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J. Biol. Chem., Vol. 280, Issue 51, 41852-41863, December 23, 2005
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1
2
From the
Department of Animal Sciences and the Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801
Received for publication, August 8, 2005 , and in revised form, October 13, 2005.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The striking similarities among the eukaryotic RFCS proteins suggest that they evolved from a single ancestral gene. However, evidence for how this might have occurred has been elusive. Our effort to systematically assemble the proteins required for replicating the genome of Methanosarcina acetivorans, a mesophilic species of Archaea, has led to several findings of interest to evolution of DNA replication proteins in the archaeal/eukaryotic sister lineages (13–15). Here, we present the results from biochemical and mutational analyses of an unusual clamp loader found in Methanosarcinales. This clamp loader type (composed of two RFCS subunits and one RFCL subunit) may represent a critical link in the evolution of complex clamp loaders from simple forms in the archaeal/eukaryotic sister lineages. In addition to the RFC complex, each subunit of the methanosarcinal RFC homolog was expressed as an individual protein and biochemically characterized. Furthermore, we used mutational analysis of residues involved in ATP binding and hydrolysis to study the contribution of each subunit to the function of the M. acetivorans clamp loader. Nucleotide binding and hydrolysis are critical to loading of the sliding clamp by the clamp loader. Clamp loaders are members of the AAA+ (ATPases associated with a variety of cellular activities) ATPase family, and their ATP-binding sites are located at subunit interfaces (10, 11, 16, 17). Critical to nucleotide binding and hydrolysis in these proteins are the P-loop (Walker A motif) and an SRC motif from an adjacent subunit (11, 16). Thus, we targeted critical residues in the P-loop and SRC motif for site-directed mutagenesis.
The organisms in which the clamp loader type described in this report is found are quite interesting among the species of Archaea. Halobacterium sp. NRC-1 harbors a genome of 
2.0 Mb in size, and in addition, it has two large extrachromosomal replicons of 350 and 200 kb (18). Members of the order Methanosarcinales have the largest known archaeal genomes (4.1–5.7 Mb) (19, 20). Furthermore, they are unique among the species of Archaea in forming complex multicellular structures (21). The discovery of this new form of RFC in these organisms is of major importance, as further biochemical and genetic analyses may provide us with critical insights into cellular developments that might have led to a requirement for more complex clamp loaders from the equally competent but simple ones found in the archaeal/eukaryotic sister lineages.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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NcoI (TABLE ONE), which introduced only a silent mutation into the gene. The gene for MacRFCS1 was cloned with NdeI and XhoI, and the gene for MacRFCS2 was digested with NcoI and XhoI and cloned into the NcoI/SalI site in pACYCDuet. The genes for the mutant subunits were used in different combinations to create different mutants of the MacRFC complex.
Production of Recombinant Proteins—Plasmids harboring each individual RFCS gene were transformed into Escherichia coli BL21-Codon-Plus(DE3)-RIL cells (Stratagene) and spread on LB plates containing 100 µg/ml ampicillin and 50 µg/ml chloramphenicol. After an overnight incubation, a single colony was picked from each plate, inoculated into LB broth containing the two antibiotics at the concentrations mentioned above, and incubated with vigorous shaking at 37 °C until the absorbance at 600 nm reached 0.3. Gene expression was induced by addition of 0.1 mM isopropyl 
