A Unique Organization of the Protein Subunits of the DNA Polymerase Clamp Loader in the Archaeon Methanobacterium thermoautotrophicum ΔH
2000; Elsevier BV; Volume: 275; Issue: 10 Linguagem: Inglês
10.1074/jbc.275.10.7327
ISSN1083-351X
Autores Tópico(s)Genomics and Chromatin Dynamics
ResumoReplication factor C (RFC, also called activator 1), in conjunction with proliferating cell nuclear antigen (PCNA), is responsible for processive DNA synthesis catalyzed by the eukaryotic replicative DNA polymerases δ and ε. Here we report the isolation and characterization of homologues of RFC and PCNA from the archaeon,Methanobacterium thermoautotrophicum ΔH. In contrast to the five subunit RFC complex isolated from eukaryotic cells, the mthRFC contains only two subunits. The two genes encoding the RFC subunits called, mthRFC1 and mthRFC3, were cloned, and the proteins (54.4 and 36.8 kDa, respectively) were overexpressed in Escherichia coli and purified individually and as a complex. The gene encoding PCNA was also cloned, and the protein was purified after overexpression in E. coli. Based on sizing column elution and subunit composition, the mthRFC complex appears to be a hexamer consisting of two mthRFC1 protomers and four mthRFC3protomers. Although mthRFC differs in organization from its eukaryotic counterpart, it was shown to be functionally similar to eukaryotic RFC in: (i) catalyzing DNA-dependent ATP hydrolysis; (ii) binding preferentially to DNA primer ends; (iii) loading mthPCNA onto singly nicked circular DNA; and (iv) supporting mthPolB-catalyzed PCNA-dependent DNA chain elongation. The importance and roles of RFC and PCNA in M. thermoautotrophicum ΔH replication are discussed. Replication factor C (RFC, also called activator 1), in conjunction with proliferating cell nuclear antigen (PCNA), is responsible for processive DNA synthesis catalyzed by the eukaryotic replicative DNA polymerases δ and ε. Here we report the isolation and characterization of homologues of RFC and PCNA from the archaeon,Methanobacterium thermoautotrophicum ΔH. In contrast to the five subunit RFC complex isolated from eukaryotic cells, the mthRFC contains only two subunits. The two genes encoding the RFC subunits called, mthRFC1 and mthRFC3, were cloned, and the proteins (54.4 and 36.8 kDa, respectively) were overexpressed in Escherichia coli and purified individually and as a complex. The gene encoding PCNA was also cloned, and the protein was purified after overexpression in E. coli. Based on sizing column elution and subunit composition, the mthRFC complex appears to be a hexamer consisting of two mthRFC1 protomers and four mthRFC3protomers. Although mthRFC differs in organization from its eukaryotic counterpart, it was shown to be functionally similar to eukaryotic RFC in: (i) catalyzing DNA-dependent ATP hydrolysis; (ii) binding preferentially to DNA primer ends; (iii) loading mthPCNA onto singly nicked circular DNA; and (iv) supporting mthPolB-catalyzed PCNA-dependent DNA chain elongation. The importance and roles of RFC and PCNA in M. thermoautotrophicum ΔH replication are discussed. replication factor C proliferating cell nuclear antigen polymerase polyacrylamide gel electrophoresis M. thermoautotrophicum ΔH single-stranded circular dithiothreitol isopropyl-1-thio-β-d-galactopyranoside bovine serum albumin single-stranded DNA S. pombe replication protein A gene product Replication factor C (RFC,1 also known as activator 1) and proliferating cell nuclear antigen (PCNA) are two accessory factors that are required for processive DNA synthesis catalyzed by the eukaryotic DNA polymerases (pol) δ and ε (1.Jonsson Z.O. Hubscher U. Bioessays. 1997; 19: 967-975Crossref PubMed Scopus (218) Google Scholar, 2.Kelman Z. Oncogene. 