The Metastasis Suppressor NM23-H1 Possesses 3′-5′ Exonuclease Activity
2004; Elsevier BV; Volume: 279; Issue: 17 Linguagem: Inglês
10.1074/jbc.m400185200
ISSN1083-351X
AutoresDeqin Ma, J. Robert McCorkle, David M. Kaetzel,
Tópico(s)Advanced biosensing and bioanalysis techniques
ResumoNM23-H1 belongs to a family of eight gene products in humans that have been implicated in cellular differentiation and development, as well as oncogenesis and tumor metastasis. We have defined NM23-H1 biochemically as a 3′-5′ exonuclease by virtue of its ability in stoichiometric amounts to excise single nucleotides in a stepwise manner from the 3′ terminus of DNA. The activity is dependent upon the presence of Mg2+, is most pronounced with single-stranded substrates or mismatched bases at the 3′ terminus of double-stranded substrates, and is inhibited by both ATP and the incorporation of cordycepin, a 2′-deoxyadenosine analogue, into the 3′-terminal position. The 3′-5′ exonuclease activity was assigned to NM23-H1 by virtue of: 1) precise coelution of enzymatic activity with wild-type and mutant forms of NM23-H1 protein during purification by hydroxylapatite and gel filtration column high performance liquid chromatography and 2) significantly diminished activity exhibited by purified recombinant mutant forms of the proteins. Lysine 12 appears to play an important role in the catalytic mechanism, as evidenced by the significant reduction in 3′-5′ exonuclease activity resulting from a Lys12 to glutamine substitution within the protein. 3′-5′ Exonucleases are believed to play an important role in DNA repair, a logical candidate function underlying the putative antimetastatic and oncogenic activities of NM23-H1. NM23-H1 belongs to a family of eight gene products in humans that have been implicated in cellular differentiation and development, as well as oncogenesis and tumor metastasis. We have defined NM23-H1 biochemically as a 3′-5′ exonuclease by virtue of its ability in stoichiometric amounts to excise single nucleotides in a stepwise manner from the 3′ terminus of DNA. The activity is dependent upon the presence of Mg2+, is most pronounced with single-stranded substrates or mismatched bases at the 3′ terminus of double-stranded substrates, and is inhibited by both ATP and the incorporation of cordycepin, a 2′-deoxyadenosine analogue, into the 3′-terminal position. The 3′-5′ exonuclease activity was assigned to NM23-H1 by virtue of: 1) precise coelution of enzymatic activity with wild-type and mutant forms of NM23-H1 protein during purification by hydroxylapatite and gel filtration column high performance liquid chromatography and 2) significantly diminished activity exhibited by purified recombinant mutant forms of the proteins. Lysine 12 appears to play an important role in the catalytic mechanism, as evidenced by the significant reduction in 3′-5′ exonuclease activity resulting from a Lys12 to glutamine substitution within the protein. 3′-5′ Exonucleases are believed to play an important role in DNA repair, a logical candidate function underlying the putative antimetastatic and oncogenic activities of NM23-H1. nm23-H1 was first classified as a metastasis suppressor gene on the basis of its reduced expression in several metastatic melanoma cell lines relative to nonmetastatic counterparts (1Steeg P.S. Bevilacqua G. Kopper L. Thorgerisson U.R. Talmadge J.E. Liotta L.A. Sobel M. J. Natl. Cancer Inst. 1988; 80: 200-205Crossref PubMed Scopus (1321) Google Scholar). Subsequently, low expression of the NM23-H1 protein has been linked to increased metastatic potential in human breast carcinoma (2Bevilacqua G. Sobel M. Liotta L.A. Steeg P.S. Cancer Res. 1989; 49: 5185-5190PubMed