Replication Protein A in Pyrococcus furiosus Is Involved in Homologous DNA Recombination*

Kayoko Komori and Yoshizumi IshinoDagger

From the Department of Molecular Biology, Biomolecular Engineering Research Institute, Suita, Osaka 565-0874, Japan

Received for publication, March 19, 2001, and in revised form, April 20, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Single-stranded DNA-binding protein in Bacteria and replication protein A (RPA) in Eukarya play crucial roles in DNA replication, repair, and recombination processes. We identified an RPA complex from the hyperthermophilic archaeon, Pyrococcus furiosus. Unlike the single-peptide RPAs from the methanogenic archaea, Methanococcus jannaschii and Methanothermobacter thermoautotrophicus, P. furiosus RPA (PfuRPA) exists as a stable hetero-oligomeric complex consisting of three subunits, RPA41, RPA14, and RPA32. The amino acid sequence of RPA41 has some similarity to those of the eukaryotic RPA70 subunit and the M. jannaschii RPA. On the other hand, RPA14 and RPA32 do not share homology with any known open reading frames from Bacteria and Eukarya. However, six of eight archaea, whose total genome sequences have been published, have the open reading frame homologous to RPA32. The PfuRPA complex, but not each subunit alone, specifically bound to a single-stranded DNA and clearly enhanced the efficiency of an in vitro strand-exchange reaction by the P. furiosus RadA protein. Moreover, immunoprecipitation analyses showed that PfuRPA interacts with the recombination proteins, RadA and Hjc, as well as replication proteins, DNA polymerases, primase, proliferating cell nuclear antigen, and replication factor C in P. furiosus cells. These results indicate that PfuRPA plays important roles in the homologous DNA recombination in P. furiosus.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Single-stranded DNA-binding protein (SSB)1 in Bacteria and replication protein A (RPA) in Eukarya play essential roles in DNA replication, recombination, and repair. (1-3). The bacterial SSB and the eukaryotic RPA bind to single-stranded DNA as a homotetramer and a heterotrimer, respectively. Although there is little amino acid sequence similarity between SSB and RPA, recent analyses of the three-dimensional structures revealed that these proteins have a structurally similar domain (4-6) containing a common fold, called the oligonucleotide/oligosaccharide binding (OB) fold (7). The presence of this common structure in SSB and RPA, and also in the RNA binding domain of Escherichia coli polynucleotide phosphorylase (8), suggests that the mechanism of single-stranded nucleic acid binding appears to be conserved in the biological domains.

Bacterial SSB is a homotetramer of a 20-kDa peptide with one OB fold. In contrast, eukaryotic RPA is a stable heterotrimer composed of 70, 32, and 14 kDa subunits. The largest subunit, RPA70, contains two tandem repeats of an OB fold that are responsible for the major interaction with a single-stranded DNA in the central region (5, 9-11). The N-terminal and C-terminal regions of RPA70 mediate interactions with many cellular or viral proteins and RPA32, respectively (12, 13). The zinc finger motif, which is important for RPA function, is strongly conserved in the C-terminal region of RPA70 (14, 15). The middle subunit, RPA32, contains an OB fold at the central region (6, 16, 17). The RPA32 interacts with other RPA subunits and various cellular proteins at the C-terminal region (12, 13, 18, 19). The smallest subunit, RPA14, also contains an OB fold (6).

Eukaryotic RPA function has been well studied in the in vitro SV40 DNA replication system. The RPA interacts with the SV40 T-antigen and the DNA polymerase alpha -primase complex, resulting in the formation of an initiation complex at the replication origin (20). The RPA also stimulates Pol alpha -primase activity and PCNA-dependent Pol delta  activity (21, 22). In homologous recombination, RecA in Bacteria and Rad51 in eukarya play a central role in homologous pairing and strand exchange between single-stranded DNA and homologous double-stranded DNA (reviewed in Refs. 23-25). In the eukaryotic recombination system, RPA strikingly stimulates the Rad51-mediated strand-exchange reaction in vitro (26-28). Furthermore, various proteins, including Rad52, Rad55, Rad57, and Rad54, stimulate the RPA-associated Rad51 reaction (29-34).

