Physical and Functional Interaction between the Mini-chromosome Maintenance-like DNA Helicase and the Single-stranded DNA Binding Protein from the Crenarchaeon Sulfolobus solfataricus
2002; Elsevier BV; Volume: 277; Issue: 14 Linguagem: Inglês
10.1074/jbc.m200091200
ISSN1083-351X
AutoresFloriana Carpentieri, Mariarita De Felice, Mariarosaria De Falco, Mosè Rossi, Francesca M. Pisani,
Tópico(s)Bacterial Genetics and Biotechnology
ResumoMini-chromosome Maintenance (MCM) proteins play an essential role in both initiation and elongation phases of DNA replication in Eukarya. Genes encoding MCM homologs are present also in the genomic sequence of Archaea and the MCM-like protein from the euryarchaeon Methanobacterium thermoautotrophicum(Mth MCM) was shown to possess a robust ATP-dependent 3′-5′ DNA helicase activity in vitro. Herein, we report the first biochemical characterization of a MCM homolog from a crenarchaeon, the thermoacidophile Sulfolobus solfataricus (Sso MCM). Gel filtration and glycerol gradient centrifugation experiments indicate that the Sso MCM forms single hexamers (470 kDa) in solution, whereas the Mth MCM assembles into double hexamers. The Sso MCM has NTPase and DNA helicase activity, which preferentially acts on DNA duplexes containing a 5′-tail and is stimulated by the single-stranded DNA binding protein from S. solfataricus (Sso SSB). In support of this functional interaction, we demonstrated by immunological methods that the Sso MCM and SSB form protein·protein complexes. These findings provide the first in vitro biochemical evidence of a physical/functional interaction between a MCM complex and another replication factor and suggest that the two proteins may function together in vivo in important DNA metabolic pathways. Mini-chromosome Maintenance (MCM) proteins play an essential role in both initiation and elongation phases of DNA replication in Eukarya. Genes encoding MCM homologs are present also in the genomic sequence of Archaea and the MCM-like protein from the euryarchaeon Methanobacterium thermoautotrophicum(Mth MCM) was shown to possess a robust ATP-dependent 3′-5′ DNA helicase activity in vitro. Herein, we report the first biochemical characterization of a MCM homolog from a crenarchaeon, the thermoacidophile Sulfolobus solfataricus (Sso MCM). Gel filtration and glycerol gradient centrifugation experiments indicate that the Sso MCM forms single hexamers (470 kDa) in solution, whereas the Mth MCM assembles into double hexamers. The Sso MCM has NTPase and DNA helicase activity, which preferentially acts on DNA duplexes containing a 5′-tail and is stimulated by the single-stranded DNA binding protein from S. solfataricus (Sso SSB). In support of this functional interaction, we demonstrated by immunological methods that the Sso MCM and SSB form protein·protein complexes. These findings provide the first in vitro biochemical evidence of a physical/functional interaction between a MCM complex and another replication factor and suggest that the two proteins may function together in vivo in important DNA metabolic pathways. Mini-chromosome Maintenance (MCM) 1The abbreviations used are: MCMMini-chromosome MaintenanceSSBsingle-stranded DNA binding proteinPMSFphenylmethylsulfonyl fluorideBSAbovine serum albuminPBSphosphate-buffered salineTBSTris-buffered saline, TBE, Tris-Borate-EDTAELISAenzyme-linked immunosorbent assayssDNAsingle-stranded DNAdsDNAdouble-stranded DNAATPγSadenosine 5′-O-(thiotriphosphate)WRNWerner's syndrome geneBLMBloom's syndrome genentnucleotide(s) genes (MCMs 2–7) were originally identified in budding and fission yeast by a genetic analysis of mutants that were unable to efficiently replicate mini-chromosomes (1.Maine G.T. Sinha P. Tye B.K. Genetics. 