-D-thiogalactopyranoside; the incubation temperature was dropped to 16 °C; and cells were cultured for 12 h. One liter of culture from each cell line was centrifuged to pellet the cells. The cells were suspended in a lysis buffer or in buffer A (50 mM sodium phosphate (pH 7.0) and 300 mM NaCl) and lysed using a French pressure cell (American Instruments Co.) to release the cell contents. Because both MacRFCS1 and MacRFCS2 were produced with an N-terminal His6 tag, the supernatants for MacRFCS1 and MacRFCS2 were each applied to a cobalt affinity resin (TALONTM, Clontech) to immobilize the His6-tagged proteins (His6-MacRFCS1 and His6-MacRFCS2). The resin was washed extensively with the lysis buffer, and the bound protein was eluted with buffer A containing 150 mM imidazole. Aliquots of samples were resolved by SDS-PAGE, and the fractions containing the respective RFCS subunit were pooled together, suspended in buffer B (50 mM Tris-HCl (pH 8.0), 10 mM MgCl2, 500 mM NaCl, 1 mM dithiothreitol (DTT), and 20% glycerol), and dialyzed against the same buffer. The samples were then loaded onto a Superose 12 HR 10/30 gel filtration column (Amersham Biosciences) equilibrated with buffer B. The chromatography was developed with buffer B at a flow rate of 0.35 ml/min, and 0.5-ml fractions were collected for analysis by SDS-PAGE. In the case of MacRFCL, the plasmid construct was transformed into E. coli BL21(DE3) cells (Stratagene). The production of the MacRFC complex and all of its derivatives and also all other combinations of the MacRFC subunits tested was carried out in E. coli BL21(DE3) cells. The cells carrying MacRFCL were selected on ampicillin-containing LB medium, whereas the cells carrying MacRFCS1/MacRFCS2 were selected on chloramphenicol-containing LB medium. The RFC complex and its derivatives were selected on ampicillinand chloramphenicol-containing LB medium. Therefore, His6-MacRFCL and the MacRFC complex (coexpressed His6-RFCL, RFCS1, and His6-RFCS2) were purified by affinity chromatography on cobalt affinity resin as described above, followed by passage through a heparin column and then gel filtration chromatography. For affinity chromatography on a His-TrapTM HP column (5 ml; Amersham Biosciences), the elution buffer contained 500 mM imidazole. In the heparin chromatography step, His6-RFCL or the MacRFC complex obtained from the affinity chromatography was dialyzed against buffer C (50 mM KH2PO4 (pH 6.8), 100 mM NaCl, 7 mM -mercaptoethanol, and 10% glycerol) and then loaded onto HiTrapTM heparin HP column (5 ml; Amersham Biosciences) equilibrated with the same buffer. The column was washed with 5 column volumes of buffer C, followed by elution of bound proteins with buffer C containing 1 M NaCl. The fractions containing the proteins were dialyzed against buffer B and applied to a gel filtration column as described above for the RFCS subunits. His6-RFCL and the MacRFC complex were each stored in buffer containing 50 mM Tris-HCl (pH 8.0), 200 mM NaCl, 5 mM MgCl2, 10% glycerol, and 1 mM DTT until used. The gene for a homolog of the eukaryotic proliferating cell nuclear antigen (PCNA) in M. acetivorans was cloned into pET21a for expression. The expression and purification of MacPCNA were performed following a method described previously (22) for Pyrococcus furiosus (Pfu) PCNA, except for the omission of the heating step because MacPCNA is from a mesophilic organism. In brief, this method involved Polymin P treatment for 30 min at 4 °C, ammonium sulfate precipitation, and anion exchange chromatography (HiTrap Q, Amersham Biosciences). The cloning, expression, and purification of M. acetivorans DNA polymerase BI (PolBI) and its truncated forms were as described in our previous reports (14, 15). The primers for the C-terminal truncation of MacPolBI are shown in TABLE ONE. All of the mutant MacRFC proteins were expressed and their products were purified as described for their wild-type counterparts.
Western Blot Analysis—Polyclonal antibodies were raised against purified recombinant MacRFCS1 and MacRFCS2 (Immunological Resource Center, University of Illinois at Urbana-Champaign). In the case of His6-MacRFCL and His6-MacRFCS2, an antibody commercially available for detection of the His6 tag (Amersham Biosciences) was purchased and used according to the manufacturer's instructions. All protein samples of interest were resolved by SDS-PAGE, followed by electroblotting onto polyvinylidene difluoride membrane (HybondTM-P, Amersham Biosciences), and analyzed as described previously (13).
Estimation of Subunit Organization by Gel Filtration—To estimate the subunit organization of individual proteins and protein complexes in solution, the RFC proteins were subjected to gel filtration analysis. Purified proteins were dialyzed against buffer B. Samples were then loaded onto a Superose 12 HR 10/30 gel filtration column equilibrated with buffer B. The chromatography was developed with buffer B at a flow rate of 0.35 ml/min, and 0.5-ml fractions were collected and analyzed by SDS-PAGE. The column was calibrated by running a set of protein standards (thyroglobulin, 669 kDa; ferritin, 440 kDa; aldolase, 158 kDa; catalase, 58 kDa; ovalbumin, 43 kDa; and ribonuclease A, 13.7 kDa) under the same conditions.