1997; 14: 629-640Crossref PubMed Scopus (720) Google Scholar, 3.Waga S. Stillman B. Annu. Rev. Biochem. 1998; 67: 721-751Crossref PubMed Scopus (663) Google Scholar). Following its association with DNA at a primer-template junction coated with a single-stranded DNA-binding protein, RFC catalyzes the assembly of the ring-shaped PCNA (also referred to as a DNA sliding clamp (4.Huang C.C. Hearst J.E. Alberts B.M. J. Biol. Chem. 1981; 256: 4087-4094Abstract Full Text PDF PubMed Google Scholar)) around the primer DNA in an ATP-dependent manner. For this reason, RFC is referred to as a clamp loader. Polδ and -ε are recruited to this protein-DNA complex and tethered to the DNA primer-template junction through their interaction with PCNA. The resulting complex (polδ or polε holoenzyme) is then capable of catalyzing highly processive DNA synthesis. The subunit structure of RFC is highly conserved in all eukaryotes from yeast to humans (5.Cullmann G. Fien K. Kobayashi R. Stillman B. Mol. Cell. Biol. 1995; 15: 4661-4671Crossref PubMed Scopus (213) Google Scholar). It contains five subunits that range in size between 36 and 140 kDa as revealed by SDS-PAGE analysis (Fig.1 A). Genes encoding each of these subunits have been cloned from both mammals and Saccharomyces cerevisiae, and each gene encoding each subunit has been shown to be essential in yeast by deletion analysis (5.Cullmann G. Fien K. Kobayashi R. Stillman B. Mol. Cell. Biol. 1995; 15: 4661-4671Crossref PubMed Scopus (213) Google Scholar). The predicted amino acid sequence of each subunit of the yeast and human RFC reveals significant homology in seven regions commonly referred to as RFC boxes (box II–VIII) (Fig. 1,A and B) (1.Jonsson Z.O. Hubscher U. Bioessays. 1997; 19: 967-975Crossref PubMed Scopus (218) Google Scholar, 5.Cullmann G. Fien K. Kobayashi R. Stillman B. Mol. Cell. Biol. 1995; 15: 4661-4671Crossref PubMed Scopus (213) Google Scholar). The large subunit (p140, RFC1) contains an additional RFC box (box I) within its N-terminal region that shares homology with prokaryotic DNA ligases. Archaea, the third domain of life (6.Woese C.R. Fox G.E. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5088-5090Crossref PubMed Scopus (2458) Google Scholar), are believed to replicate DNA in a eukaryotic-like fashion. This conclusion is based in large part on the amino acid sequences of several members of this domain (7.Kawarabayasi Y. Sawada M. Horikawa H. Haikawa Y. Hino Y. Yamamoto S. Sekine M. Baba S. Kosugi H. Hosoyama A. Nagai Y. Sakai M. Ogura K. Otsuka R. Nakazawa H. Takamiya M. Ohfuku Y. Funahashi T. Tanaka T. Kudoh Y. Yamazaki J. Kushida N. Oguchi A. Aoki K. Kikuchi H. DNA Res. 1998; 5: 55-76Crossref PubMed Scopus (554) Google Scholar, 8.Klenk H.P. Clayton R.A. Tomb J.F. White O. Nelson K.E. Ketchum K.A. Dodson R.J. Gwinn M. Hickey E.K. Peterson J.D. Richardson D.L. Kerlavage A.R. Graham D.E. Kyrpides N.C. Fleischmann R.D. Quackenbush J. Lee N.H. Sutton G.G. Gill S. Kirkness E.F. Dougherty B.A. McKenney K. Adams M.D. Loftus B. Peterson S. Reich C.I. McNeil L.K. Badger J.H. Glodek A. Zhou L. Overbeek R. Gocayne J.D. Weidman J.F. McDonald L. Utterback T. Cotton M.D. Spriggs T. Artiach P. Kaine B.P. Sykes S.M. Sadow P.W. D'Andrea K.P. Bowman C. Fujii C. Garland S. Mason T. Olsen G.J. Fraser C.M. Smith H.O. Woese C.R. Venter J.C. Nature. 1997; 390: 364-370Crossref PubMed Scopus (1204) Google Scholar, 9.Smith D.R. Doucette-Stamm L.A. Deloughery C. Lee H. Dubois J. Aldredge T. Bashirzadeh R. Blakely D. Cook R. Gilbert K. Harrison D. Hoang L. Keagle P. Lumm W. Pothier B. Qiu D. Spadafora R. Vicaire R. Wang Y. Wierzbowski J. Gibson R. Jiwani N. Caruso A. Bush D. Safer H. Patwell D. Prabhakar S. McDougall S. Shimer G. Goyal A. Pietrokovski S. Church G.M. Daniels C.J. Mao J-I. Rice P. Nölling J. Reeve J.N. J. Bacteriol. 