1984; 106: 365-385Crossref PubMed Google Scholar, 2.Takahashi K. Yamada H. Yanagida M. Mol. Biol. Cell. 1994; 5: 1145-1158Crossref PubMed Scopus (186) Google Scholar). Homologs of the six yeast MCM genes were subsequently identified in various other eukaryotic organisms, from Drosophila melanogaster to Homo sapiens and found to code for proteins (ranging in length from 776 to 1017 amino acidic residues), which are evolutionarily conserved especially in the central third of their polypeptide chain (3.Kearsey S.E. Labib K. Biochim. Biophys. Acta. 1998; 1398: 113-136Crossref PubMed Scopus (230) Google Scholar, 4.Tye B.K. Annu. Rev. Biochem. 1999; 68: 649-686Crossref PubMed Scopus (515) Google Scholar). In fact, this region contains the four sequence motifs typically found in DNA helicases, including the Walker A and B boxes that are critical for nucleotide binding and hydrolysis (5.Neuwald A.L.A. Spouge J. Koonin E.V. Genome Res. 1999; 9: 27-43Crossref PubMed Google Scholar). The MCM proteins are relatively abundant in proliferating cells and were purified from cell extracts of various organisms either as hetero-hexameric complexes containing all six polypeptides or as sub-assemblies of various subunit composition (such as MCM 2/4/6/7 and MCM 4/6/7 (6.Thommes P. Kubota Y. Takisawa H. Blow J.J. EMBO J. 1997; 16: 3312-3319Crossref PubMed Scopus (117) Google Scholar, 7.Ishimi Y. Ichinose S. Omori A. Sato K. Kimura H. J. Biol. Chem. 1996; 271: 24115-24122Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 8.Fujita M. Kiyono T. Hayashi Y. Ishibashi M. J. Biol. Chem. 1997; 272: 10928-10935Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 9.Adachi Y. Usukura J. Yanagida M. Genes Cells. 1997; 2: 467-479Crossref PubMed Scopus (108) Google Scholar, 10.Sherman D.A. Forsburg S.L. Nucleic Acids Res. 1998; 26: 3955-3960Crossref PubMed Scopus (40) Google Scholar)). However, among all these multimeric complexes only the MCM 4/6/7 hexamer was demonstrated to have a weak and non-processive DNA helicase activity (11.Ishimi Y. J. Biol. Chem. 1997; 272: 24508-24513Abstract Full Text Full Text PDF PubMed Scopus (461) Google Scholar, 12.You Z. Komamura Y. Ishimi Y. Mol. Cell. Biol. 1999; 19: 8003-8015Crossref PubMed Scopus (171) Google Scholar, 13.Lee J.K. Hurwitz J. J. Biol. Chem. 2000; 275: 18871-18878Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). The MCM 4/6/7 complex is dis-assembled in vitro upon addition of MCM 2 or MCM 3/5, and this causes inhibition of its DNA unwinding activity (13.Lee J.K. Hurwitz J. J. Biol. Chem. 2000; 275: 18871-18878Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 14.Ishimi Y. Komamura Y. You Z. Kimura H. J. Biol. Chem. 1998; 273: 8639-8675Abstract Full Text Full Text PDF Scopus (115) Google Scholar). Based on these findings, it was proposed that the MCM 4/6/7 assembly could act as DNA unwinding factor at the replication origins, whereas the other MCM subunits could play regulatory functions. However, due to the limited processivity of their DNA unwinding activity the MCM proteins were considered poor candidates for the helicase associated with the DNA replication fork. In addition, several genetic studies have evidenced that the MCM proteins in vivo could interact with the Origin Recognition Complex, Cdc6, Cdc45, and Cdc7/Dbf4 kinase (15.Tanaka T. Knapp D. Nasmyth K. Cell. 1997; 90: 649-660Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar, 16.Duha A. Bell S.P. Annu. Rev. Cell Dev. Biol. 1997; 13: 293-332Crossref PubMed Scopus (340) Google Scholar, 17.Donovan S. Harwood J. Drury L.S. Diffley J.F.X. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5611-5616Crossref PubMed Scopus (433) Google Scholar, 18.Zou L. Stillman B. Science. 