Electrophoretic Mobility Shift Assay—The MacRFC complex and its individual subunits were tested for their ability to bind to single-stranded DNA (ssDNA) and singly primed ssDNA (pDNA). The pDNA was made by end labeling the pDNA primer (38-mer) and annealing it to the partially complementary pDNA template (76-mer). The nucleotide sequences of the primer and template are shown in TABLE ONE. Increasing amounts of proteins were incubated with 0.5 pmol of 
-32P-end-labeled ssDNA (CDC6-1F (40-mer oligonucleotide) in TABLE ONE) or -32P-labeled pDNA in 10 µl of binding buffer (20 mM Tris acetate (pH 8.0) and 0.5 mM magnesium acetate) at 37 °C for 30 min. Two microliters of loading dye (20 mM Tris acetate (pH 8.0) 10% glycerol, and 0.1% bromphenol blue) were added to the product of the reaction and resolved by 1% agarose gel electrophoresis in 0.1x Tris acetate/EDTA buffer (15). The gels were dried, and the products were visualized by autoradiography.
ATPase Activity—ATPase activity was measured in a reaction mixture (20 µl) containing 25 mM Tris-HCl (pH 7.5), 0.1 mM DTT, 6 mM MgCl2, 0.5 µg/µl bovine serum albumin, 0.034 µM [-32P] ATP (6000 Ci/mmol), 10 µM unlabeled ATP, and one of the following: 2.4 µg of MacRFCS1, 1.2 µg of MacRFCS2, 2.1 µg of MacRFCL, or 0.45 µg of MacRFC complex. The reaction mixture was incubated at 37 °C for 40 min and then terminated by addition of 2 µl of a 400 mM EDTA solution. When dATPase activity was investigated, the radioactive and non-radioactive ATPs were substituted with radioactive and nonradioactive dATPs, respectively. One microliter of products from each reaction mixture was spotted on polyethyleneimine-cellulose thin layer plates (Merck, Darmstadt, Germany) and then developed in a solution containing 1.0 M formic acid and 0.5 M LiCl for 10 min at room temperature. When their effects were assessed, cofactors or effectors were added as follows: ssDNA (39-mer, 130 ng), double-stranded DNA (dsDNA; 375 bp, 50 ng), pDNA (56 ng), or MacPCNA (12 pmol). The method used to make the pDNA is described above under "Electrophoretic Mobility Shift Assay." Note that, for this purpose, the primer was not end-labeled. The sequences of the nucleic acids are shown in TABLE ONE. The thin layer chromatography plates were dried and exposed for autoradiography, and the results were quantified using a BAS-1800 II bio-imaging analyzer (Fuji Photo Film Co., Ltd.).
Primer Extension Analysis—We used a primer extension assay to compare the capacity of the MacRFC proteins and their mutants to stimulate MacPCNA-dependent DNA synthesis of MacPolBI. An oligonucleotide that is complementary to positions 6205–6234 of the M13mp18 (+)-strand was 5'-end-labeled with [-32P]ATP and T4 polynucleotide kinase (New England Biolabs Inc.). Two picomoles of the end-labeled primer were annealed to 1 µg of template (M13mp18 (+)-strand circular ssDNA) as described previously (15). The primer extension reaction (20 µl) contained 20 mM Tris-HCl (pH 8.8), 100 mM NaCl, 5 mM MgCl2, 2 mM -mercaptoethanol, 250 µM each dNTP, 20 mM ATP, and 0.5 µg of MacPolBI. When accessory factors were added, they were included as follows: MacRFCS1, MacRFCS2, and MacRFCL at 2.4, 1.2, and 2.1 µg/reaction, respectively; MacRFC complex or its mutants at 0.34 µg/reaction; and MacPCNA at 6 pmol/reaction. Each reaction mixture was incubated at 37 °C for 5 min, followed by termination by addition of 6 µl of stop solution (98% formamide, 1 mM EDTA, 0.1% xylene cyanol, and 0.1% bromphenol blue). After heating to 95 °C for 5 min, the samples were resolved on 1% alkali-agarose gel in 50 mM sodium hydroxide and 1 mM EDTA.