Recently, several laboratories have reported a RecA/Rad51 family protein, called RadA, in Archaea, the third domain of life (35-38). The archaeal RadA protein sequence is more similar to the eukaryotic Rad51 than to the bacterial RecA and can mediate the strand-exchange in vitro. Although the RadA-mediated strand-exchange ability is very weak, like the case of eukaryotic Rad51, stimulating factors have not yet been identified in Archaea. The RPAs from Methanococcus jannaschii and Methanothermobacter thermoautotrophicus (Methanobacterium thermoautotrophicum) (MJ1159 and MTH1385, respectively) have been reported (39-41). These proteins have amino acid sequence similarity to the eukaryotic RPA70 and contain four or five repeated domains containing the putative OB fold and one putative zinc finger motif. The M. jannaschii RPA exists as a monomer in solution and has single-strand DNA binding activity. However, it is not yet known whether the archaeal RPA is required for DNA replication and recombination. To understand the biological functions of the archaeal RPA, especially in DNA replication and recombination, we have characterized the RPA from the hyperthermophilic archaeon, Pyrococcus furiosus. Similar to the eukaryotic RPA, P. furiosus RPA (PfuRPA) forms a complex consisting of three distinct subunits. The PfuRPA strikingly stimulated the RadA-promoted strand-exchange reaction in vitro. These analyses will contribute to the complete understanding of the molecular mechanism of homologous recombination in Archaea.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Preparation of P. furiosus RPA Subunit Proteins-- The structural genes of rpa1, rpa2, and rpa3 were amplified by polymerase chain reaction directly from P. furiosus genomic DNA. Pfu DNA polymerase (Stratagene) was used to maintain the accuracy of amplification. To adjust the translational initiation codon, ATG, at the NdeI site (for rpa1 and rpa2) or the NcoI site (for rpa3) for the expression vector pET21a or pET21d (Novagen), the recognition sequences were included in each forward primer. In the reverse primers of rpa1 and rpa2, a SalI site was made just after the termination codon. To fuse a His6 tag at the C-terminus of the rpa3 gene product, an XhoI site was generated in frame with a His6-tag coding sequence in the reverse primer. The polymerase chain reaction products were digested by NdeI-SalI or NcoI-XhoI and were inserted into the corresponding sites of pET21a or pET21d. The nucleotide sequence of each plasmid was confirmed by DNA sequencing. The resultant plasmids were designated as pPFRPA1, pPFRPA2, and pPFRPA3, respectively. E. coli JM109 (DE3) carrying pPFRPA1 was cultivated in 1 liter of LB medium containing 100 µg/ml ampicillin to an A600 of 0.3 and then isopropyl-1-thio-beta -D-galactopyranoside was added to a final concentration of 1 mM for the induction of the rpa1 gene. After an incubation for 5 h at 37 °C, the cells were harvested by centrifugation. The cells were disrupted by sonication in HI buffer (30 mM HEPES-NaOH, pH7.8, 1 mM DTT, 0.25 mM EDTA, 0.01% (v/v) Nonidet P-40, 0.25% (w/v) inositol, 50 mM KCl) containing 1 mM phenylmethanesulfonyl fluoride (PMSF), and the soluble extract was incubated at 80 °C for 20 min. The heat-resistant fraction was collected by centrifugation and was applied onto a HiTrap Blue column (Amersham Pharmacia Biotech). The HiTrap Blue-bound fraction that eluted at 0.1-1 M KCl was dialyzed against Buffer A (50 mM Tris-HCl, pH 8.0, 0.5 mM DTT, 0.1 mM EDTA, 10% glycerol), and the dialysate was loaded on a HiTrap Q column (Amersham Pharmacia Biotech). RPA41 was eluted at 0.38-0.45 M NaCl. E. coli BL21-CodonPlusTM-(DE3)-RIL (Stratagene) carrying pPFRPA2 was cultivated in 0.5 liter of LB medium containing 100 µg/ml ampicillin and 20 ng/ml chloramphenicol to an A600 of 0.5, and the isopropyl-1-thio-beta -D-galactopyranoside was added to a final concentration of 1 mM. After an incubation for 6 h at 37 °C, the cells were harvested by centrifugation. The heat-resistant extract was prepared by sonication in sonication buffer (50 mM Tris-HCl, pH 8.0, 0.5 mM DTT, 0.1 mM EDTA, 1 mM PMSF) and heat treatment at 75 °C for 20 min. The extract was treated with 0.1% polyethyleneimine, and the precipitate was suspended in 0.3 M ammonium sulfate in Buffer A to elute the RPA14 protein. The proteins were precipitated with ammonium sulfate (80% saturation), and the precipitate was dissolved in and dialyzed against Buffer A. The dialysate was applied onto a HiTrap Q column, and RPA14 was eluted at 0.2-0.3 M NaCl. The fraction was diluted with two volumes of Buffer A and was loaded on a Mono Q HR 5/5 column (Amersham Pharmacia Biotech). RPA14 was eluted at 0.23-0.26 M NaCl. E. coli JM109 (DE3) carrying pPFRPA3 was cultivated in 1.2 liters of LB-ampicillin medium to an A600 of 0.4, and the expression of rpa3 was induced by adding isopropyl-1-thio-beta -D-galactopyranoside to a final concentration of 0.2 mM. After an incubation for 17 h at 20 °C, the cells were collected by centrifugation. The cells were disrupted by sonication in Buffer B (50 mM Tris-HCl, pH 7.5, 0.3 M NaCl) containing 1 mM PMSF, and the soluble extract was incubated at 80 °C for 20 min. The heat-resistant fraction was applied onto a TALON metal affinity resin (CLONTECH) column equilibrated in Buffer B. RPA32 was eluted with Buffer B containing 150 mM imidazole and was dialyzed against Buffer A containing 0.1 M NaCl. RPA32 was precipitated during dialysis, and therefore, the precipitate was dissolved again with Buffer B and applied onto a HiTrap Q column. RPA32 was eluted at 0.35-0.45 M NaCl. Various combinations of purified RPA subunits were mixed in Buffer C (Buffer A containing 0.3 M NaCl), and the complexes were separated from the free subunits on a Superdex 200 PC 3.2/30 column (Amersham Pharmacia Biotech) equilibrated with Buffer C.

