption in a Dounce glass homogenizer (15 strokes). Nuclei were collected by low speed centrifugation, and a protein extract was prepared in the presence of 0.3 m KCl as described previously (25Masutani C. Sugasawa K. Yanagisawa J. Sonoyama T. Ui M. Enomoto T. Takio K. Tanaka K. van der Spek P.J. Bootsma D. Hoeijmakers J.H.J. Hanaoka F. EMBO J. 1994; 13: 1831-1843Crossref PubMed Scopus (333) Google Scholar). Sequential chromatography of the extract on a phosphocellulose P11 column (Whatman, Clifton, NJ) and an Ultrogel AcA34 gel filtration column (Sepracor/IBF, Marlborough, MA), and ammonium sulfate precipitation steps were carried out as described by Robins and Lindahl (26Robins P. Lindahl T. J. Biol. Chem. 1996; 271: 24257-24261Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar), except that the elution buffer for the first column contained 0.6 m KCl and the elution buffer for the second column contained 0.5 m NaCl. The active fractions in the second peak from the gel filtration column were pooled (63 mg of protein in a total volume of 54 ml), dialyzed against buffer A (50 mm Tris-HCl, pH 7.5, 100 mm NaCl, 1 mm dithiothreitol, 1 mm potassium phosphate, and 10% glycerol). The specific activity at this stage of purification was approximately 0.06 units/mg of protein, where one unit of enzyme is the amount required to incorporate 1 nmol of phosphate from ATP into micrococcal nuclease-treated DNA in 30 min at 37 °C (27Richardson C.C. Cantoni G.L. Davies D.R. Procedures in Nucleic Acid Research. 2. Harper and Row, New York1971: 815-828Google Scholar). The pooled material was loaded onto a column (2.5 × 5.0 cm) of Bio-Gel HT hydroxyapatite (Bio-Rad) pre-equilibrated with buffer A. The column was washed with five volumes of buffer A before eluting bound protein with a 200-ml linear gradient of 50–500 mm potassium phosphate in buffer A collecting in 5-ml fractions. The active fractions, 28–33, were pooled and dialyzed against buffer B (10 mm potassium phosphate, pH 6.8, 4 mm 2-mercaptoethanol, and 10% glycerol) containing 50 mm KCl. The material was loaded onto a 1-ml HiTrap SP column (Amersham Pharmacia Biotech), washed with 10 column volumes of buffer B and eluted with a 30-ml linear gradient of 50–600 mm KCl in 30 1-ml fractions. A peak of kinase activity eluted at fractions 10–12. The contents of fraction 11 were dialyzed against buffer C (50 mm Tris-HCl, pH 7.5, 1 mm dithiothreitol, 1 mm potassium phosphate, and 10% glycerol) containing 50 mm NaCl, and loaded onto a Mono S PC 1.6/5 column attached to a SMART micropurification chromatography system (Amersham Pharmacia Biotech). Protein was eluted with a 2-ml linear salt gradient of 50–450 mm NaCl at a flow rate of 100 μl/min in 20 100-μl fractions. After assaying the fractions for DNA kinase activity, a small quantity of each fraction was examined by SDS-PAGE to determine which polypeptide correlated with activity. The remaining contents of the fraction with the peak of kinase activity (fraction 12) was further fractionated by gel electrophoresis and electroblotted onto polyvinylidene difluoride membrane. The electroblotted HeLa protein was stained with sulforhodamine B (0.05% w/v in 30% v/v aqueous methanol, 0.1% v/v acetic acid) using a rapid-staining protocol (28Coull J.M. Pappin D.J.C. J. Protein Chem. 1990; 9: 259-260Google Scholar). The dried, stained protein was then digested in situ on the polyvinylidene difluoride membrane with trypsin (Roche Molecular Biochemicals, modified) for 18 h at 30 °C and the peptides extracted with 1:1 v/v formic acid/ethanol (29Sutton C.W. Pemberton K.S. Cottrell J.S. Corbett J.M. Wheeler C.H. Dunn M.J. Pappin D.J.C. Electrophoresis. 