1997; 179: 7135-7155Crossref PubMed Scopus (1038) Google Scholar, 10.Bult C.J. White O. Olsen G.J. Zhou L. Fleischmann R.D. Sutton G.G. Blake J.A. FitzGerald L.M. Clayton R.A. Gocayne J.D. Kerlavage A.R. Dougherty B.A. Tomb J.F. Adams M.D. Reich C.I. Overbeek R. Kirkness E.F. Weinstock K.G. Merrick J.M. Glodek A. Scott J.L. Geoghagen N.S.M. Venter J.C. Science. 1996; 273: 1058-1073Crossref PubMed Scopus (2285) Google Scholar). Interestingly, only two genes with homology to RFC have been identified within these genomes. These two putative archaeal RFC subunits are similar to the eukaryotic RFC3 and the C-terminal region of RFC1 (Fig.1, A and B). The archaea RFC1 homologue, however, lacks box I, the DNA ligase domain, at its N-terminal region (Fig.1 A). One of the archaea for which the complete sequence has been reported isMethanobacterium thermoautotrophicum ΔH (9.Smith D.R. Doucette-Stamm L.A. Deloughery C. Lee H. Dubois J. Aldredge T. Bashirzadeh R. Blakely D. Cook R. Gilbert K. Harrison D. Hoang L. Keagle P. Lumm W. Pothier B. Qiu D. Spadafora R. Vicaire R. Wang Y. Wierzbowski J. Gibson R. Jiwani N. Caruso A. Bush D. Safer H. Patwell D. Prabhakar S. McDougall S. Shimer G. Goyal A. Pietrokovski S. Church G.M. Daniels C.J. Mao J-I. Rice P. Nölling J. Reeve J.N. J. Bacteriol. 1997; 179: 7135-7155Crossref PubMed Scopus (1038) Google Scholar). M. thermoautotrophicum ΔH is an obligatory anaerobic thermophilic microorganism with an optimal growth temperature of 65–70 °C and a generation time of about 5 h (11.Zeikus J.G. Wolfe R.S. J. Bacteriol. 1972; 109: 707-715Crossref PubMed Google Scholar). Based on sequence similarities to known RFCs, two putative RFC homologues have been identified within its genome: a RFC1 homologue with a calculated molecular mass of 54.4 kDa and 35% identity to the corresponding region in human RFC1 and a homologue of human RFC3 with a calculated molecular mass of 36.8 kDa and 22% identity to the human protein (data not shown). A homologue of PCNA has also been identified in the genome of M. thermoautotrophicum ΔH with a calculated molecular mass of 28.8 kDa and 29% identity to the human protein. In contrast to RFC homologues from other archaea (7.Kawarabayasi Y. Sawada M. Horikawa H. Haikawa Y. Hino Y. Yamamoto S. Sekine M. Baba S. Kosugi H. Hosoyama A. Nagai Y. Sakai M. Ogura K. Otsuka R. Nakazawa H. Takamiya M. Ohfuku Y. Funahashi T. Tanaka T. Kudoh Y. Yamazaki J. Kushida N. Oguchi A. Aoki K. Kikuchi H. DNA Res. 1998; 5: 55-76Crossref PubMed Scopus (554) Google Scholar, 10.Bult C.J. White O. Olsen G.J. Zhou L. Fleischmann R.D. Sutton G.G. Blake J.A. FitzGerald L.M. Clayton R.A. Gocayne J.D. Kerlavage A.R. Dougherty B.A. Tomb J.F. Adams M.D. Reich C.I. Overbeek R. Kirkness E.F. Weinstock K.G. Merrick J.M. Glodek A. Scott J.L. Geoghagen N.S.M. Venter J.C. Science. 1996; 273: 1058-1073Crossref PubMed Scopus (2285) Google Scholar), no inteins are found in the RFC proteins from M. thermoautotrophicum ΔH. In this report, we describe the isolation and the biochemical characterization of the RFC complex from the archaeon, M. thermoautotrophicum ΔH. Recombinant proteins were expressed and purified from Escherichia coli cells, and the properties of the isolated RFC were studied in vitro. mthRFC contains two subunits that appear to form a heterohexamer consisting of two copies of the RFC1 subunit and four copies of the RFC3 subunit. As with RFC isolated from eukaryotic cells, mthRFC possesses a DNA-dependent ATPase activity that is stimulated by PCNA. As expected of a clamp loader, mthRFC can load the M. thermoautotrophicum ΔH homologue of PCNA around DNA in an ATP-dependent manner. It is also demonstrated that mthRFC and mthPCNA can jointly stimulate the activity of a