Protein Concentrations-- The concentration of each purified protein was calculated by measuring the absorbance at 280 nm after denaturation by 6 M guanidine chloride. The theoretical molar absorption coefficient of each molecule was calculated based on its number of tryptophans and tyrosines, as described earlier (42). The molar extinction coefficients are 34070, 34990, and 19630 M-1 cm-1 for RPA41, RPA32, and RPA14, respectively.

In Vitro Interaction Analysis-- Rabbit polyclonal antibodies were raised against homogenous RPA41 as reported previously for Hjc (43). All subsequent steps were performed at room temperature. Various combinations of purified RPA subunit proteins (each at 2 µM) were mixed in TBS-T (20 mM Tris-HCl, pH 7.5, 0.3 M NaCl, 0.1% Triton X-100) and incubated at 60 °C for 5 min. After cooling down at room temperature, anti-RPA41 serum was added to the mixture, which was incubated for 30 min. An aliquot of protein A-Sepharose (Amersham Pharmacia Biotech) was added to the mixture, which was further incubated for 30 min with a rotary shaker. Protein A-Sepharose-bound antibody-antigen complexes were separated from free proteins by centrifugation and washing three times in TBS-T. The precipitates were analyzed by 12.5% SDS-PAGE and Coomassie Brilliant Blue staining. The gel filtration chromatography was performed as described above, using a buffer containing 0.3 M sodium chloride, because RPA32 immediately precipitated in a solution with a low salt concentration.

Gel-retardation Assay-- Various combinations of RPA complexes or subunits were incubated with 5 nM of a 5'-32P-labeled oligo(dT)30 for 10 min at 70 °C in binding buffer (20 mM Tris-HCl, pH 8.8, 15 mM MgCl2, 2 mM DTT, 50 µg/ml bovine serum albumin). The reaction products were analyzed by 1% agarose gel electrophoresis in 0.1× TAE (4 mM Tris-acetate, 0.1 mM EDTA, (pH 8.4) buffer, and the bands were detected by autoradiography.

Strand-exchange Reaction-- RadA (7.5 µM) was incubated with single-stranded pUC118 (18 µM nucleotide) in exchange buffer (20 mM Tris-HCl, pH 8.8, 15 mM MgCl2, 2 mM DTT, 50 µg/ml bovine serum albumin, 2.5 mM ATP) at 70 °C for 10 min. Then, various concentrations of RPA complexes were added to the mixture, which was further incubated for 10 min. The strand-exchange reaction was started by the addition of a PstI-digested and 3'-32P-labeled double-stranded pUC118 (14.5 µM nucleotide) to the mixture and was incubated at 70 °C for 1 h. The reaction mixtures were deproteinized at 37 °C for 30 min in 0.1 mg/ml of proteinase K and 0.5% SDS and were separated by 1.2% agarose gel electrophoresis in 1× TAE buffer. The bands were detected by autoradiography.