Google Scholar), hepatoma (3Nakayama T. Ohtsuru A. Nakao K. Shima M. Nakata K. Watanabe K. Ishii N. Kimura N. Nagataki S. J. Natl. Cancer Inst. 1992; 84: 1349-1354Crossref PubMed Scopus (164) Google Scholar), and gastric carcinoma (4Nakayama H. Yasui W. Yokozaki H. Tahara E. Jpn. J. Cancer Res. 1993; 84: 184-190Crossref PubMed Scopus (133) Google Scholar). NM23-H1 overexpression has been shown experimentally to inhibit the metastatic phenotype and/or promote differentiation in melanoma (5Leone A. Flatow U. King C.R. Sandeen M.A. Margulies I.M. Liotta L.A. Steeg P.S. Cell. 1991; 65: 25-35Abstract Full Text PDF PubMed Scopus (557) Google Scholar), breast carcinoma (6Leone A. Flatow U. VanHoutte K. Steeg P.S. Oncogene. 1993; 8: 2325-2333PubMed Google Scholar), and transformed neural cell lines (7Lombardi D. Palescandolo E. Giordano A. Paggi M.G. Cell Death Differ. 2001; 8: 470-476Crossref PubMed Scopus (29) Google Scholar, 8Backer M.V. Kamel N. Sandoval C. Jayabose S. Mendola C.E. Backer J.M. Anticancer Res. 2000; 20: 1743-1749PubMed Google Scholar). The human nm23-H1 gene is one of eight related NM23 family members identified to date (reviewed in Ref. 9Lacombe M.-L. Milon L. Mehus J.G. Lambeth D.O. J. Bioenerg. Biomembr. 2000; 32: 247-258Crossref PubMed Scopus (298) Google Scholar). Each exhibits nucleoside-diphosphate kinase (NDPK) 1The abbreviations used are: NDPK, nucleoside-diphosphate kinase; AP, apurinic; H1, NM23-H1; HPLC, high pressure liquid chromatography; HTP, hydroxyapatite; NHE, nuclease hypersensitive element; nt, nucleotide(s); PDGF, platelet-derived growth factor; WRN, Werner syndrome protein; 5′-SHS, 5′-S1 hypersensitive sequence; ATPγS, adenosine-5′-O-3-thiotriphosphate. activity, catalyzing the transfer of γ-phosphate between nucleoside triphosphate and nucleoside diphosphate via a "ping-pong" mechanism (10Agarwal R.P. Robinson B. Parks R.E. Methods Enzymol. 1978; 51: 376-386Crossref PubMed Scopus (130) Google Scholar). NDPK activity does not appear to be relevant to metastasis suppression, however, as catalytically inactive mutants retain metastasis suppressor activity (11Lee H.Y. Lee H. Cancer Lett. 1999; 145: 93-99Crossref PubMed Scopus (47) Google Scholar, 12Hamby C.V. Abbi R. Prasad N. Stauffer C. Thomson J. Mendola C.E. Sidorov V. Backer J.M. Int. J. Cancer. 2000; 88: 547-553Crossref PubMed Scopus (24) Google Scholar). In addition to NDPK, a number of other biological activities have been reported for NM23 proteins, some of which have been proposed to underlie metastasis suppression. Two spontaneous mutations in NM23-H1, a serine 120 to glycine (S120G) substitution seen frequently in aggressive neuroblastomas (13Chang C.L. Zhu X.X. Thoraval D.H. Ungar D. Rawwas J. Hora N. Strahler J.R. Hanash S.M. Radany E. Nature. 1994; 370: 335-336Crossref PubMed Scopus (135) Google Scholar), and a proline 96 to serine mutation that corresponds to the killer of prune mutation (Kpn; P96S) in the Drosophila homologue of NM23 (awd), both result in loss of antimetastatic activity (14MacDonald N.J. Freije J.M. Stracke M.L. Manrow R.E. Steeg P.S. J. Biol. Chem. 1996; 271: 25107-25116Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Interestingly, both mutations also abrogate a histidine-dependent protein kinase activity of NM23-H1 (15Wagner P.D. Vu N.D. J. Biol. Chem. 1995; 270: 21758-21764Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). This activity has been implicated more recently in the serine phosphorylation of the kinase suppressor of ras (KSR), suggesting antimetastatic activity may arise via the suppression of ras-initiated growth signals (16Hartsough M.T. Morrison D.K. Salerno M. Palmieri D. Ouatas T. Mair M. Patrick J. Steeg P.S. J. Biol. Chem. 2002; 277: 32389-32399Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). Considerable evidence has also been presented to indicate that NM23 proteins interact with DNA. NM23-H2 has been shown to bind and activate the nuclease-hypersensitive element (NHE) of the c-myc promoter (17Postel E.H. Berberich S.J. Flint S.J. Ferrone C.A. Science. 