B-type DNA polymerase of M. thermoautotrophicum ΔH. Labeled deoxy- and ribonucleoside triphosphates were obtained from Amersham Pharmacia Biotech. Unlabeled deoxyribonucleoside triphosphates were from Amersham Pharmacia Biotech. Single-stranded circular (ssc) M13mp19 was from Life Technologies, Inc., φx174 sscDNA was from New England Biolabs, poly(dA)300 was from Amersham Pharmacia Biotech, oligonucleotides were synthesized by Gene Link (Hawthorne, NY), and polynucleotides were from Supertechs (Bethesda, MD). The various pET vectors used were from Novagene. Rabbit polyclonal antibodies were generated by Cocalico Biologicals Inc. (Reamstown, PA).Schizosaccharomyces pombe PCNA and singly primed M13 ssDNA were prepared as described previously (12.Kelman Z. Zuo S. Arroyo M.P. Wang T.S. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9515-9520Crossref PubMed Scopus (17) Google Scholar) as was singly nicked pBS DNA (13.Gibbs E. Kelman Z. Gulbis J.M. O'Donnell M. Kuriyan J. Burgers P.M.J. Hurwitz J. J. Biol. Chem. 1997; 272: 2373-2381Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Briefly, singly nicked pBluescript DNA was prepared by DNase I treatment in the presence of ethidium bromide. Analysis of the product by alkaline agarose gel electrophoresis indicated the presence of an equal mixture of linear and circular DNA molecules; neutral agarose gel electrophoresis in the presence of ethidium bromide indicated that the DNA had been quantitatively converted to an RF II structure. mthPolB and mthRPA were purified as described (14.Kelman Z. Pietrokovski S. Hurwitz J. J. Biol. Chem. 1999; 274: 28751-28761Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). The buffers used and their composition were: buffer A, which contained 20 mm Tris-HCl (pH 7.5), 2 mm DTT, 0.5 mm EDTA, and 10% glycerol; buffer B, which contained 25 mm potassium phosphate (pH 7.5), 500 mm KCl, 2 mm DTT, and 10% glycerol; buffer C, which contained 50 mm Tris-HCl (pH 8.0), 500 mm NaCl, and 10% glycerol; and buffer D, which contained 50 mm Tris-HCl (pH 8.0), 500 mm KCl, and 10% glycerol. RFC1, RFC3, and PCNA genes (MTH240, MTH241, andMTH1312, respectively) were amplified by polymerase chain reaction from M. thermoautotrophicum ΔH DNA (kindly provided by John Reeve, Ohio State University) and were cloned, after sequencing, between the NdeI and XhoI (RFC1) or the NdeI and BamHI (RFC3 and PCNA) sites of the bacterial expression vector pET-16b (Novagene; called pET16-RFC1, pET16-RFC3, and pET16-PCNA). The oligonucleotide primers used in the polymerase chain reaction reactions were as follow: for the cloning of RFC1, the oligonucleotides Z102 (5′-GGGTCGACCATATGTCATGGACAGAGAAATACCGGCC-3′) and Z103 (5′-GGAAGCTTCTCGAGTTATGAGAACTGGAAGAGTGACGTC-3′), which contained NdeI and XhoI sites (underlined), respectively; for the cloning of RFC3, the oligonucleotides Z104 (5′-GGGGATCCCATATGATCATTATGAACGGACCTTGGG-3′) and Z105 (5′-GGGGATCCGTCGACTTAGGCGTGTTCAAGGAACCTTGCAAGG-3′), which contained NdeI and BamHI sites (underlined), respectively; for the cloning of PCNA, the oligonucleotides Z100 (5′-GGGGATCCCATATGTTCAAGGCAGAATTGAATGACCC-3′) and Z101 (5′-GGGGATCCGTCGACTTATTCCTCTGCCTCTATTCTTGGAGC-3′), which contained NdeI and BamHI sites (underlined), respectively. The NdeI site contains the initiation codon ATG and XhoI orBamHI sites are immediately after the stop codon. These constructs contained a His10 tag at the N terminus of their respective proteins. RFC3 was also cloned into pET-21a (Novagene; called pET21-RFC3) using the same restriction sites. A vector that expressed both subunits of RFC, RFC1, and RFC3 was generated as follows. A BglII-XhoI fragment of pET16-RFC1 that contained the entire coding region and the upstream regulatory sequences (the T7 promoter and the ribosome binding sites) was cloned between the BamHI and XhoI sites of pET21-RFC3. Thus, though the new vector (called pET21-RFC) expressed both subunits of RFC, only the large subunit, RFC1, contained a His10tag. The PCNA gene was also cloned between the NdeI andBamHI sites of pHKEp vector (15.Kelman Z. Yao N. O'Donnell M. Gene (Amst.). 