Immunoprecipitation Analysis-- P. furiosusstrain Vcl, DSM3638T (44), was cultured as described earlier (45). The cells from 1 liter of culture were suspended in 20 ml of Buffer A containing 1 mM of PMSF and were disrupted by sonication. The soluble extract was obtained by centrifugation and was used for the immunoprecipitation analysis. All subsequent steps were carried out at room temperature, as described earlier (46). Polyclonal antisera that were raised independently by immunizing rabbits (anti-RPA41, anti-RPA14 (this study), anti-RadA (38), anti-RadB (47), anti-Hjc (43), anti-Pol I (46), anti-DNA polymerase II small subunit (46), anti-RFC small subunit (48), anti-PCNA (49), anti-primase p41 subunit (50), and anti-PI-PfuI (51)) were used to precipitate proteins from a P. furiosus cell extract. The precipitates were analyzed by Western blotting by using an enhanced chemiluminescence system (Amersham Pharmacia Biotech), according to the supplier's method.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Identification of the Genes for RPA Proteins-- To identify and to characterize the RPA of P. furiosus, we searched for the candidate open reading frame (ORF) in the P. furiosus genome by using the amino acid sequence of the E. coli SSB or the human RPA70 subunit as a query. One ORF with a sequence similar to the eukaryotic RPA70 was found and was designated RPA41 from its estimated molecular mass (41 kDa). The RPA41 protein contains two distinct regions, which are closely related to the OB fold domain and the zinc finger motif in the human RPA70 (Fig. 1). This protein shares some sequence similarity with M. jannaschii RPA, which contains four tandem repeats of the putative OB fold domains and the zinc finger motif. M. jannaschii RPA exists as a monomer form in solution and can bind to a single-stranded DNA without any other factors (40). To examine whether RPA41 binds to a single-stranded DNA, the gene for this ORF, designated rpa1, was cloned, and the gene product was characterized. The purified protein that was consistent with the predicted size on SDS-PAGE was confirmed by N-terminal amino acid sequencing. However, a gel-retardation assay showed that RPA41 did not bind to a single-stranded DNA, as shown below. This result suggested two possibilities. First, RPA41 needs to form a complex with other subunits to exhibit the single-stranded DNA binding, and second, the true RPA in P. furiosus is a different protein with very weak similarity to the bacterial SSB and eukaryotic RPA proteins. The similarity search was carried out further, using the sequence of the putative OB fold domain of RPA41 as a query, and one candidate ORF was found. Interestingly, the gene for the candidate ORF was located downstream of the rpa1 gene, with one more small ORF between them (Fig. 2). The genes for the three ORFs seem likely to compose an operon based on their gene organization. The entire amino acid sequences of the two ORFs beside RPA41 are not similar to those of any ORFs in the currently available data base; however, they have a OB fold-like sequence, as shown in Fig. 1A. Therefore, the genes were designated as rpa2 and rpa3, respectively, and the gene products were designated as RPA14 and RPA32, respectively, from their molecular masses.


View larger version (58K):
[in this window]
[in a new window]
 
Fig. 1.   Amino acid sequence comparison. A, comparison of the putative OB fold sequences of the P. furiosus RPA subunits with the M. jannaschii RPA (first domain) and the human RPA70 (domain B), 32, and 14 subunits. The identical and conserved residues are shaded black and gray, respectively. The residues marked with asterisks are those that make contact with the single-stranded DNA in the crystal structure of human RPA70 (5). The open bar and the black arrows indicate the position of an alpha  helix and the beta  sheets in the conserved OB fold of human RPA70, respectively. B, alignment of the zinc finger domains of the P. furiosus RPA41, the M. jannaschii RPA, and the human RPA70. Cysteine residues thought to be part of a zinc finger domain are indicated by asterisks.


View larger version (6K):
[in this window]
[in a new window]
 
Fig. 2.   Restriction enzyme map of the rpa genes and their flanking region. The ORFs found from the nucleotide sequences are indicated by arrowheads. The abbreviations are as follows: B, BamHI; Bg, BglII; C, ClaI; EV, EcoRV; E22, EcoT22I; H, HindIII; Hc, HincII; Nd, NdeI; Sa, SacI; Sh, SphI; Xb, XbaI; and Xh, XhoI.