1993; 261: 478-480Crossref PubMed Scopus (486) Google Scholar, 18Berberich S.J. Postel E.H. Oncogene. 1995; 10: 2343-2347PubMed Google Scholar), suggesting a molecular mechanism of oncogenesis and malignant progression. NM23-H2 also cleaves the NHE sequence in vitro when presented in either linear or supercoiled plasmid form (19Postel E.H. J. Biol. Chem. 1999; 274: 22821-22829Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), suggesting a role in modulating transcription via the remodeling of regulatory elements that exhibit non-B-form, or paranemic, DNA conformations (for a review, see Ref. 20Postel E. Berberich S.J. Rooney J.W. Kaetzel D.M. J. Bioenerg. Biomembr. 2000; 32: 277-284Crossref PubMed Scopus (123) Google Scholar). Each of these interactions with the NHE were independent of NDPK activity, as they were retained with an NDPK-defective mutant form (H118F) of the protein (21Postel E. Ferrone C.A. J. Biol. Chem. 1994; 269: 8627-8630Abstract Full Text PDF PubMed Google Scholar). The DNA cleaving activity of NM23-H2 was further shown to occur via a DNA glycosylase/lyase-like mechanism (22Postel E. Abramczyk B.M. Levit M.N. Kyin S. Proc. Natl. Acad. Sci. U. S. A. 2001; 97: 14194-14199Crossref Scopus (80) Google Scholar), a hallmark of base excision DNA repair enzymes. Both NM23-H1 and NM23-H2 repress transcription via interactions with paranemic elements in the promoter region of the platelet-derived growth factor-A (PDGF-A) gene (23Ma D. Xing Z. Liu B. Pedigo N. Zimmer S. Bai Z. Postel E. Kaetzel D.M. J. Biol. Chem. 2002; 277: 1560-1567Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 24Kaetzel D.M. Cytokine Growth Factor Rev. 2003; 14: 427-446Crossref PubMed Scopus (19) Google Scholar). Repression of this oncogenic and metastasis-promoting growth factor (25Anan K. Morisaki T. Katano M. Ikubo A. Kitsuki H. Uchiyama A. Kuroki S. Tanaka M. Torisu M. Surgery. 1996; 119: 333-339Abstract Full Text PDF PubMed Scopus (154) Google Scholar) is consistent with a potential antimetastatic function of NM23 proteins. Interestingly, NM23-H1 and NM23-H2 also cleaved the PDGF-A regulatory elements in vitro; NM23-H1 appeared to excise nucleotides progressively from the 3′ terminus of single-stranded oligodeoxynucleotides, whereas NM23-H2 appeared to cleave internally, as observed previously with the c-myc NHE sequence. Interestingly, the DNA cleavage function of NM23 is conserved from the primordial NDPK gene in Escherichia coli (26Levit M.N. Abramczyk B.M. Stock J.B. Postel E.H. J. Biol. Chem. 2002; 277: 5163-5167Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). A recent study has also shown that NM23-H1 is the DNA-cleaving component of a latent protein complex (SET) that is activated during cytotoxic T lymphocyte-mediated apoptosis (27Fan Z. Beresford P.J. Oh D.Y. Zhang D. Lieberman J. Cell. 2003; 112: 659-672Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar). While observed in the context of the immune response, this finding suggests that proapoptotic or immunosurveillance functions may underlie the antimetastatic activity of NM23-H1. In this report we show that human NM23-H1 that is expressed in E. coli and purified to apparent homogeneity exhibits biochemical characteristics consistent with 3′-5′ exonuclease activity. 