1995; 166: 177-178Crossref PubMed Scopus (43) Google Scholar). This construct contained a His10 tag, a cAMP-dependent protein kinase recognition motif, and a hemagglutinin epitope at the N terminus. M. thermoautotrophicum ΔH RFC1, RFC3, and PCNA proteins were overexpressed as follows: 2 liters of E. coli cells BL21(DE3) pLysS (Novagene), harboring the different plasmids, were grown at 37 °C in Luria-Bertani (LB) medium in the presence of appropriate antibiotics. When the culture reached anA 600 of 0.6, protein expression was induced by incubation in the presence of 2 mm IPTG for 3 h after which time the cells were harvested. All subsequent steps used for the isolation of proteins were carried out at 4 °C. The isolation of the RFC3 subunit was carried out with 2 liters of cells (7.2 g wet weight) expressing the RFC3 subunit. Bacterial lysates were prepared by sonication (five 1-min pulses using the Sonic Dismembrator 500 (Fisher Scientific)) of cells in 25 ml of buffer C. After centrifugation for 20 min at 36,000 × g, the extract (350 mg of protein) was mixed with 1 ml of nickel chelate (ProBound resin, Invitrogen) for 2 h with gentle shaking. The mixture was then poured onto a column and washed with 25 ml of buffer C containing 20 mmimidazole, and bound protein was eluted with 3 ml of buffer C containing 500 mm imidazole. The latter fraction (20 mg of protein) was dialyzed for 3–4 h against 2 liters of buffer A containing 500 mm NaCl. Longer periods of dialysis (12 h) resulted in marked precipitation of the protein. When the salt concentration of the dialyzing fluid was reduced to 0.3 mNaCl, the RFC3 subunit precipitated from solution after 2 h of dialysis. The isolation of the RFC1 subunit was carried out with 2 liters of cells (6.8 g wet weight) expressing the RFC1 subunit. In contrast to the expression of RFC3, RFC1 was not soluble under the conditions that were used for the extraction of RFC3 from cells. For this reason, the isolation of RFC1 was carried out in the presence of urea as follows: bacterial lysates were prepared by sonication in 25 ml of buffer C containing 6m urea as described for the isolation of RFC3. After centrifugation for 20 min at 36,000 × g, the extract (300 mg of protein) was mixed with 1 ml of nickel chelate (ProBound resin, Invitrogen) for 2 h with gentle shaking. The mixture was then loaded onto a column, washed with 2.5 ml of buffer C containing 6m urea and 20 mm imidazole, and eluted with 3 ml of buffer C containing 6 m urea plus 500 mmimidazole. The protein eluted in this step (3 mg) was dialyzed overnight against 2 liters of buffer A containing 6 murea. The two-subunit RFC complex was isolated from 12 liters of E. coli cells (14.4 g wet weight) containing the plasmid pET21-RFC. The simultaneous expression of both RFC1 and RFC3 resulted in the detection of soluble RFC1 as well as soluble RFC3. Cells were suspended in 50 ml of buffer C and sonicated as described above. After centrifugation for 20 min at 36,000 × g, the soluble extract (1.02 g of protein) was mixed with 2 ml of nickel chelate for 2 h with gentle shaking. The mixture was poured onto a column, washed with 25 ml of buffer C containing 20 mm imidazole and then washed with 10 ml of buffer C containing 500 mmimidazole. The protein eluted after the latter step (25 mg) was dialyzed 12 h against 2 liters of buffer A containing 500 mm KCl. No visible precipitation occurred during the dialysis step. The