1998; 280: 593-596Crossref PubMed Scopus (275) Google Scholar), but no direct evidence for their physical and/or functional interaction with any replication factor was reported so far. Mini-chromosome Maintenance single-stranded DNA binding protein phenylmethylsulfonyl fluoride bovine serum albumin phosphate-buffered saline Tris-buffered saline, TBE, Tris-Borate-EDTA enzyme-linked immunosorbent assay single-stranded DNA double-stranded DNA adenosine 5′-O-(thiotriphosphate) Werner's syndrome gene Bloom's syndrome gene nucleotide(s) More recently, it was clearly demonstrated that in Saccharomyces cerevisiae all six MCM genes are essential not only for the initiation but also for the elongation phase of chromosome replication (19.Labib K. Tercero J.A. Diffley J.F.X. Science. 2000; 288: 1643-1647Crossref PubMed Scopus (523) Google Scholar). Furthermore, an important clue to the in vivo function of the MCM proteins derived from the recent biochemical characterization of the single MCM homolog from the euryarchaeon Methanobacterium thermoautotrophicum (Mth MCM). In three reports, Mth MCM was demonstrated to form a ring-shaped double hexamer and to possess a robust and processive 3′-5′ DNA helicase activity in vitro (20.Kelman Z. Lee J.-K. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14783-14788Crossref PubMed Scopus (197) Google Scholar, 21.Chong J.P.J. Hayashi M.K. Simon M.N. Xu R.-M. Stillman B. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1530-1535Crossref PubMed Scopus (259) Google Scholar, 22.Shechter D.F. Ying C.Y. Gautier J. J. Biol. Chem. 2000; 275: 15049-15059Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). These findings reinforced the hypothesis that the MCM proteins may act as the helicase associated to the replication fork, although there is no direct evidence that Mth MCM is required in vivo for chromosome duplication (23.Labib K. Diffley J.F.X. Curr. Opin. Genet. Dev. 2001; 11: 64-70Crossref PubMed Scopus (125) Google Scholar). In addition, these studies once again pointed out that Archaea possess a replication machinery that is in several instances a simplified version of the eukaryotic counterpart and the biochemical characterization of the replication proteins from these peculiar organisms could provide an useful model to elucidate the molecular mechanisms of replication initiation, as well as replisome assembly and progression, in a context devoid of the eukaryotic regulatory complexities (24.Tye B.K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2399-2401Crossref PubMed Scopus (54) Google Scholar). The Archaea domain is composed of two subdomains: Euryarchaeotes (including M. thermoautotrophicum and Pyrococcus species) and Crenarchaeotes (including Sulfolobus solfataricus and Aeropyrum pernix (25.Woese C.R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8392-8396Crossref PubMed Scopus (435) Google Scholar)). The two groups show important differences at the molecular level: only the euryarchaeal species were found to possess the hetero-dimeric family D DNA polymerase, whereas the Crenarchaeotes are believed to utilize DNA polymerases of the family B as the chromosomal replicases; the Euryarchaeotes have histone-like proteins, whereas the crenarchaeal chromatin contains a different kind of DNA binding protein (26.Bernander R. Trends Microbiol. 2000; 8: 278-283Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). We recently produced in recombinant form and biochemically characterized some putative components of the replisome from the thermoacidophilic crenarchaeon S. solfataricus (27.She Q. Singh R.K. Confalonieri F. Zivanovic Y. Allard G. Awayez M.J. Chan-Weiher C.C. Clausen I.G. Curtis B.A. De Moors A. Erauso G. Fletcher C. Gordon P.M. Heikamp-de Jong I. Jeffries A.C. Kozera C.J. Medina N. Peng X. Thi-Ngoc H.P. Redder P. Schenk M.E. Theriault C. Tolstrup N. Charlebois R.L. Doolittle W.F. Duguet M. Gaasterland T. Garrett R.A. Ragan M.A. Sensen C.W. Van der Oost J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7835-7840Crossref PubMed Scopus (672) Google Scholar), including a single-subunit family B DNA polymerase (28.Pisani F.M. Manco G. Carratore V. Rossi M. Biochemistry. 