To investigate whether the three ORFs form a complex in P. furiosus and act as a single-stranded DNA-binding protein, rpa2 and rpa3 were also independently cloned, and each protein was produced in E. coli cells. Although RPA14 was produced as a soluble form in E. coli cells, like the case of RPA41, most of the RPA32 proteins were precipitated in the insoluble fraction when it was overproduced in E. coli cells. Therefore, to purify the small amount of soluble RPA32 protein, it was fused to a hexahistidine tag at the C terminus and was purified by metal affinity chromatography, as described under "Experimental Procedures." Each protein was purified to homogeneity, as shown in Fig. 3A, and was confirmed by N-terminal amino acid sequencing. From 1 liter of E. coli culture, about 4.8 mg of RPA41, 0.5 mg of RPA32, and 7.6 mg of RPA14 were purified.


View larger version (25K):
[in this window]
[in a new window]
 
Fig. 3.   Purification and complex formation of PfuRPA. A, purification of RPA proteins. Purified RPA subunit proteins (0.5 µg) were subjected to 12.5% SDS-PAGE and were stained with Coomassie Brilliant Blue. B, reconstitution of PfuRPA in vitro. Purified proteins (each at 2 µM) were mixed at the indicated combinations and were precipitated by anti-RPA41 or anti-Hjc (as a negative control) antibodies. Protein A-Sepharose-bound antibody-antigen complexes were analyzed by 12.5% SDS-PAGE and Coomassie Brilliant Blue staining.

Complex Formation by PfuRPA Proteins-- Immunoprecipitation analyses were carried out to test whether RPA41, RPA32, and RPA14 form a complex. The three purified proteins were mixed in the various combinations indicated in Fig. 3B and then were precipitated by the anti-RPA41 antibody. The precipitates were separated by SDS-PAGE, and the bands were detected by Coomassie Brilliant Blue staining (Fig. 3B). RPA41 and RPA32 were co-precipitated by anti-RPA41, suggesting that RPA32 directly interacts with RPA41. On the other hand, RPA14 was not precipitated with RPA41. When RPA32 was mixed together with RPA41 and RPA14, RPA14 was co-precipitated with RPA41 and RPA32 by anti-RPA41 antibody. Furthermore, to confirm the complex formation, the three proteins were subjected to gel-filtration chromatography. When each protein was independently applied to the column, the peaks of the eluted fractions of RPA41, RPA14, and RPA32 corresponded to 46, 12, and 44 kDa, respectively, indicating that each protein exists as a monomer in solution (data not shown). The three sets of mixtures, RPA41 and RPA32 (RPA41/32), RPA14 and RPA32 (RPA14/32), and RPA41, RPA14, and RPA32 (RPA41/14/32), eluted at positions corresponding to about 190, 100, and 250 kDa, respectively, clearly showing the formation of the complex (data not shown). In the case of the mixture of RPA41 and RPA14, the proteins eluted independently, although both proteins were pre-mixed before loading (data not shown). According to these observations, RPA32 directly interacts with both RPA41 and RPA14 and forms the stable complex together with RPA41 and RPA14. Therefore, the RPA41/32/14 complex was designated as PfuRPA.

DNA Binding Properties of PfuRPA-- The eukaryotic RPA complex has been reported to bind tightly to single-stranded DNA, although the binding activity of each subunit is quite low. To investigate whether PfuPRA binds to a single-stranded DNA, a gel retardation assay was performed by using 32P-labeled oligo(dT)30 DNA (Fig. 4A). As shown in Fig. 4A, binding of the RPA41/14/32 complex (10 ng) to oligo(dT)30 DNA was detected. The DNA band was not shifted with each subunit of the RPA, even though an excess amount of protein (2 µg) was present in the reaction. Interestingly, the RPA41/32 complex also binds to the DNA with the same affinity as that of the RPA41/32/14 complex. However, the RPA41/32-DNA complex seems to be less stable, as compared with the three-subunit complex because of the smeared shift band on the gel retardation assay. When two hundred nanograms of the RPA14/32 complex were incubated with oligo(dT)30, a very faint shift band appeared.


View larger version (43K):
[in this window]
[in a new window]
 
Fig. 4.   Single-stranded DNA binding activity of RPA complexes. A, a fixed quantity of 32P-labeled oligo(dT)30 (5 nM) was incubated with the indicated amounts of RPA41/14/32, RPA41/32, RPA14/32 complex, or each subunit protein in a 10-µl reaction mixture at 70 °C for 10 min. Protein-DNA complexes were analyzed by 1% agarose gel electrophoresis in 0.1× TAE buffer, and the bands were visualized by autoradiography. B, competition analysis of the single-stranded DNA binding activity of RPA complexes using single-stranded or double-stranded M13 DNA as a competitor. A 32P-labeled oligo(dT)30 (5 nM) was premixed with the indicated amount of single (ss)- or double (ds)-stranded M13 DNA and was then incubated with 50 ng of RPA41/14/32 or RPA41/32 complex in a 10-µl reaction mixture at 70 °C for 10 min.