dialyzed material was loaded onto a 5-ml Econo-Pac CHT-II column (Bio-Rad) preequilibrated with buffer B. The column was washed with 25 ml of buffer B and developed with a 50-ml linear gradient of potassium phosphate buffer (pH 7.5) from 25 to 300 mm in buffer B. The protein peak, which eluted at 200 mm potassium phosphate, was pooled and dialyzed overnight against 2 liters of buffer A containing 500 mm NaCl (see Fig. 2 C) yielding 3 mg of protein (which hydrolyzed a total of 8850 nmol of ATP/30 min at 50 °C in the presence of φX174 sscDNA in the ATPase assay described below). Dialysis against buffers containing lower salt concentrations resulted in substantial protein precipitation and loss of enzymatic activity. This fraction, which was used in all experiments described below, was aliquoted and stored at −80 °C. The activity associated with this fraction (measured either in the DNA-dependent ATPase or in the elongation of a singly primed M13 ssDNA template assays as described below) was stable to repeated freezing and thawing and retained its full activity after 1 year of storage at −80 °C. PCNA was purified as described above using the procedure described for the isolation of RFC3 using (7.4 g wet weight) of E. coli cells. The eluted protein fraction from the nickel chelate column (30 mg of protein) was diluted 3-fold with buffer A and loaded onto a 5-ml HiTrap-Q column (Amersham Pharmacia Biotech) equilibrated with buffer A containing 100 mm NaCl. The column was washed with 25 ml of buffer A containing 100 mm NaCl and then developed with a 50-ml linear gradient of NaCl from 100 to 600 mm in buffer A. The pooled protein peak, which eluted at 480 mm NaCl (15.5 mg), was dialyzed overnight against 2 liters of buffer A containing 100 mm NaCl (see Fig. 4 A). Protein concentrations were determined by the Bradford assay (Bio-Rad) using bovine serum albumin (BSA) as the standard. mthPCNA containing a cAMP-dependent protein kinase recognition site at its N terminus was phosphorylated with [γ-32P]ATP as described previously (16.Kelman Z. Naktinis V. O'Donnell M. Methods Enzymol. 1995; 262: 430-442Crossref PubMed Scopus (47) Google Scholar). The mthRFC-catalyzed loading of mthPCNA onto DNA was carried out in reaction mixtures (50 μl) containing 20 mm Tris-HCl (pH 7.5), 8 mm MgCl2, 5 mm DTT, 0.1 mm EDTA, 80 μg/ml BSA, 4% glycerol, 1.5 μg (0.8 pmol as molecules) of singly nicked pBluescript DNA, 4 mm ATP, 0.3 μg of mthRFC, and 3.3 pmol of32P-labeled PCNA. After 10 min of incubation at the temperature indicated in the figure legend, reaction mixtures were filtered at 4 °C through a 5-ml Bio-Gel A15 m (Bio-Rad) column preequilibrated with buffer containing 20 mm Tris-HCl (pH 7.5), 100 mm NaCl, 2 mm DTT, 0.5 mmEDTA, 50 μg/ml BSA, and 5% glycerol. The column resolved PCNA bound to DNA (eluted in the excluded volume) from PCNA in solution (eluted in the included volume). Fractions (210 μl) were collected, and the presence of PCNA was quantitated by Cerenkov counting. Under the conditions used, the loading of PCNA onto DNA was proportional to the amount of protein up to 1.0 μg of mthRFC. Above this amount of protein, PCNA loading decreased probably because of the unloading of PCNA by RFC (17.Yao N. Turner J. Kelman Z. Stukenberg P.T. Dean F. Shechter D. Pan Z.Q. Hurwitz J. O'Donnell M. Genes Cells. 