Growth of M. acetivorans Cells—M. acetivorans cells were grown in HS/methanol/acetate broth medium (400 mM NaCl, 54 mM MgCl2·6H2O, 45 mM NaHCO3, 19 mM NH4Cl, 13 mM KCl, 5 mM KH2PO4, 2 mM CaCl2·2H2O, 4 µM resazurin, 2.8 mM cysteine HCl, and 0.4 mM Na2S·9H2O) at 35 °C under strictly anaerobic conditions (23). The cells at mid-exponential phase (A600 nm 0.3) were harvested by centrifugation at 6000 x g for 15 min. Next, 1.5 g of wet cell pellet were resuspended in 2.25 ml of buffer containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, and 1 mM DTT. The cells were lysed using a French pressure cell, and the cell debris was removed by centrifugation at 10,000 x g for 20 min. The supernatant was collected and stored at 4 °C until used for Western blot analysis.
N-terminal Sequencing—An aliquot of the MacRFC complex was fractionated by SDS-PAGE (7.5% polyacrylamide gel) and electroblotted onto a membrane optimized for protein transfer (HybondTM-P). The membrane was stained with Coomassie Brilliant Blue R-250 (0.02% in 40% methanol) and destained with 5% methanol. The protein bands were excised and subjected to automated Edman degradation in an Applied Biosystems 494/HT Procise sequencing system (Biomolecular Resource Facility Core, University of Texas Medical Branch, Galveston, TX).
Amino Acid Sequence Alignments—All alignments were carried out with the multiple alignment program ClustalW (available at www.ebi.ac.uk/clustalw/), and the shading was manually carried out.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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67 and 38 kDa by Western blot analysis (data not shown). The sizes of the two proteins corresponded well with those of His6-tagged MacRFCL and MacRFCS2, respectively. A polyclonal antibody raised against MacRFCS2 reacted with the 38-kDa band, but not with the 67-kDa band (data not shown). The presence of MacRFCS1 in the complex was detected by N-terminal sequencing. The band in Fig. 2 (lane 5) at 35 kDa has an N-terminal amino acid sequence of MQALMEDS, which is an exact match for MacRFCS1. The N-terminal sequence of the band beneath MacRFCS1 is MKEEIIIE. We used this sequence to search the E. coli genomes deposited in the GenBankTM Data Bank, and we did not find a perfect match. However, the sequence corresponded well with an internal sequence of MacRFCS1 (KEEIWIE). A protein harboring this sequence at the N terminus will lack the first 10 amino acids of MacRFCS1. Furthermore, polyclonal antibodies raised against MacRFCS1 reacted with both RFCS1 and the smaller band (data not shown). The protein may be either an aberrantly translated product or a truncated product. We intend to investigate this further in future experiments. To determine the capacity of the MacRFC proteins to enhance DNA synthesis by MacPolBI in the presence of the cognate sliding clamp, we cloned and expressed the gene coding for a PCNA homolog in M. acetivorans (MacPCNA, GenBankTM accession number AAM03564
[GenBank]
). Following previous protocols (22), MacPCNA was purified to homogeneity, as shown in Fig. 2 (lane 6). In addition, we expressed the gene coding for MacPolBI and purified it (Fig. 2, lane 7) as described in our previous work (14). Gel filtration analysis was used to estimate the subunit organization of the MacRFC complex and its subunits in solution. MacRFCS1 eluted as a protein with a relative molecular mass of 72.4 ± 1.7 kDa with or without the His6 tag, and because the predicted molecular mass of the protein was 37.9 kDa, the results suggest a protein that forms dimers in solution (supplemental Fig. 1). On the other hand, MacRFCS2 eluted as a protein of 44.8 ± 3.7 kDa. The calculated molecular mass of MacRFCS2 was 38.3 kDa. Thus, the elution volume suggests that MacRFCS2 exists as monomers in solution. MacRFCL eluted in the void volume, suggesting a protein with a very large relative molecular mass or an aggregated product. Note, however, that aliquots of samples from the peak elution of MacRFCL exhibited robust ATP hydrolysis that was stimulated by effectors, as described below. A sample of the MacRFC complex that eluted from the heparin column was analyzed to estimate its subunit organization. The elution volume of the MacRFC complex upon gel filtration suggested a relative molecular mass of 344.5 ± 6.3 kDa; and due to the multiple proteins in the RFC complex, we could not readily estimate its subunit composition in solution (supplemental Fig. 1).