1996; 35: 9158-9166Crossref PubMed Scopus (17) Google Scholar), two proliferating cell nuclear antigen-like sliding clamps (29.De Felice M. Sensen C.W. Charlebois R.L. Rossi M. Pisani F.M. J. Mol. Biol. 1999; 291: 47-57Crossref PubMed Scopus (49) Google Scholar), and a replication factor C-like clamp-loader (30.Pisani F.M. De Felice M. Carpentieri F. Rossi M. J. Mol. Biol. 2000; 301: 61-73Crossref PubMed Scopus (49) Google Scholar). In addition, a SSB protein from this species (Sso SSB) has been recently identified and characterized (31.Wadsworth R.I.M. White F.M. Nucleic Acids Res. 2001; 29: 914-920Crossref PubMed Scopus (115) Google Scholar). The Sso SSB (16,184 Da) is an abundant protein that exists as a monomer in solution and multimerizes upon DNA binding, forming probably tetramers. Each monomer contains a single "OB-fold" (oligonucleotide/oligosaccharide binding fold) and is able to bind 4–5 nucleotides of ssDNA. Herein we report the biochemical characterization of the single MCM homolog from the crenarchaeon S. solfataricus(Sso MCM). This protein forms hexamers in solution and has ATPase and DNA helicase activity. This latter preferentially melts 5′-tailed oligonucleotides and is stimulated by the Sso SSB. In addition, we demonstrated that Sso MCM and Sso SSB physically interact each other. These findings provide the first direct biochemical evidence that a MCM complex functionally and physically interacts with another replication factor and have implications for other MCM proteins from higher organisms including humans. All chemicals were of reagent grade. Restriction and modification enzymes were from Roche Molecular Biochemicals. Radioactive nucleotides were purchased from Amersham Biosciences, Inc. Oligonucleotides were synthesized by Primm (Milan, Italy). The anti-SsoMCM rabbit antiserum was kindly provided by Dr. Stephen D. Bell (Cambridge, United Kingdom). The Escherichia coli expression vector pET19b-SsoMCM was constructed by Dr. M. F. White (Saint Andrews, United Kingdom). This plasmid harbors the Sso MCM gene cloned between the NcoI and BamHI restriction sites of the polylinker. The Sso MCM was mutated at lysine 346 to alanine by PCR-based mutagenesis (32.Ho S.N. Hunt D.H. Horton R.M. Pullen J.K. Pease L.R. Gene. 1989; 77: 51-59Crossref PubMed Scopus (6849) Google Scholar) using the following synthetic oligonucleotides: MCM-Kpn-for (5′-ACCAGAAGAGGTACCCTCAGGTCAGTTACC-3′), MCM-Spe-rev (AGATTCTTAGCCTCACTAGTAATTTTTGGT), MCM-K346A-for (AATAGGTGATCCCGGTACTGCCGCATCACAAATGCTACAGTTTAT), MCM-K346A-rev (ATAAACTGTAGCATTTGTGATGCGGCAGTACCGGGATCACCTATT). The final PCR product was subcloned back into the SsoMCM-pET19b vector at the unique KpnI and SpeI sites present in the Sso MCM sequence. The presence of the desired mutation was checked by sequencing the KpnI-SpeI amplified DNA fragment. This allowed us to rule out the presence of additional mutations in this construction. The E. coli plasmid pET19b-SsoSSB expressing the recombinant S. solfataricus SSB was a generous gift of Dr. M. F. White (Saint Andrews, United Kingdom (31.Wadsworth R.I.M. White F.M. Nucleic Acids Res. 2001; 29: 914-920Crossref PubMed Scopus (115) Google Scholar)). E. coliBL21-CodonPlus(DE3)-RIL cells (Novagen) transformed with the plasmid pET19b-SsoMCM were grown at 37 °C in 2 liters of LB medium containing 100 μg/ml ampicillin and 100 μg/ml chloramphenicol. When the culture reached an A600 nm of 1 OD, protein expression was induced by addition of