3′-5′ Exonucleases are associated generally with DNA proofreading (reviewed in Ref. 28Shevelev I.V. Hübscher U. Nat. Rev. Mol. Cell. Biol. 2002; 3: 1-12Crossref Scopus (216) Google Scholar), with loss of expression and/or function often associated with mutator phenotypes, and increased potential for cancer progression (29Jackson A. Loeb L.A. Genetics. 1998; 148: 1483-1490Crossref PubMed Google Scholar). Thus, the 3′-5′ exonuclease activity of NM23-H1 demonstrated in this report would appear to represent a logical candidate function underlying its antimetastatic activity. DNA and Site-directed Mutagenesis—E. coli plasmids (pET3c, New England Biolabs) for expression of wild-type NM23-H1 and its mutant variants, R34A and H118F, were kindly provided by E. Postel (Princeton). K12Q was generated by the overlap extension modification of the polymerase chain reaction (30Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 51-59Crossref PubMed Scopus (6963) Google Scholar) using the sequences 5′-cgggatcccatatggccaactgtga-3′ and 5′-ccggatccccgaattctcattcatagatccagt-3′ as 5′- and 3′-end primers, respectively, and the mutagenic primers 5′-cccatctggctggatcgcaatgaaggtac-3′ and 5′-cattgcgatccagccagatggggtcca-3′ (codons encoding the mutant glutamine residue are in underlined bold font). The cDNA sequence encoding K12Q was inserted in-frame between the NdeI and BamHI sites of pET3c. The cloned PCR product was sequenced to ensure that only the desired mutation was present. Overexpression and Purification of Recombinant Human NM23-H1—NM23-H1 and mutant proteins were expressed in E. coli essentially as described (23Ma D. Xing Z. Liu B. Pedigo N. Zimmer S. Bai Z. Postel E. Kaetzel D.M. J. Biol. Chem. 2002; 277: 1560-1567Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Briefly, pET3c vectors containing wild-type and variants of NM23-H1 were used to transform E. coli (BL21 plysS; Promega). For protein expression, freshly transformed bacteria were grown overnight at 37 °C in LB-ampicillin medium. Two ml of the overnight culture was used to inoculate 200 ml of LB medium containing ampicillin. When the A600 reached 0.6, protein expression was induced by the addition of isopropyl-1-thio-β-d-galactopyranoside (final concentration, 0.4 mm). Three hours following induction, cells were harvested, resuspended in 30 ml of lysis buffer (50 mm Tris, pH 8.0) containing 1 mm EDTA and dithiothreitol, 1 mm leupeptin and pepstatin A, and 0.1 mm phenylmethylsulfonyl fluoride (Sigma) and lysed by sonication. Cell lysates were cleared by centrifugation at 12,000 × g for 30 min and the proteins were precipitated with ammonium sulfate (60-90% fractions). The 60-90% fraction was prepared for application to a DEAE-Sephacel column by dialysis into 50 mm Tris buffer (pH 7.5) containing a mixture of protease inhibitors. Under these conditions, NM23-H1 bound to the column and was eluted with a 0-1 m NaCl gradient with the peak fraction at ∼350 mm NaCl. Peak fractions containing NM23 proteins were equilibrated in 10 mm phosphate buffer (pH 7.0) by centrifugal filtration (Centricon-10, Millipore) and loaded onto a hydroxyapatite (HTP) column. NM23-H1 was eluted with a 80-ml phosphate gradient of 10-800 mm. Purity of recombinant proteins was evaluated by SDS-PAGE and Commassie Blue and/or silver staining. Unless otherwise indicated, only the 1-ml peak fraction was employed for cleavage assays. DNA Cleavage Assays—A number of the oligodeoxyribonucleotide sequences employed for cleavage assays were derived from either the 5′-SHS silencer or the NHE elements found in the 5′-flanking region of the PDGF-A promoter (Table I; Refs. 23Ma D. Xing Z. Liu B. Pedigo N. Zimmer S. Bai Z. Postel E. Kaetzel D.M. J. Biol. Chem. 2002; 277: 