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Clamp loaders couple ATP hydrolysis to loading of their cognate sliding clamps. The ATP hydrolysis is inherent to the clamp loader subunits, which are all members of the AAA+ ATPase family (24). We therefore investigated the capacity of the individual subunits of MacRFC to hydrolyze ATP. Both MacRFCS subunits exhibited very low intrinsic ATPase activities (Fig. 4A). Nucleic acids (ssDNA or dsDNA) as effectors stimulated the ATPase activities of the MacRFCS subunits. Addition of either ssDNA or dsDNA together with MacPCNA to the reaction mixture resulted in further increases in the ATPase activities of both MacRFCS subunits. The highest stimulation of the ATPase activities of the RFCS subunits was achieved in the presence of both ssDNA and MacPCNA in the reaction mixture. Under these conditions, the ATPase activities of MacRFCS1 and MacRFCS2 were stimulated by 10- and 15-fold, respectively (Fig. 4A). MacRFCL exhibited 15-fold higher intrinsic ATPase activity compared with the MacRFCS subunits (Fig. 4A). The ATPase activity of MacRFCL (unlike that of the small subunits) was stimulated only in the presence of both nucleic acid and MacPCNA. The MacRFC complex (a protein complex of MacRFCS1, MacRFCS2, and MacRFCL) also possessed very low intrinsic ATPase activity. This activity was stimulated 33-fold by ssDNA, 12-fold by dsDNA, 58-fold by pDNA, and 3-fold by MacPCNA (Fig. 4B). The ATPase activity of MacRFC was further stimulated by addition of both nucleic acid and MacPCNA to the reaction mixture. However, addition of MacPCNA to MacRFC in the presence of pDNA did not yield higher ATP hydrolysis compared with pDNA alone as the effector. Interestingly, however, addition of both dsDNA and MacPCNA to the reaction mixture resulted in ATPase activity that was about four times that of MacRFC in the presence of only dsDNA. Mutation of the conserved lysine in the Walker A motif (RFC box III) (Fig. 1) to alanine resulted, in general, to decreases in the capacity of the MacRFC subunits to hydrolyze ATP (supplemental Fig. 2). Mutation of the conserved arginine in the SRC motifs (RFC box VII) (Fig. 1) of the RFCS subunits resulted in further drastic decreases in their capacity to hydrolyze ATP (supplemental Fig. 2, A and B). We also made MacRFC complexes harboring several combinations of mutations in the Walker A and SRC motifs. As observed for the MacRFCS subunits, mutations in the Walker A motif of individual subunits in the MacRFC complex led to decreases in ATP hydrolysis, with mutations in multiple subunits leading to further drastic decreases in ATPase activity (supplemental Fig. 3A). In this case also, mutant RFC complex proteins harboring arginine-to-alanine mutations in the SRC motif of either small subunit or both exhibited further drastic decreases in ATP hydrolysis (supplemental Fig. 3B).