isopropyl-1-thio-β-d-galactopyranoside to 0.2 mm. The bacterial culture was incubated at 37 °C for an additional 1.5 h. Then cells were harvested by centrifugation, and the pellet was stored at −20 °C until use. The pellet was thawed and resuspended in 30 ml of buffer A (25 mm Tris-HCl, pH 7.0, 2.5 mm MgCl2) supplemented with some protease inhibitors (50 μg/ml phenylmethylsulfonyl fluoride, 0.2 μg/ml benzamidine, 1 μg/ml aprotinin). Cells were broken by two consecutive passages through a French pressure cell apparatus (Aminco Co., Silver Spring, MD) at 2000 p.s.i. The resulting lysate was centrifuged for 30 min at 30,000 rpm (Sorvall rotor 50.2 Ti) at 10 °C. The supernatant was subjected to heat treatment at 80 °C for 10 min, then incubated in ice for 10 min. The thermoprecipitated proteins were removed by centrifugation for 30 min at 30,000 rpm (Sorvall rotor 50,2 Ti) at 10 °C. The supernatant was passed through a 0.45-μm filter and loaded onto a Heparin Sepharose™ column (vol: 10 ml) pre-equilibrated in buffer A. After a washing step in buffer A, elution was carried out with 200 ml of a 0–1 mNaCl linear gradient in buffer A. 2.5-ml fractions were collected and analyzed by SDS-PAGE to detect the Sso MCM. Fractions containing the recombinant protein were pooled, and the pool was dialyzed overnight against buffer B (25 mm Tris-HCl, pH 8.5, 2.5 mm MgCl2, 50 mm NaCl). The dialyzed sample was then loaded onto a MonoQ HR 10/10 column pre-equilibrated in buffer B. A linear gradient from 0.05 to 1m NaCl in buffer B (volume, 80 ml) was applied to elute the protein. 1-ml fractions were collected and analyzed by SDS-PAGE to detect the Sso MCM. The peak fractions were pooled, concentrated using a Centricon 10 system (Millipore), and dialyzed overnight against buffer C (25 mm Tris-HCl, pH 8.09, 2.5 mm MgCl2, 100 mm NaCl). The dialyzed sample was aliquoted and stored at −20 °C. The final yield of the recombinant protein after this purification procedure was of about 30 mg. The K346A mutant Sso MCM was purified using the above protocol. The recombinant Sso SSB was purified as described (31.Wadsworth R.I.M. White F.M. Nucleic Acids Res. 2001; 29: 914-920Crossref PubMed Scopus (115) Google Scholar). Samples of the purified Sso MCM (263 μg in 50 μl) were subjected to analytical gel filtration chromatography on a Superdex 200 HR 26/30 fast protein liquid chromatography column (Amersham Biosciences, Inc.) equilibrated with buffer 50 mm Tris-HCl, pH 8.0, 20 mmMgCl2, 200 mm NaCl. The column was run at 0.25 ml/min at room temperature. 1-ml fractions were collected and analyzed by SDS-PAGE. Two sets of globular protein standards were run in the same conditions as the experiment. The first set included tyroglobulin (670 kDa), catalase (232 kDa), BSA (67 kDa). The second set included ferritin (440 kDa), aldolase (158 kDa), and ovalbumin (43 kDa). Samples of the purified Sso MCM (263 μg in 50 μl) were injected into a Superose 6 HR 10/30 fast protein liquid chromatography column (Amersham Biosciences, Inc.), equilibrated in 50 mmTris-HCl, pH 8.0, 10 mm MgCl2, 100 mm NaCl. The chromatographic run was carried out at a flow rate of 0.25 ml/min at room temperature. The column was calibrated by running a set of gel filtration markers that included tyroglobulin (670 kDa), ferritin (440 kDa), and BSA (67 kDa). A sample of the purified Sso MCM (186 μg in 100 μl) was applied to a 5-ml 15–35% glycerol gradient in 50 mm Tris-HCl, pH 8.0, 10 mm MgCl2, 100 mm NaCl. After centrifugation at 48,000 rpm for 15 h and 20 min in a Beckman SW 65 rotor at 10 °C, fractions (250 μl) were collected from the bottom of the tube. The distribution of the Sso MCM protein was detected after SDS/10% PAGE and staining with Coomassie Brilliant Blue (R-250). A mixture of protein markers (tyroglobulin, 670 