To confirm the binding specificity of the PfuRPA to single-stranded DNA, binding competition analyses were performed. A fixed amount of PfuRPA and 32P-labeled oligo(dT)30 DNA were incubated with increasing amounts of unlabeled single- or double-stranded M13 DNA (Fig. 4B). When 20 nanograms of unlabeled single-stranded DNA were added as a competitor, the binding of PfuRPA to oligo(dT)30 DNA was completely inhibited. On the other hand, this binding did not decrease in the presence of an excess amount of unlabeled double-stranded DNA. The binding activity of RPA41/32 to oligo(dT)30 DNA was also inhibited by unlabeled single-stranded, but not double-stranded, M13 DNA. These data clearly show that the RPA41/14/32 and RPA41/32 complexes both have binding specificity to single-stranded DNA.

Stimulation of the RadA-mediated Strand-exchange Reaction by PfuPRA-- Both bacterial SSB and eukaryotic RPA have been reported to stimulate the strand-exchange reaction promoted by the recombination proteins RecA and Rad51, respectively, as described above. Previously, we demonstrated that P. furiosus RadA, the eukaryotic Rad51 homologue in archaea, mediated the three-strand-exchange reaction using two types of plasmid DNA as the substrate, and therefore, RadA was an actual recombinase in P. furiosus cells (38). To determine the involvement of PfuRPA in homologous recombination, we investigated whether RPA complexes could stimulate the RadA-promoted strand-exchange reaction, by using the assay system shown in Fig. 5A. When PfuRPA was added to the reaction mixtures after RadA, the formation efficiency of the joint molecule was about three times higher than that by RadA alone (Fig. 5B). Moreover, a distinct band of nicked circular DNA, the final product of the exchange reaction, was observed by the addition of PfuRPA, although RadA alone produced a small amount of nicked circular DNA. Interestingly, RPA41/32 was more effective for the stimulation of the strand-exchange reaction, and it stimulated this activity at least 5-fold higher than RadA alone. This result suggests that RPA14 is not an essential factor for the RPA function in the recombination reaction. We further examined whether RPA14 forms an exact RPA complex together with the other subunits in P. furiosus cells by using an immunoprecipitation analysis. A distinct amount of RPA14 was co-precipitated with RPA41, suggesting that RPA14 tightly bound to other subunits and formed the RPA complex in P. furiosus cells (Fig. 6A).


View larger version (31K):
[in this window]
[in a new window]
 
Fig. 5.   Both RPA41/14/32 and RPA41/32 complexes enhance the RadA-promoted strand-exchange reaction. A, schematic drawing of the strand-exchange reaction. B, RadA (7.5 µM) was preincubated with the single-stranded DNA (18 µM nucleotide) for 10 min at 70 °C and then RPA41/14/32 (lane 3, 40 ng/µl; lane 4, 60 ng/µl; lane 5, 100 ng/µl) or RPA41/32 (lane 6, 30 ng/µl; lane 7, 45 ng/µl; lane 8, 75 ng/µl) complex was added to the mixtures, which were further incubated for 10 min. The strand-exchange reaction was started by adding 32P-labeled double-stranded DNA (14.5 µM nucleotide) to the mixture and was incubated at 70 °C for 1 h. The reaction products were analyzed by 1.2% agarose gel electrophoresis after deproteinizing. The bands were detected by autoradiography.


View larger version (37K):
[in this window]
[in a new window]
 
Fig. 6.   RPA interacts with RadA and various replication proteins in P. furiosus cells. A, the cell extract was precipitated with anti-RPA41 (RPA41), anti-RPA14 (RPA14), and anti-PI-PfuI (PI-PfuI; as a negative control). Cell extracts without immunoprecipitation (Pfu extract) or precipitated with phosphate-buffered saline-treated protein A-Sepharose (PBS) were loaded as controls. The precipitates were analyzed by Western blotting using anti-RPA41 and anti-RPA14 antibodies. B, the cell extract was precipitated with anti-RPA41 (RPA41), anti-RadA (RadA), anti-RadB (RadB), anti-Hjc (Hjc), anti-Pol I (PolI), anti-Pol II small subunit (DP1), anti-RFC small subunit (RFCs), anti-PCNA (PCNA), anti-primase p41 subunit (primase), and anti-PI-PfuI (PI-PfuI). The precipitates were detected by Western blotting using anit-RPA41 and anti-RadA antibodies.