1995; 16: 308-316Crossref PubMed Scopus (99) Google Scholar). Aliquots were sampled and directly analyzed by matrix-assisted laser desorption ionization (MALDI) time-of-flight mass spectrometry using a LaserMat 2000 mass spectrometer (Thermo Bioanalysis, UK) (30Mock K.K. Sutton C.W. Cottrell J.S. Rapid Commun. Mass Spectrom. 1992; 6: 233-238Crossref PubMed Scopus (137) Google Scholar). Additional aliquots were quantitatively esterified using 1% v/v thionyl chloride in methanol and also analyzed by MALDI to provide acidic residue composition (31Pappin D.J.C. Rahman D. Hansen H.F. Bartlet-Jones M. Jeffery W.A. Bleasby A.J. Burlingame A.L. Carr S.A. Mass Spectrometry in the Biological Sciences. Humana Press, Totowa, NJ1996: 135-150Crossref Google Scholar). Native and esterified peptide masses were then screened against the MOWSE peptide mass fingerprint data base (32Pappin D.J.C. Hojrup P. Bleasby A.J. Curr. Biol. 1993; 3: 327-332Abstract Full Text PDF PubMed Scopus (1416) Google Scholar). The remaining digested peptides (>90% of total digest) were then reacted with N-succinimidyl-2-morpholine acetate (SMA) in order to enhance b-ion abundance and facilitate sequence analysis by tandem mass spectrometry (33Sherman, N. E., Yates, N. A., Shabanowitz, J., Hunt, D. F., Jeffery, W. A., Bartlet-Jones, M., and Pappin, D. J. C. (1995) Proceedings of the 43rd ASMS Conference on Mass Spectrometry and Allied Topics, May 21–26, 1995, Atlanta, GAGoogle Scholar). Dried peptide fractions were treated with 7 μl of freshly prepared, ice-cold 1% w/v N-succinimidyl-2-morpholine acetate in 1.0 mHEPES (pH 7.8 with NaOH) containing 2% v/v acetonitrile. Following reaction for 20 min on ice, the reaction was terminated by the addition of 1 μl of heptafluorobutyric acid and diluted with an equal volume of water. The solution was then injected in three 5-μl aliquots onto a capillary reverse-phase column (300 μm x 15 cm) packed with POROS R2/H material (Perseptive Biosystems, MA) equilibrated with 2% v/v methanol, 0.05% v/v trifluoroacetic acid running at 3 μl/min. The adsorbed peptides were washed isocratically with 15% v/v methanol, 0.05% v/v trifluoroacetic acid for 30 min at 3 μl/min to elute the excess reagent and HEPES buffer. Derivatized peptides were eluted with a single step gradient to 75% v/v methanol, 0.1% v/v formic acid and collected in two 3-μl fractions. The derivatized peptides were then sequenced by low energy collision-activated dissociation using a Finnigan MAT TSQ7000 tandem triple quadrupole mass spectrometer and a Finnigan MAT LCQ ion-trap mass spectrometer, both instruments fitted with nanoelectrospray sources (34Hunt D.F. Yates J.R. Shabanowitz J. Winston S. Hauer C.R. Proc. Natl. Acad. Sci. U. S. A. 1986; 84: 6233-6237Crossref Scopus (1073) Google Scholar, 35Wilm M. Mann M. Anal. Chem. 1996; 68: 1-8Crossref PubMed Scopus (1696) Google Scholar). Collision-activated dissociation was typically performed with collisional offset voltages between −18 and −30 V. Two tryptic peptides from previously purified calf thymus PNK (11Karimi-Busheri F. Weinfeld M. J. Cell. Biochem. 1997; 64: 258-272Crossref PubMed Scopus (34) Google Scholar) were sequenced by the Harvard Microchemistry Facility (Cambridge, MA) using either an ABI 477A protein sequencer (Applied Biosystems, Foster City, CA) or an HP G1000A (Hewlett Packard, Palo Alto, CA). Confirmation of sequence was obtained by MALDI time-of-flight mass spectrometry on a LaserMat 2000 mass spectrometer. DNA sequences derived from the peptide sequences were used to screen the dbEST data base (NIH). A cDNA clone from infant brain (clone number 32798 inserted in lafmid BA) was identified and obtained from the I.M.A.G.E. Consortium. The cDNA insert (1548 bp) was fully sequenced, using an automated ABI Prism 377 DNA analysis system (Applied Biosystems), and confirmed the presence of the poly(A) tail, and