kDa; ferritin, 440 kDa; BSA, 67 kDa) was applied to a parallel gradient. Standard ATPase assay reaction mixture (10 μl) contained 25 mm Hepes-NaOH, pH 7.5, 5 mmMgCl2, 50 mm sodium acetate, 2.5 mm 2-mercaptoethanol, 100 μm[γ-32P]ATP (0.5–1 μCi). Incubations were performed for 1 h at 60 °C in a heated-top PCR machine to prevent evaporation and stopped in ice. A 1-μl aliquot of each mixture was spotted onto a polyethyleneimine-cellulose thin layer plate (Merck), pre-run with 1 m formic acid and developed in 0.5m LiCl, 1 m formic acid. The amounts of [γ-32P]ATP hydrolyzed to [32P]orthophosphate were quantitated using a PhosphorImager (Molecular Dynamics, Inc.). The rate of ATP hydrolysis was determined in the linear range of reaction time and protein concentration dependence. The amount of spontaneously hydrolyzed ATP was determined using blank reactions without enzyme and subtracted from the reaction rate values calculated as above. Three DNA oligomers were synthesized and used for the preparation of helicase substrates. These included the following: a 55-mer (5′-CTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCGGGTACCGAGCTCGAATTCG-3′), which was fully complementary to the M13mp18(+) strand; a 64-mer (5′-TCACTTCTACTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCGGGTACCGAGCTCGAATTCG-3′), which had a 9-nt 5′-tail (the tail is underlined) not complementary to the M13mp18(+) strand; an 85-mer (5′-TTGAACCACCCCCTTGTTAAATCACTTCTACTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCGGGTACCGAGCTCGAATTCG), which had a 30-nt 5′-tail (the tail is underlined) not complementary to the M13mp18(+) strand. The oligonucleotides were labeled with [γ-32P]ATP and T4 polynucleotide kinase, and, after the labeling reaction, they were purified using Quantum Prep PCR Kleen Spin columns (Bio-Rad Laboratories), according to the manufacturer's instructions. To prepare partial duplexes, DNA molecules and mixtures containing equal molar amounts of each oligonucleotide and the M13mp18(+) strand were incubated for 5 min at 95 °C and then slowly cooled at room temperature. Helicase assay reaction mixtures (20 μl) contained 25 mm Hepes-NaOH, pH 7.5, 5 mm MgCl2, 50 mm sodium acetate, 2.5 mm 2-mercaptoethanol, 5 mm ATP, 50 fmol of32P-labeled substrate (about 1 × 103cpm/fmol). The reactions were incubated for 30 min at 70 °C in a heated-top PCR machine to prevent evaporation and stopped by addition of 5 μl of 5× stop solution (0.5% SDS, 40 mm EDTA, 0.5 mg/ml proteinase K, 20% glycerol, 0.1% bromphenol blue), then run on a 8% polyacrylamide gel in TBE containing 0.1% SDS at constant voltage of 150 V. After the electrophoresis the gel was soaked in 20% trichloroacetic acid and analyzed by means of a PhosphorImager (Molecular Dynamics, Inc.). The reaction products were quantitated, and any free oligonucleotide in the absence of enzyme was subtracted. The Sso SSB was diluted to a concentration of 1.4 ng/μl in carbonate buffer (16 mmNa2CO3, 34 mm NaHCO3, pH 9.6). This solution containing the Sso SSB was then added to the appropriate wells of a 96-well ELISA plate (100 μl/well) and allowed to incubate for 2 h at room temperature. For control experiments, BSA was substituted for the Sso SSB in the coating step. Wells were then aspirated and washed three times with wash Buffer (PBS, 0.5% Tween 20). Blocking buffer (PBS, 0.5% Tween 20, 3% BSA) was added to appropriate wells and allowed to incubate at room temperature. Wells were aspirated and washed one time with blocking buffer. The Sso MCM was diluted to 1.8 ng/μl in binding buffer (25 mm Hepes-NaOH, pH 7.5, 50 mmNaCl, 2.5 mm 2-mercaptoethanol, 5 mmMgCl2). The diluted Sso MCM was then added to appropriate wells of the ELISA plate (100 μl/well) and allowed to incubate for 30 min at room temperature. Wells were