1560-1567Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar and 31Liu B. Maul R.S. Kaetzel Jr., D.M. J. Biol. Chem. 1996; 271: 26281-26290Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). Oligodeoxyribonucleotides 12-15 were substrates of the WRN helicase/exonuclease provided by David Orren (University of Kentucky). Radiolabeling of 5′ termini was performed in a 40-μl reaction mixture at 37 °C for 30 min using [γ-32P]ATP and T4 polynucleotide kinase. Double-stranded probes were annealed by incubation of the radiolabeled single strand of interest with its complement at 95 °C for 5 min followed by gradual cooling to ambient room temperature over a 3-h interval. For 3′-end labeling, the strand to be labeled was first annealed to a complementary strand designed to provide a duplex with a 5′-overhang. The recessed 3′ terminus was filled in with [α-32P]dCTP in the presence of the three remaining unlabeled nucleotides and the Klenow fragment of DNA polymerase I. To prepare a doubled-stranded oligodeoxyribonucleotide probe with the 2′-deoxyadenosine analogue cordycepin at the 3′ terminus, the purine-rich strand of NHE was first 5′-labeled with γ-32P and T4 polynucleotide kinase, then annealed to its complementary pyrimidine-rich strand (9-bp mismatch, oligonucleotide number 3). This duplex was incubated with 10 mm cordycepin 5′-triphosphate and terminal transferase at 37 °C for 1 h (specific activity: ∼1.0 × 106 cpm/pmol). Routine DNA cleavage assays were performed in a 15-μl reaction mixture containing 20 mm Hepes buffer (pH 7.9), 10-50 fmol of 5′-32P-labeled oligonucleotide, 2 mm MgCl2, and 100 mm KCl as described previously (23Ma D. Xing Z. Liu B. Pedigo N. Zimmer S. Bai Z. Postel E. Kaetzel D.M. J. Biol. Chem. 2002; 277: 1560-1567Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Standard reactions were initiated by the addition of 0.5-1 μg of protein and carried out at room temperature. Reactions were terminated by adding an equal volume of sequencing loading dye consisting of 80% deionized formamide (w/v), 10 mm EDTA (pH 8.0), xylene cyanol FF (1 mg/ml), and bromphenol blue (1 mg/ml) followed by heating at 95 °C for 5 min. Cleavage products were resolved on sequencing gels ranging in polyacrylamide concentration between 10 and 15% as indicated in the text and visualized by phosphorimaging (Storm 860, Amersham Biosciences).Table IOligodeaxyribonucleotides used in cleavage assays#NameSourceLengthSequence1C345′-SHSas34nt5′-TCCACACACCTGCAGGCCCCCTCCCCACGTCTCT-3′2G28NHEs28nt5′-GGGGGCGGGGGCGGGGGCGGGGGAGGGG-3′3C28NHEas28nt5′-CCCCTCCCCCGCCCCCGCCCCCGCCCCC-3′4G22NHEs28nt5′-GGGGGCGGGGGCGGGGGAGGGG-3′5C22NHEas22nt5′-CCCCTCCCCCGCCCCCGCCCCC-3′6C20NHEas20nt5′-CCGCCCCCGCCCCCGCCCCC3′7M1NHEs40nt5′-TTTTTTTTTTTTTGGGGCGGGGGCGGGGGCGGGGGAGGGG-3′8M2NHEs40nt5′-TTTTTTTTTTTTTTGGGCGGGGGCGGGGGCGGGGGAGGGG-3′9M3NHEs40nt5′-TTTTTTTTTTTTTTTGGCGGGGGCGGGGGCGGGGGAGGGG-3′10M9NHEs40nt5′-TTTTTTTTTTTTTTTTTTGGGGGCGGGGGCGGGGGAGGGG-3′11C80WRNaWRN refers to oligodeoxyribonucleotide sequences employed previously as substrates in the study of the Werner syndrome protein, WRN (39). WRN substrate G80b (oligodeoxyribonucleotide 15) contains 21 nucleotides of mismatched sequence (bold letters) in relation to the partially complementary oligodeoxyribonucleotide, C80 (oligodeoxyribonucleotide 14). Annealing of these sequences yield an internal "bubble" structure that serves as a high affinity substrate for the WRN protein.80nt5′-GCTGATCAACCCTACATGTGTAGGTAACCCTAACCCTAACCCTAAGGACAACCCTAGTGAAGCTTGTAA CCCTAGGAGCT-3′12G80WRN80nt5′-AGCTCCTAGGGTTACAAGCTTCACTAGGGTTGTCCTTAGGGTTAGGGTTAGGGTTACCTACACATGTAGG GTTGATCAGC-3′13G80bWRN80nt5′-AGCTCCTAGGGTTACAAGCTTCACTAGGGTTGTCCGTCACAGTCAGAGTCACAGTCCTACACATGTAGGG