Interaction of RPA with RadA and Other Replication Proteins in P. furiosus Cells-- Eukaryotic RPA is known to interact with various proteins involved in DNA replication, recombination, and repair in cells, and these physical interactions affect the various activities mediated by these proteins. To investigate the physical interactions between RPA41 and various proteins involved in recombination and replication in P. furiosus, immunoprecipitation analyses were carried out using various antisera including anti-PI-PfuI, which is the antiserum against a homing endonuclease working for the double-stranded DNA cleavage (51), as a negative control (Fig. 6B). From the P. furiosus cell extract, RPA41 was co-precipitated with RadA by the anti-RadA antibody, and RadA was also co-precipitated with RPA41 by the anti-RPA41 antibody. These results agree with the direct interaction of eukaryotic RPA with Rad51 (52). RPA41 was also co-precipitated with Hjc, which resolves the Holliday junction intermediate, by the anti-Hjc antibody. RPA41 was not precipitated with RadB by the anti-RadB. When antibodies against replication proteins, including Pol I, DNA polymerase II small subunit, RFCS, PCNA, and primase p41 subunit, were used for precipitation, RPA41 was detected in all of the antibody-bound fractions (Fig. 6B). RPA41 was not precipitated with PI-PfuI, a DNA-binding protein. These results indicate that PfuRPA is also involved in the DNA replication machinery in P. furiosus cells.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this paper, we identified the RPA from P. furiosus and showed that PfuRPA forms a stable complex consisting of three proteins, RPA41, RPA14, and RPA32. In contrast to the M. jannaschii RPA, which has four repeats of the putative OB fold domain, the P. furiosus RPA41 has only one corresponding sequence. Eukaryotic RPA and bacterial SSB, which have stable binding ability to single-stranded DNA, contain at least four OB fold domains in their complexes. Indeed, RPA41 by itself did not show stable binding to single-stranded DNA. The RPA41/32 complex bound to single-stranded DNA, and furthermore, the RPA41/14/32 complex shows more stable binding to the DNA (Fig. 4A). P. furiosus RPA14 and RPA32 are not similar to any known ORFs in the current data base. However, a putative OB fold domain is present in both proteins, as shown in Fig. 1A. These domains may be necessary for stable DNA binding. The zinc finger motif, which was recently shown to be important for RPA to modulate its DNA binding (14), is also conserved between the eukaryotic RPA70 and archaeal RPA41 homologues. However, the fourth residue of four repeats of cysteine in the putative zinc finger motif is substituted with histidine in the archaeal RPA homologs, except for those of M. jannaschii and M. thermoautotrophicus. Currently, the genome sequences of seven euryarcheaotes and one crenarchaeote have been published. We found RPA41 and RPA32 homologues in seven and six euryarchaeal genomes, respectively. Pyrococcus abyssi and Pyrococcus horikoshii contain an operon corresponding to RPA41, RPA14, and RPA32, like that in P. furiosus. In Archaeoglobus fulgidus and Thermoplasma acidophilum, two RPA41 homologues, one with a putative zinc finger motif (AF0780 and Ta0387) and the other without the motif (AF0382 and Ta1149), were found, in addition to one RPA32 homologue (AF0779 and Ta0388). Interestingly, the RPA41 homologue containing the zinc finger motif and the RPA32 homologue are arranged in tandem in both genomes, suggesting that these proteins functionally associate. Furthermore, Halobacterium sp. NRC1 has four RPA41 homologues, two with a zinc finger motif (Vng1255c and Vng2160c) and the other two without it (Vng0133 g and Vng6403 h). Two homologs were also found for RPA32 (Vng1253c and Vng2162c) in the genome of this haloarchaea, and they were arranged in tandem with each of the RPA41 homologs containing a zinc finger motif. These findings imply that in euryarchaeotes, except for methanogens, the RPA complex consists of three subunits, one subunit with a zinc finger motif and two subunits without a zinc finger motif. Interestingly, we found an ORF (MJ1654), with a sequence similar to that of RPA32, in M. jannaschii. To compare it with that RPA in M. thermoautotrophicus, which contains five repeats of the OB fold domain (39), MJ1654 may associate with MJ1159 (RPA; see Ref. 40), resulting in five repeats of the domain in the M. jannaschii RPA molecule. We found the RPA41 homolog in the two crenarchaeal genomes, Aeropyrum pernix and Sulfolobus solfataricus; however, no RPA32 homolog has been found in these crenarchaeotes. During the preparation of this manuscript, the S. solfataricus RPA41 was published (53). It would be very interesting to understand the evolutional relationships of the archaeal RPA subunits. Further detailed analyses will give us some interesting insight regarding this matter.