aspirated and washed three times with binding buffer. Primary antibody (rabbit polyclonal antiserum against the Sso MCM protein) was diluted 1:2000 in blocking buffer, added to appropriate wells, and allowed to incubate overnight at 4 °C. Wells were then aspirated and washed four times with blocking buffer. Secondary antibody (goat anti-rabbit IgG-horseradish peroxidase) was diluted 1:3000 in conjugate buffer (50 mm Tris-HCl, pH 8.0, 150 mm NaCl, 0.05% Tween 20, 1% BSA), added to appropriate wells, and allowed to incubate 30 min at room temperature. Wells were aspirated and washed five times with conjugate buffer. Complexes were detected using a colorimetric reaction with o-phenylenediamine dihydrochloride (Sigma Chemical Co.). The reaction was terminated after 1 min with 2.5 m sulfuric acid. Absorbance readings were taken at 490 nm. The A490 values, corrected for background signal in the presence of BSA, are expressed as the mean of three independent determinations. Far Western blotting analysis was conducted as described by Wu et al. (33.Wu L. Davies S.L. North P.S. Goulaouic H. Riou J.F. Turley H. Gatter K.C. Hickson I.D. J. Biol. Chem. 2000; 275: 9636-9646Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar). Each polypeptide was subjected to SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (Schleicher and Schuell). All subsequent steps were performed at 4 °C. Filters were immersed twice in denaturation buffer (6 m guanidine-HCl in TBS) for 10 min followed by six times for 10 min in serial dilutions (1:1) of denaturation buffer supplemented with 1 mm dithiothreitol. Filters were blocked in TBS containing 3% BSA, 0.3% Tween 20 for 30 min before being incubated in the Sso MCM (6 μg/ml) in TBS supplemented with 0.1% BSA, 0.3% Tween 20, 1 mmdithiothreitol overnight at 4 °C. Filters were washed four times for 10 min in TBS containing 0.3% Tween 20, 0.1% BSA. The second wash contained 0.0001% glutaraldehyde. Conventional Western analysis was then performed to detect the presence of the Sso MCM using rabbit polyclonal antiserum against the Sso MCM as primary antibody. Anti-rabbit IgG-horseradish peroxidase conjugate was used as secondary antibody and detected by a colorimetric reaction. The analysis of the S. solfataricus genomic sequence revealed the presence of a single open reading frame coding for a putative homolog of the eukaryotic MCM proteins (Sso MCM (27.She Q. Singh R.K. Confalonieri F. Zivanovic Y. Allard G. Awayez M.J. Chan-Weiher C.C. Clausen I.G. Curtis B.A. De Moors A. Erauso G. Fletcher C. Gordon P.M. Heikamp-de Jong I. Jeffries A.C. Kozera C.J. Medina N. Peng X. Thi-Ngoc H.P. Redder P. Schenk M.E. Theriault C. Tolstrup N. Charlebois R.L. Doolittle W.F. Duguet M. Gaasterland T. Garrett R.A. Ragan M.A. Sensen C.W. Van der Oost J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7835-7840Crossref PubMed Scopus (672) Google Scholar)). Using the computer program ClustalW (34.Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (55958) Google Scholar) we aligned the sequence of the single MCM protein from the Archaea S. solfataricus, A. pernix (Ape MCM), and M. thermoautotrophicum (Mth MCM), and the sequence of the MCM 4 protein from the eukaryotes H. sapiens(Hsa MCM) and Schizosaccharomyces pombe(Spo MCM) and found that the Sso MCM is 48, 43, 33, and 34% identical to Ape, Mth, Hsa, and Spo MCM, respectively. As schematically depicted in Fig. 1, the SsoMCM lacks the N-terminal extension of about 160 amino acidic residues found in the eukaryotic counterparts, and it is devoid of the cyclin-dependent kinase phosphorylation sites that are clustered in the N-terminal region of the eukaryotic MCM 4, as also found in the Ape and Mth MCM (3.Kearsey S.E. Labib K. Biochim. Biophys. Acta. 1998; 1398: 113-136Crossref PubMed Scopus (230) Google Scholar, 4.Tye B.K. Annu. Rev. Biochem. 