TTGATCA-3′14G24WRN24nt5′-CCTACACATGTAGGGTTGATCAGC-3′a WRN refers to oligodeoxyribonucleotide sequences employed previously as substrates in the study of the Werner syndrome protein, WRN (39Machwe A. Xiao L. Theodore S. Orren D.K. J. Biol. Chem. 2002; 277: 4492-4504Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). WRN substrate G80b (oligodeoxyribonucleotide 15) contains 21 nucleotides of mismatched sequence (bold letters) in relation to the partially complementary oligodeoxyribonucleotide, C80 (oligodeoxyribonucleotide 14). Annealing of these sequences yield an internal "bubble" structure that serves as a high affinity substrate for the WRN protein. Open table in a new tab Nucleoside-diphosphate Kinase Assays—NDPK activity was measured as described (10Agarwal R.P. Robinson B. Parks R.E. Methods Enzymol. 1978; 51: 376-386Crossref PubMed Scopus (130) Google Scholar) in a coupled pyruvate-lactate dehydrogenase assay in which ATP acted as a phosphate donor and dTDP as an acceptor. The procedure was modified for the current study to accommodate the use of 96-well plates and microplate reader. NDPK reactions were initiated by the addition of 10 μl of purified NM23-H1 in 20 mm Hepes buffer (pH 7.9) into individual wells of a 96-well plate, each containing 140 μl of reaction mixture consisting of 1 mm pyruvate enol phosphate, 5 mm ATP, 2.5 mm dTDP, 6 mm MgCl2, 50 mm KCl, 1000 units of pyruvate kinase and lactate dehydrogenase. NAD+ formation, which reflected ADP production, was measured every 25 s at 340 nm by a HTS 7000 plus Bioassay Plate Reader (PerkinElmer Life Sciences). Specific activity was expressed as units/mg of protein, with 1 unit defined as the amount of enzyme converting 1 μmol of ADP/min at room temperature (ϵ340 of NADH = 6.22 × 103m-1 cm-1). Circular Dichroism Analysis—Far UV-CD spectra were recorded from 260 to 190 nm using a Jasco J-810 spectrometer, with each individual spectrum representing the average of 30 replicate measurements. Prior to analysis, purified NM23-H1 proteins were diluted to a concentration of 0.12 mg/ml in 5 mm phosphate buffer (pH 7.0) (ϵ280 = 1.35 for a 1 mg/ml solution; Ref. 32Lascu I. Schaertl S. Wang C. Sarger C. Giartosio A. Briand G. Lacombe M.L. Konrad M. J. Biol. Chem. 1997; 272: 15599-15602Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). CD spectra were recorded at 5 °C in a quartz cuvette with a 0.1-cm path-length. CD data were converted to mean residual ellipticity, assuming a hexameric structure and mean residue molecular mass of 113 Da, the latter calculated from a 152-amino acid monomer with a molecular mass of 17,180 Da (33Rosengard A.M. Krutzch H.C. Shearn A. Biggs J.R. Barker E. Margulies I.M. King C.R. Liotta L.A. Steeg P.S. Nature. 1989; 342: 177-180Crossref PubMed Scopus (490) Google Scholar). Secondary structure estimates were derived from the 250-190-nm region of the recorded CD spectra using the programs CONTILL, SELCON3, and CDSSTR and their reference set of 43 proteins (34Sreerama N. Woody R.W. Anal. Biochem. 2000; 287: 252-260Crossref PubMed Scopus (2584) Google Scholar) from the CDPro software package. 2lamar.colostate/edu~sreeram/CDPro/main.html. High Performance Liquid Chromatography (HPLC) Gel Filtration—HTP-purified wild-type or mutant forms of NM23-H1 were loaded on a Shodex gel filtration HPLC column (Shodex Protein KW-800, Showa Denko) pre-equilibrated in 50 mm Tris (pH 7.5), 0.1 m KCl. Molecular masses of NM23-H1 were estimated relative to a standard curve generated with a commercially available molecular mass standards kit (Sigma) containing cytochrome c (12.4 kDa), carbonic anhydrase (29 kDa), bovine serum albumin (66 kDa), alcohol dehydrogenase (150 kDa), β-amylase (200 kDa), and blue