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500 nucleotides in length (Fig. 5A, panels i–iv, lanes 2). Addition of only MacPCNA to MacPolBI failed to elicit an effect on the length of products synthesized by the DNA polymerase (Fig. 5A, panels i–iv, lanes 3). Addition of each of the MacRFC subunits to the reaction mixture in the presence of MacPCNA led to synthesis of shorter products by MacPolBI (Fig. 5A, panels i–iii, lanes 4). It is likely that MacPCNA increases the affinity of MacRFC subunits for ssDNA. The increase in protein binding to the M13mp18 ssDNA template then impeded synthesis by the DNA polymerase. In contrast, addition of MacRFC and MacPCNA to the reaction mixture stimulated primer extension, and full-length products (7.2 kb) were synthesized (Fig. 5A, panel iv, lane 4). In the absence of MacPCNA, neither the MacRFC complex nor its subunits could stimulate an increase in primer extension by MacPolBI (Fig. 5A, panels i–iv, lanes 5). MacRFCS1 instead suppressed the length of the product synthesized (Fig. 5A, panel i, lane 5), which may reflect its higher affinity for ssDNA (Fig. 3A, lane 6). The results thus suggest that only the MacRFC complex has the capacity to enhance MacPCNA-dependent primer extension by MacPolBI. We created several mutant MacRFC complexes harboring lysine-toalanine mutations in the Walker A motif as described above. The first MacRFC mutant investigated was RFC-S1K/S2/L. This MacRFC mutant contained a single lysine-to-alanine mutation in MacRFCS1, and it exhibited appreciable ATPase activity in the presence of effectors, although not as much as the wild-type MacRFC complex (supplemental Fig. 3A). As shown in Fig. 5B (panel i, lane 4), this mutant MacRFC complex failed to enhance MacPCNA-dependent primer extension by MacPolBI. The next mutant investigated was RFC-S1/S2K/L. This mutant harbored a lysine-to-alanine mutation in only MacRFCS2. This mutant MacRFC complex maintained strong ATPase activity; and in the presence of some effectors, it even exhibited higher activity compared with the wild-type MacRFC complex (supplemental Fig. 3A). Interestingly, this mutant MacRFC complex was capable of stimulating MacPCNA-dependent primer extension by MacPolBI (Fig. 5B, panel ii, lane 4). The next mutant MacRFC complex (designated RFC-S1/S2/LK) contained a single lysine-to-alanine mutation in only MacRFCL; and interestingly, this mutation led to further reduced ATPase activity compared with the mutant MacRFC complex harboring a similar mutation in either MacRFCS1 or MacRFCS2 (supplemental Fig. 3A). As shown in Fig. 5B (panel iii, lane 4), this mutant MacRFC complex also stimulated MacPCNA-dependent primer extension by MacPolBI. The mutant MacRFC complexes containing the lysine-to-alanine mutations in both MacRFCS subunits (RFC-S1K/S2K/L) and also in all three subunits (RFC-S1K/S2K/LK) failed to stimulate primer extension by MacPolBI in the presence of MacPCNA (Fig. 5B, panels iv and v, lanes 4). These two mutants exhibited low ATPase activity and also failed, in general, to show any response to nucleic acids and MacPCNA in the ATP hydrolysis experiments described above.
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The PfuRFC complex can be reconstituted by expressing the small subunit (PfuRFCS) and the large subunit (PfuRFCL) separately and then dialyzing excess amounts of PfuRFCS together with PfuRFCL (9). We investigated whether a functional MacRFC complex can be reconstituted by the same process. The mixtures tested were RFCS1/RFCS2, RFCL/RFCS1, RFCL/RFCS2, and RFCL/RFCS1/RFCS2. The products of RFCS1/RFCS2, RFCL/RFCS1, and RFCL/RFCS2 did not possess the capacity to stimulate primer extension by MacPolBI in the presence of MacPCNA (data not shown). Primer extension instead decreased in each case, which may be due to ssDNA binding by RFC subunits in the presence of PCNA and nucleotides, as described above. On the other hand, a similar experiment with the dialysate of RFCL/RFCS1/RFCS2 showed some increase in the length of the products synthesized by MacPolBI in the presence of MacPCNA. However, the efficiency of this stimulation was very low because most of the products were similar in length to those synthesized by the DNA polymerase alone (data not shown). Also, several archaeal clamp loaders have been shown to function without ATP in the reaction mixture. In a comparative analysis, we determined that the ATPase activities of MacRFC alone, MacRFC in the presence of pDNA, and MacRFC in the presence of both pDNA and MacPCNA with either ATP or dATP as the substrate were almost identical (data not shown). This observation suggests that MacRFC may use dATP, as observed in some archaeal clamp loaders, to load its cognate sliding clamp (9, 25). As expected, with or without ATP in the reaction mixture, MacRFC stimulated the primer extension capacity of MacPolBI in the presence of MacPCNA (data not shown).