1999; 68: 649-686Crossref PubMed Scopus (515) Google Scholar, 22.Shechter D.F. Ying C.Y. Gautier J. J. Biol. Chem. 2000; 275: 15049-15059Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). On the other hand, the core region of the archaeal and eukaryal sequences shows a higher level of similarity, because it contains the four amino acidic motifs typically found in the DNA helicases (35.Koonin E.V. Nucleic Acids Res. 1993; 21: 2541-2547Crossref PubMed Scopus (341) Google Scholar). As evidenced in Fig. 1, the sequence boxes A and B correspond to the Walker A and B motifs that are responsible for nucleotide binding and hydrolysis, respectively, in the large family of ATPases associated with a variety of cellular activities (AAA+ super-family (5.Neuwald A.L.A. Spouge J. Koonin E.V. Genome Res. 1999; 9: 27-43Crossref PubMed Google Scholar)). In addition, the Sulfolobus and Aeropyrum MCM sequences seem to contain a zinc finger of the His-Cys3type, whereas the MCM proteins from eukaryotic organisms (3.Kearsey S.E. Labib K. Biochim. Biophys. Acta. 1998; 1398: 113-136Crossref PubMed Scopus (230) Google Scholar, 4.Tye B.K. Annu. Rev. Biochem. 1999; 68: 649-686Crossref PubMed Scopus (515) Google Scholar), Methanobacterium, and other euryarchaeal species (36.Poplawski A. Grabowski B. Long S.E. Kelman Z. J. Biol. Chem. 2001; 276: 49371-49377Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) possess a zinc finger of the Cys4 type. The gene encoding the Sso MCM was produced in E. coli using the pET19b plasmid vector. The recombinant protein was found to be expressed at high level in soluble form and was purified by a procedure that included a thermal treatment of the cell extracts and chromatographic steps on heparin-Sepharose and MonoQ columns, as described under "Experimental Procedures." The purified Sso MCM migrated as a Coomassie Blue-stained protein band of the predicted size (77 kDa) in a 10% SDS-polyacrylamide gel (see Fig. 2A). An additional tiny band of about 60 kDa is observed in the sample collected from the MonoQ column. N-terminal sequence analyses after electrotransfer onto a polyvinylidene difluoride membrane revealed that the 77-kDa polypeptide corresponds to the Sso MCM, whereas the 60-kDa band is a C-terminally truncated proteolytic fragment of the intact protein. 2F. Carpentieri et al., unpublished observations. To assess the oligomeric state of the recombinant Sso MCM, we carried out gel filtration experiments onto two columns: a Superose 62 and a Superdex 200 (Fig. 2, B and C). By either experiment we estimated a molecular mass of about 470 kDa for the Sso MCM and hypothesized that it could form hexamers in solution. However, as shown in Fig. 2B, the peak eluted from the Superdex 200 column was quite broad and a portion of the protein was detected in fractions that corresponded to a molecular mass of about 75 kDa, as expected for the monomeric form of the Sso MCM. This result suggested that the SsoMCM could exist in equilibrium between an hexameric and a monomeric state. Consistent results were obtained also by glycerol gradient centrifugation experiments: as shown in Fig. 2D, the Sso MCM was detected by SDS-polyacrylamide gel electrophoresis in the fractions from 8 to 13 of the gradient indicating that the protein assembles into hexamers that dissociate into monomers. The oligomeric state of the Sso MCM was not found to be affected by addition of ATP (at 100 μm) in the buffer used to prepare the gradient or by preincubating the protein sample with forked double- or single-stranded DNA molecules before starting the ultracentrifugation.2 As shown in Fig. 1, Walker A and B motifs are present in the primary
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