dextran (2,000 kDa). NM23-H1 and DNA Cleavage Activity Coelute following Gel Filtration HPLC—DNA cleaving activity co-purifies with NM23-H1 following a three-step protocol consisting of ammonium sulfate precipitation and sequential chromatographic steps of DEAE-Sephacel and hydroxylapatite (Fig. 1A; Ref. 23Ma D. Xing Z. Liu B. Pedigo N. Zimmer S. Bai Z. Postel E. Kaetzel D.M. J. Biol. Chem. 2002; 277: 1560-1567Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). When NM23-H1-containing fractions were incubated with a 5′-end labeled oligodeoxyribonucleotide corresponding to the noncoding strand of the 5′-SHS silencer (5′-SHS antisense strand, or 5′-SHSas) from the PDGF-A gene (31Liu B. Maul R.S. Kaetzel Jr., D.M. J. Biol. Chem. 1996; 271: 26281-26290Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar), progressively smaller fragments were obtained with increasing amounts of protein. Oligodeoxyribonucleotide substrates employed in this study are summarized in Tables I and II. This DNA cleavage pattern strongly suggested that NM23-H1 was excising nucleotides progressively from the 3′ terminus of this single-stranded DNA molecule, consistent with a 3′-5′ exonuclease activity. To rule out further the possibility that the 3′-5′ exonuclease-like activity was catalyzed by nuclease contamination from the E. coli expression host, the peak fraction of NM23-H1 from the hydroxylapatite column (82-83 min) was further analyzed by gel filtration HPLC. Two peaks of NM23-H1 protein were observed (Fig. 2, top), a primary peak at approximately 18.8 min with an estimated molecular mass of 82 kDa (Table III) and a minor peak of an (∼200 kDa) aggregate. No peaks corresponding to monomeric or dimeric NM23-H1 were observed. The derived molecular mass estimate of 82 kDa is somewhat less than the predicted molecular mass of 105-120 kDa reported for hexameric NM23-H1 and NM23-H2, based on a monomeric molecular mass of 17-19 kDa (35Schaertl S. FEBS Lett. 1996; 394: 316-320Crossref PubMed Scopus (10) Google Scholar, 36Webb P.A. Perisic O. Mendola C.E. Backer J.M. Williams R.L. J. Mol. Biol. 1995; 251: 574-587Crossref PubMed Scopus (127) Google Scholar, 37Milon L. Meyer P. Chiadmi M. Munier A. Johansson M. Karlsson A. Lascu I. Capeau J. Janin J. Lacombe M.L. J. Biol. Chem. 2000; 275: 14264-14272Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). This difference could be the result of anomalous interactions with the gel filtration matrix, as has been observed by others. 3I. Lascu, personal communication. It should also be noted that tetrameric structures have been reported for NM23-H2 based on gel filtration analysis, as well as for Myxococcus xanthus NDPK by direct assessment of the crystal structure (36Webb P.A. Perisic O. Mendola C.E. Backer J.M. Williams R.L. J. Mol. Biol. 1995; 251: 574-587Crossref PubMed Scopus (127) Google Scholar). Nevertheless, DNA cleavage activity (Fig. 2, bottom) coeluted precisely with the primary peak of oligomerized NM23-H1 protein (middle), providing additional evidence that the activity is not because of copurifying or nonspecifically associated nuclease contaminants.Table IIDNA substrates used in this study1 Asterisk denotes the presence of 32P at the 5′ - or 3′ -terminus of the listed oligodeoxyribonucleotides, as identified in representations under the DNA substrate column. Number assignments are based on the compilation in Table 1. Shaded ovals represent unpaired nucleotide residues. Open table in a new tab Fig. 2NM23-H1 and DNA cleavage activity coelute during gel filtration HPLC. One mg of NM23-H1, purified as described in the legend to Fig. 1, was loaded on a Shodex gel filtration HPLC column (Shodex Protein KW-800) pre-equilibrated