We examined the amino acid sequences of the methanosarcinal RFC subunits for any salient differences from their known archaeal counterparts. As shown in Fig. 6A, MacRFCS2 and its orthologs in three other members of the order Methanosarcinales as well as an ortholog in Halobacterium sp. NRC-1 contain an insertion of 26 amino acids immediately after their RFC box IV. Interestingly, the polypeptides of the RFCL subunit of Methanosarcinales are also longer than those of other archaeal polypeptides as stated above. Sequence analysis suggested the insertion of a peptide at the C-terminal end of the methanosarcinal RFCL subunit. As shown in Fig. 6B, most archaeal RFCL proteins terminate with a peptide similar to the PCNA-interacting peptide (PIP) box (6). In the methanosarcinal RFCL polypeptides, there appear to be two PIP boxes, one that was likely to be the original PIP box in the polypeptide (methanosarcinal PIP box I) (Fig. 6B) and another (methanosarcinal PIP box II) that was likely acquired together with the extended C terminus of the methanosarcinal RFCL subunit. The PIP boxes in the other archaeal organisms are the single highlighted PIP boxes found in the RFCL subunits of these organisms (Fig. 6B).
We deduced that if the stimulation of primer extension by MacPolBI in the presence of MacPCNA and MacRFC is due to the interaction of the DNA polymerase and the sliding clamp (after being loaded by the clamp loader), then if we deleted the PIP box of the DNA polymerase, the stimulation in the presence of the sliding clamp (MacPCNA) and the clamp loader (MacRFC) should be abolished. Similar to the archaeal RFCL proteins, archaeal PolBI proteins usually harbor a PIP box-like sequence at their C termini, as shown in Fig. 7A (22). Surprisingly, we found a second putative PIP box in MacPolBI, as in the methanosarcinal RFCL subunit. We deleted PIP box II to create MacPolBIC1 and tested this polypeptide for response to MacPCNA and MacRFC. The deletion did not abolish MacPCNA/MacRFC-dependent enhancement of DNA synthesis by MacPolBI (Fig. 6B, panel i). A second deletion that removed PIP box I resulted in a polypeptide (MacPolBIC2) that was unresponsive to MacPCNA and MacRFC.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The elution volume of the small subunit of PfuRFC suggests a protein that exists in solution as a homotetramer (9), and a similar subunit organization was also determined for the RFCS subunit of another hyperthermophile, Sulfolobus solfataricus (8). In the case of the Archaeoglobus fulgidus RFCS subunit, the protein eluted as a heterogeneous complex (26). Our gel filtration analysis (supplemental Fig. 1) resulted in a single symmetrical peak for each of the MacRFCS subunits, and the elution volumes suggested a homodimer and a monomer for MacRFCS1 and MacRFCS2, respectively. Thus, the subunit organizations of the MacRFCS subunits are very dissimilar to those of known archaeal RFCS subunits. MacRFCL was expressed and purified as a His6-tagged protein, and although it eluted in the void volume, the highly purified protein (Fig. 2) exhibited both ATP hydrolysis and DNA binding activities. It is possible, however, that the aggregated RFCL subunit might have occluded, from the host cells, proteins that contributed to its ATPase and DNA binding activities. The elution volume of the MacRFC complex suggested a protein with a relative molecular mass 341 kDa. The stoichiometry of the subunits in the MacRFC complex was not readily evident, and experiments aimed at obtaining this information are currently under way in our laboratory.
It has been reported that the A. fulgidus RFCS subunit does not bind to DNA significantly, whereas the RFCL subunit binds to DNA (26). The protein/DNA interaction of A. fulgidus RFC was therefore attributed mainly to the large subunit. In the PfuRFC proteins, however, the RFCS subunit clearly binds to pDNA, whereas the RFCL subunit shows only very weak pDNA binding activity (9). Interestingly, S. solfataricus RFC (8) does not bind to ssDNA, and Methanothermobacter thermoautotrophicus (7) RFC binds to ssDNA only weakly. In contrast, the two proteins bind to ssDNA annealed with a primer (pDNA). These findings point to subtle differences in DNA binding activities in the archaeal RFC proteins and their subunits. A possible reason may be differences in the methods used in preparing the archaeal RFCS subunits and also differences in the stability of these proteins in solution in the absence of their partners. However, it is clear that the archaeal functional clamp loaders bind to pDNA, the substrate onto which the clamp is loaded. Among the three MacRFC subunits, only MacRFCL bound to pDNA, which suggests that the recognition of this structure for loading of the sliding clamp is dependent on this subunit. This finding is similar to that obtained with the human RFC subunits in that only the large subunit was demonstrated to bind to primer·template DNA (27).