1996; 1: 101-113Crossref PubMed Scopus (181) Google Scholar). mthPolB catalyzed elongation of singly primed M13 single-stranded DNA was carried out in reaction mixtures (20 μl) containing 40 mm Tris-HCl (pH 7.5), 0.5 mm DTT, 0.01% BSA, 7 mm magnesium acetate, 2 mm ATP, 100 μm each of dCTP, dGTP, and dTTP, 20 μm[α-32P]dATP (0.5–2 × 104 cpm/pmol), 12 fmol of singly primed M13 DNA (primed with M13–1 primer, map position 5999–6033), either 0.25 or 0.3 m NaCl (as indicated) and other proteins as indicated in the figure legend. Reaction mixtures were incubated for 30 min at 55 °C, stopped with 10 mm EDTA, and separated by electrophoresis through an alkaline agarose gel (1.5%) followed by autoradiography. For quantitation, an aliquot (2 μl) of the reaction mixture was removed, and the amount of DNA synthesis was measured by adsorption to DE81 paper. ATPase assays were carried out in reaction mixtures (30 μl) containing 20 mm Hepes-NaOH (pH 7.5), 2 mm DTT, 50 μg/ml BSA, 2 mm MgCl2, 100 mm NaCl, 50 μm [γ-32P]ATP (3–5 × 103 cpm/pmol) and DNAs and proteins as indicated in the figure legends. Reaction mixtures were incubated at 50 °C and analyzed by thin layer chromatography on polyethyleneimine (PEI) Cellulose F plates (EM Sciences, Gibbstown, NJ) using the solvent 0.5 m LiCl plus 1 m HCOOH, which readily separated Pi from ATP. The hydrolysis of different nucleoside triphosphates by mthRFC was carried out under similar assay conditions in the presence or absence of 110 ng of φx174 sscDNA and 50 μm [α-32P]ribo- or deoxynucleoside triphosphates. To demonstrate that the enzymatic activities observed with preparations of mthRFC were not because of contamination with E. coli proteins, a portion of the purified protein fraction (HiTrap-Q column fraction, 90 μg in 100 μl buffer A containing 500 mm NaCl) was applied to a Superdex-200 gel-filtration column (Amersham Pharmacia Biotech) equilibrated with buffer A containing 500 mm NaCl. Fractions (250 μl) were collected and analyzed for the presence of RFC protein and activity. The distribution of RFC was detected following 12% SDS-PAGE and staining with Coomassie Brilliant Blue (R-250). Two assays were used to followed the elution of RFC. One assay measured the RFC and PCNA-dependent replication of singly primed M13 ssDNA by mthPolB (using 0.5 μl of each fraction diluted 10 and 100-fold in buffer A containing 500 mm NaCl). Reaction mixtures were as described for the elongation of singly primed M13 DNA template by mthPolB and contained 250 mm NaCl, 250 ng PCNA, 0.65 μg mthRPA, 12 fmol of singly primed M13 ssDNA, and 0.576 pmol of mthPolB. Reactions were incubated at 50 °C for 30 min and analyzed as described above. The second enzymatic assay used measured the RFC-catalyzed hydrolysis of [γ-32P]ATP (RFC-catalyzed hydrolysis of nucleoside triphosphates). An aliquot (1 μl) of each fraction from the Superdex-200 column was incubated in a reaction mixture (30 μl) containing 110 ng of φx174 sscDNA for 30 min at 50 °C. Gel-filtration analysis was also used to determine the oligomeric structure of mthPCNA. Ninety μg of mthPCNA or S. pombePCNA in 100 μl of buffer A containing 100 mm NaCl (10 μm PCNA as trimer) was applied to a Superdex-200 gel filtration column equilibrated with buffer A containing 100 mm NaCl. Fractions (250 μl) were collected, and the elution profile of PCNA was determined following 12% SDS-PAGE analysis of each fraction (20 μl) and staining with Coomassie Brilliant Blue (R-250). To determine whether the dimeric mthRFC isolated was an enzymatically active complex and whether its biochemical properties are similar to those of RFC isolated from eukaryotic cells, its subunits (RFC1 and RFC3) as well as the complex were purified and characterized. The PCNA homologue from M. thermoautotrophicum ΔH was also purified. The genes encoding RFC1 and RFC3 (open reading frames MTH240 and MTH241, respectively (9.Smith D.R. Doucette-Stamm L.A. Deloughery C. Lee H. Dubois J. Aldredge T. Bashirzadeh R. Blakely D. Cook R. Gilbert K. Harrison D. Hoang L. Keagle P. Lumm W. Pothier B. Qiu D. Spadafora R. Vicaire R. Wang Y. Wierzbowski J. Gibson R. Jiwani N. Caruso A. Bush D. Safer H. Patwell D. Prabhakar S. McDougall S. Shimer G. Goyal A. Pietrokovski S. Church G.M. Daniels C.J. Mao J-I. Rice P. Nölling J. Reeve J.N. J. Bacteriol. 