a large open reading frame, but no clearly identifiable start codon. A 609-bp probe, prepared by digestion of clone 32798 with Hin dIII and Pst I (New England Biolabs, Beverley, MA), was subsequently used to screen a λgt11 HeLa cell 5′-STRETCH PLUS cDNA library (CLONTECH, Palo Alto, CA) by a standard protocol (36Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Ten positive clones were isolated, none of which contained a poly(A) tail. The largest insert (1.5 kilobase pairs) was amplified by PCR using the λ forward and reverse primers with Pfu DNA polymerase (Stratagene, La Jolla, CA), and then sequenced. Putative full-length cDNA was reconstituted as follows: (i) the PCR-amplified product was digested with Sac I and shrimp alkaline phosphatase (Amersham Pharmacia Biotech), and the larger fragment (1.1 kilobase pairs) isolated by agarose gel electrophoresis, (ii) the DNA of clone 32798 was digested with Sac I, (iii) the DNA molecules were ligated using phage T4 DNA ligase (Amersham Pharmacia Biotech), and (iv) the ligation product was digested with Eco RI. The cDNA was amplified by PCR using Pfu DNA polymerase and primers with tails that provided cleavage sites for Nde I (5′-TTTGAATTCCCATATGGGCGAGGTGGAGCCCCCGGGC-3′) and Bam HI (5′-CGCGGATCCTCAGCCCTCGGAGAACTGGCAG-3′) and then subcloned into the expression plasmid pET-16b (Novagen Inc., Madison, WI). The new plasmid (pPNK-His), which codes for a His-tagged derivative of PNK, was transfected into host E. coli bacterial strain BLR(DE3) (Novagen). The bacteria were grown at 37 °C to an OD600 of 0.6 in 100 ml of LB medium containing 50 μg/ml ampicillin and 12.5 μg/ml tetracycline. Zinc chloride was then added to the medium to a final concentration of 0.015 mm, and PNK expression was induced at 30 °C for 3 h by addition of 0.4 mm (final concentration) isopropyl-1-thio-β-d-galactopyranoside (Sigma). After harvesting the cells by centrifugation at 5000 × g at 4 °C for 5 min, they were resuspended in 10 ml of extraction buffer (50 mm Tris-HCl, pH 7.5, 0.015 mmZnCl2, 6 mm mercaptoethanol). Lysozyme was added to a final concentration of 100 μg/ml together with Triton X-100 (final concentration, 0.1%), and, after incubation at 30 °C for 15 min, the bacteria were disrupted by sonication. The soluble fraction was separated from the insoluble fraction by centrifugation at 12,000 × g for 15 min at 4 °C. The insoluble fraction was resuspended in 1 ml of extraction buffer. The 609-bp Hin dIII/Pst I fragment used to screen the HeLa cDNA library was also used to probe a human multiple tissue Northern blot (CLONTECH) containing 2 μg (per lane) of polyadenylated RNA isolated from eight different human tissues. Hybridization was performed at 68 °C for 1 h under conditions described by the manufacturer. As a control for the amounts of mRNA in each lane, the membrane was reprobed with a sequence of β-actin cDNA provided by CLONTECH. A synthetic peptide antigen was prepared commercially (SSPEQ, Quebec) from the first 17 amino acids of peptide sequence 1 (Table I) conjugated to a four-branch multiple antigenic peptide carrier. Rabbit polyclonal antibodies were raised by standard protocol (37Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988Google Scholar).Table IAmino acid sequences obtained for tryptic peptidesNumberSequence1-aLeucine and isoleucine [LI] cannot be distinguished by low energy collision-activated dissociation as they are isomers.Source of PNK1LWLESPAGGAPPIFLPTGGQALVLGRcalf thymus2EMTDSSHVPVcalf thymus3VAGFD[LI]DGT[LI][LI]TTRHeLa cells4VA[LI]DNTNPDAASRHeLa cells5E[LI]EAEGYKHeLa cells6GP[LI]TQVTDRHeLa cells7TQVE[LI]VADPETRHeLa cells8[LI][LI]YPE[LI]PRHeLa cells1-a Leucine and isoleucine [LI] cannot be distinguished by low energy collision-activated dissociation as they are