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Interestingly, the MacRFC homolog may be analogous to the E. coli -complex (clamp loader), which is made up of three different subunits: , 
, and '. The subunits are arranged as '3. Thus, there is one '-subunit known as the stator, three -subunits referred to as the motor, and one -subunit referred to as the wrench. The -wrench does not bind ATP, but is capable of cracking open the sliding clamp. The '-subunit is the stator or the part of the -complex that is stationary, and 3 (1, 2, and 3) directs ATP-induced conformational changes in the clamp loader (17). Similar to the -wrench, Saccharomyces cerevisiae Rfc1 (human p140) interacts with S. cerevisiae PCNA. In the present study, MacRFCL contained the PCNA-binding motif, and its homolog in P. furiosus has been shown to interact with PfuPCNA (22). In addition, similar to the -wrench and yeast Rfc1, MacRFCL lacks the SRC motif. Thus, MacRFCL may represent the wrench in the MacRFC complex; MacRFCS2, which has an altered Walker A motif like the '-subunit, may represent the stator; and MacRFCS1 is then the motor. The model for clamp loading suggests that the motor (3) binds ATP to induce the conformational change that makes the N-terminal domains of the -wrench available for interaction with and thus opening the sliding clamp (-subunit) (17). It is therefore reasonable that, in this study, mutations in either the Walker A or SRC motif of MacRFCS1 (motor) abolished clamp loading activity.
The capacity of a sliding clamp to enhance DNA synthesis by its cognate DNA polymerase requires interaction of the two proteins. Unlike other archaeal PolBI proteins, the methanosarcinal orthologs have gained an extra PIP box, as seen in their RFCL subunits. Archaeal PolBI is likely to play a role in chromosomal replication (7, 22, 28), and it is our hypothesis that the dual PIP boxes in methanosarcinal PolBI are an adaptation to ensure survival in the event of a deleterious mutation in this critical motif.
An obvious inference that can be drawn from the discovery of the existence of an RFC homolog with two small subunits and one large subunit is an intermediate complex that bridges the transition from the one small/one large subunit form to the four small/one large subunit clamp loaders found in the archaeal/eukaryotic sister lineages. Thus, the gene for the ancestral RFCS subunit might have gone through several gene duplications to arrive at the genes encoding the four different small subunits found in extant eukaryotes. We are eagerly analyzing more genomes for further evidence to support this hypothesis. Thus, although we were unable to answer the critical question of the stoichiometry of the MacRFC complex, our report of the existence of an RFC complex composed of three different polypeptides has critical implication on the evolution of complex clamp loaders. M. acetivorans has a proven genetic system, and by coupling this tool with biochemical analysis, we may shed light on the requirement for more complex clamp loaders as more complex organisms evolved in the archaeal/eukaryotic sister lineages.
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FOOTNOTES |
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The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1–3.
1 Supported by Agricultural Genome Sciences and Public Policy Training Grant 2001-52100-11527.
2 To whom correspondence should be addressed: Dept. of Animal Sciences, University of Illinois at Urbana-Champaign, 1207 West Gregory Dr., Urbana, IL 61801. Tel.: 217-333-2090; Fax: 217-333-8286; E-mail: icann{at}uiuc.edu.
3 The abbreviations used are: RFC, replication factor C; RFCS, replication factor C small subunit; RFCL, replication factor C large subunit; Mac, M. acetivorans; DTT, dithiothreitol; PCNA, proliferating cell nuclear antigen; Pfu, P. furiosus; PolBI, DNA polymerase BI; ssDNA, single-stranded DNA; pDNA, singly primed single-stranded DNA; dsDNA, double-stranded DNA; Mma, M. mazei; Mba, M. barkeri; PIP, proliferating cell nuclear antigen-interacting peptide.
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