1997; 179: 7135-7155Crossref PubMed Scopus (1038) Google Scholar)) were inserted individually and together into E. coli expression vectors and expressed as fusion proteins containing N-terminal His10 tags (see "Experimental Procedures"). The RFC complex, containing both subunits, was soluble and was purified to near homogeneity by affinity chromatography onto Ni-chelate and hydroxyapatite columns (see Fig.2 C). RFC3 alone was marginally soluble in the presence of 500 mm NaCl, though it precipitated following prolonged dialysis (see "Experimental Procedures"). Nevertheless, RFC3 was purified by affinity chromatography on nickel chelate beads, which resulted in the isolation of a single protein band of 39 kDa (Fig. 2 A, lane 5). This purified protein fraction was used to generate polyclonal antibodies against RFC3. E. coli-expressed RFC1, on the other hand, was completely insoluble and could be extracted from cells only in the presence of 6 m urea. RFC1 was purified to near homogeneity following chromatography on nickel chelate beads in the presence of 6 m urea and yielded a protein of 57 kDa (Fig.2 B, lane 6). Attempts to solubilize RFC1 by dialysis against decreasing levels of urea (2 h periods each against 4 and 2 m urea) were unsuccessful. Whereas soluble material was observed after dialysis against 4 m urea, marked precipitation occurred during dialysis against 2 m urea. Attempts to form a soluble complex by mixing RFC1 and RFC3 in 6m urea, followed by dialysis against decreasing levels of urea were unsuccessful. The observations that the two individual subunits were either not soluble or marginally soluble when each was expressed alone, but were soluble as a complex, support the idea that they work together. The coexpression of both mthRFC subunits resulted in a two-subunit complex that could be purified to homogeneity. Gel filtration analysis of the pooled hydroxyapatite fraction of the RFC complex yielded a single protein peak that contained both ATPase activity and DNA synthetic activity, which depended upon mthPCNA and mthPolB (both activities peaked at fraction 45 (Fig. 3 A)). These two activities coeluted from the sizing column at a position corresponding to a complex of molecular mass of 260 kDa (Fig. 3 B) and a Stokes radius of 60Å based on comparison to globular standards. SDS-PAGE analysis of the gel filtration fractions revealed that the peak of RFC activity described above eluted at a position coincidental with both RFC subunits (Fig. 3 C). Antibodies prepared against the RFC3 subunit were used to verify that the observed 37-kDa protein band was RFC3 and not a breakdown product derived from the 57-kDa RFC1 protein (data not presented). Glycerol gradient centrifugation of the pooled hydroxyapatite fraction of the mthRFC complex also indicated that both subunits sedimented coincidentally with the enzymatic activities described above. The peak of mthRFC protein and its associated enzymatic activities sedimented at 11.4 S (data not presented). Based on comparisons to globular standards, this S value corresponded to a molecular mass of about 250 kDa. Using the Stokes radius, the S value, and assuming that the mthRFC complex possesses a partial specific volume of 0.725 ml/g, the molecular mass was estimated to be 290 kDa with a frictional ratio of 1.4 (18.Siegel L.M. Monty K.J. Biochim. Biophys. Acta. 1966; 112: 346-362Crossref PubMed Scopus (1547) Google Scholar). The latter calculation suggests a roughly globular conformation for the mthRFC complex. The Coomassie Blue-stained gel shown in Fig. 2 C, was scanned, and the relative concentration of each subunit present in the purified mthRF
Referência(s)