isomers. Open table in a new tab The human malignant lung cell line A549 (ATCC no. CCL-185) was grown as a monolayer on glass microscope slides to 80% confluence. Following rinsing in PBS, the cells were fixed in 95% ethanol at −20 °C for 15 min. The slides were allowed to dry, and were incubated for 1 h at room temperature with 1% skim milk powder in PBS to minimize nonspecific binding of the immunoreagents. Following extensive PBS rinsing, the slides were incubated overnight at 4 °C in the rabbit polyclonal antiserum (diluted 1/30 in PBS), in a humidified atmosphere. The cells were then rinsed extensively with PBS, and rhodamine-conjugated goat anti-rabbit IgG (H+L, Cappel Laboratories, Durham, NC) was applied at a dilution of 1/30 in PBS for a 1-h incubation at 37 °C in a water-saturated atmosphere. The unbound fluorescent antibody was removed by extensive washing in PBS, and the slides were covered with coverslips for confocal microscopy using PBS/glycerol, 1:1 as a mounting medium. The instrumentation and the procedures for the confocal laser scanning microscopy have been described previously (38Miller G.G. Brown K. Moore R.B. Diwu Z.J. Liu J. Huang L. Lown J.W. Begg D.A. Chlumecky V. Tulip J. McPhee M.S. Photochem. Photobiol. 1995; 61: 632-638Crossref PubMed Scopus (52) Google Scholar). Fluorescence in situ hybridization was performed as described previously (39Rowley J.D. Diaz M.O. Espinosa R. Patel Y.D. van Melle E. Ziemin S. Taillon-Miller P. Lichter P. Evans G.A. Kersey J.D. Ward D.C. Domer P.H. Le Beau M.M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9358-9362Crossref PubMed Scopus (254) Google Scholar). Human metaphase cells were prepared from phytohemagglutinin-stimulated peripheral blood lymphocytes. Biotin-labeled probes were prepared by nick translation using Bio-16-dUTP (Enzo Diagnostics, Farmingdale, NY). One PNK probe was clone 32798 (including the plasmid vector). A second probe, which provided a 440-bp sequence stretching from the 5′-untranslated region into the 5′-end of the translated sequence, was generated by PCR amplification of the HeLa cDNA clone using the λgt11 forward primer and a reverse primer, 5′-GTGGAGGCCATTGACCAAATA-3′. The two clones were labeled and co-hybridized to the chromosome preparations. Hybridization was detected with fluorescein-conjugated avidin (Vector Laboratories, Burlingame, CA), and chromosomes were identified by staining with 4,6-diamidino-2-phenylindole-dihydrochloride. Fractions of a crude extract of HeLa cells that was passed down an AcA 34 Ultragel size exclusion column in the presence of 0.5m NaCl were shown to contain DNA kinase activity (Fig.1). Two peaks of activity were apparent, the first migrating with the bulk of the higher molecular weight protein, which may suggest that PNK is bound in a complex to other proteins, and the second eluting with proteins in the range of 40–100 kDa. Initial steps in the purification were carried out by conventional chromatography using gel filtration, hydroxyapatite, and cation exchange media. For the final step, the protein was applied on a SMART system precision column and eluted in 20 100-μl fractions with a 2-ml salt gradient (50–450 mm NaCl). The kinase assay revealed a peak of activity centering on fraction 12 (Fig.2 A). Correlation of the intensities of the protein bands in fractions 10–14 (Fig.2 B) with kinase activity strongly suggested that the ∼60-kDa band (topmost of the three major bands in fraction 12, marked by an arrow) was responsible for the PNK activity. Accordingly, this band was chosen for amino acid sequencing.F" @default.
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• W2127698844 title "Molecular Characterization of a Human DNA Kinase" @default.
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