in 50 mm Tris (pH 7.5) and 0.1 m KCl at a flow rate of 0.5 ml/min. Elution times of the molecular mass standards (Sigma) cytochrome c (12.4 kDa), carbonic anhydrase (29 kDa), bovine serum albumin (66 kDa), alcohol dehydrogenase (160 kDa), β-amylase (200 kDa), and blue dextran (2000 kDa) are indicated with arrows in the top panel. The primary peak of wild-type NM23-H1 protein as indicated by A280 was observed at 18.76 min. Fifty-μl fractions were collected and a 6-μl aliquot from each fraction was analyzed by either SDS-polyacrylamide gel electrophoresis and Coomassie Blue staining (middle, ∼2 μg in peak fraction) or DNA cleavage assay (bottom, ∼1 μg in peak fraction). Indicated above the middle and bottom panels are the elution times of the fractions analyzed, with each number representing the beginning of the collection interval. Cleavage assays were conducted with 10 fmol of 32P-labeled 5′-SHSas probe in the presence of 2 mm MgCl2 and 100 mm KCl at room temperature for 1 h. Cleavage products were separated on a denaturing 10% polyacrylamide gel and visualized by phosphorimaging. Mobility of the 32P-labeled 5′-SHSas probe (arrow) and cleaved products (bracket) are identified at the right.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IIIMolecular mass estimates and nucleoside-diphosphate kinase activities for NM23-H1 variantsProteinEstimated molecular massaResults are the average ± S.E. in kDa of three replicate determinations of molecular mass by gel filtration HPLC.NDPK activitybNDPK activity was measured as described under "Experimental Procedures" and is expressed as units per mg of recombinant NM23-H1 protein. Results are the average ± S.E. of six replicate measurements.WT82.1 ± 1.3570cMean(s) within a column is significantly different (p ≤ 0.01). ± 25R34A80.5 ± 1.9332cMean(s) within a column is significantly different (p ≤ 0.01). ± 12K12Q76.0cMean(s) within a column is significantly different (p ≤ 0.01). ± 0.613.6cMean(s) within a column is significantly different (p ≤ 0.01). ± 0.9H118F81.0 ± 0.4B.D.dBelow the detection limits of the NDPK assay (<5 units/mg).a Results are the average ± S.E. in kDa of three replicate determinations of molecular mass by gel filtration HPLC.b NDPK activity was measured as described under "Experimental Procedures" and is expressed as units per mg of recombinant NM23-H1 protein. Results are the average ± S.E. of six replicate measurements.c Mean(s) within a column is significantly different (p ≤ 0.01).d Below the detection limits of the NDPK assay (<5 units/mg). Open table in a new tab 1 Asterisk denotes the presence of 32P at the 5′ - or 3′ -terminus of the listed oligodeoxyribonucleotides, as identified in representations under the DNA substrate column. Number assignments are based on the compilation in Table 1. Shaded ovals represent unpaired nucleotide residues. DNA Cleaving Activity of NM23-H1 Requires Mg2+ and Is Inhibited by KCl and ATP—As was previously shown with NM23-H2 (19Postel E.H. J. Biol. Chem. 1999; 274: 22821-22829Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), the DNA cleaving activity of NM23-H1 was dependent upon Mg2+ ions (Fig. 3A, lane 2 versus lanes 10-16). Measurable activity was detected at the lowest Mg2+ concentration administered (0.1 mm, lane 10), with an optimal concentration of 1-2 mm. Significant cleavage activity was also observed upon addition of Mn2+ (lanes 25-30), although the stimulation was less potent than that achieved with Mg2+. Ca2+ failed to support any measurable DNA cleavage (lanes 3-9), whereas Zn2+ elicited only trace activity (lanes 17-24). Cleavage activity was also inhibited by KCl, with the o
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