The in vitro strand-exchange ability of the archaeal RadA is weak, as also reported for the eukaryotic Rad51 proteins. SSB and RPA are thought to help the DNA strand exchange by removing the secondary structure of the single-stranded DNA. However, E. coli SSB and yeast RPA failed to stimulate the reaction mediated by S. solfataricus RadA, probably because of the high temperature used for the reaction (35). Our results show that the RPA complex from P. furiosus clearly stimulates the strand-exchange reaction promoted by P. furiosus RadA (Fig. 5). Interestingly, the RPA41/32 complex stimulated the strand exchange better than the RPA41/14/32 complex. It is possible that the two-subunit complex dissociates from the DNA more easily than the three-subunit complex, because the RPA41/32-DNA complex is less stable than the RPA41/14/32-DNA complex, as shown in Fig. 4A. This less stable interaction of RPA41/32 may be favorable for the reaction, because it may interfere with the processing of the strand pairing and exchange by RadA. The role of the eukaryotic RPA14 in DNA binding is still not clear, and it is now believed that the eukaryotic RPA14 has a structural role in the assembly of the RPA heterotrimer. Further analyses of P. furiosus RPA14 will help to reveal the exact function of the smallest subunit of RPA. In this study, we purified three subunits independently and prepared PfuRPA by mixing. As described above, soluble RPA32 was difficult to purify, and therefore, it was prepared as the histidine-tagged form. Construction of the co-expression system of the three genes in one cell may provide a better PfuRPA complex without the His-tag on RPA32. The PfuRPA produced by the co-expression system might stimulate the RadA-mediated strand-exchange reaction with more extent.

It is interesting that PfuRPA interacts with Hjc, the Holliday junction resolvase that we identified recently in P. furiosus (43). Our preliminary analysis showed that PfuRPA neither stimulated nor inhibited the junction-cleaving activity of Hjc in vitro. If a direct interaction with Hjc occurs in the cells, then PfuRPA is probably involved in the archaeal resolvasome. We are now searching for the branch-migrating enzyme in P. furiosus, and further progress will provide many clues to elucidate the complete molecular mechanism of the homologous recombination.

According to immunoprecipitation experiments, RPA interacts with various proteins related to DNA replication in P. furiosus cells (Fig. 6B). Studies on the in vitro SV40 replication system have shown that RPA interacts with SV40 T-antigen (which has helicase activity) and works in the denaturation of the replication origin (54). Then, the DNA polymerase alpha -primase complex is recruited to synthesize primers. The interaction between RPA and the DNA polymerase alpha -primase complex is direct, via the primase (20). As shown in Fig. 6, as well as in our previous report (50), RPA41 was significantly co-precipitated with primase when we used an anti-primase antibody from P. furiosus cells. In the elongation step of eukaryotic replication, the Pol alpha -primase remains attached to its priming site by contacting RPA, and RFC replaces it via a competitive interaction with RPA. RFC assembles PCNA onto the primed site, which Pol delta  can access by contacting RPA (55). Our immunoprecipitation experiments support the idea that PfuRPA interacts with RFC and two DNA polymerases (Pol I and Pol II), respectively. These findings suggest that PfuRPA probably associates with these replication proteins and plays important roles at the initiation and elongation steps of DNA replication in P. furiosus cells.

RPA is one of the key proteins in DNA replication and recombination. Identification of the RPA complex in P. furiosus will contribute to the progress of archaeal molecular recognition in these essential processes in DNA metabolism. Furthermore, the similarity of its molecular composition to that of the eukaryotic RPA will let us use PfuRPA as a good model to understand the structure-function relationships of eukaryotic proteins in more detail.

    ACKNOWLEDGEMENTS

We thank Drs. I. Cann for help with sequence search and H. Shinagawa and K. Morikawa for discussions. We are grateful to Dr. Y. Shimura, the director of Biomolecular Engineering Research Institute, for continuous encouragement.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Molecular Biology, Biomolecular Engineering Research Inst., 6-2-3 Furuedai, Suita, Osaka 565-0874, Japan. Tel.: 81-6-6872-8208; Fax: 81-6-6872-8219; E-mail: ishino@beri.co.jp.

Published, JBC Papers in Press, May 7, 2001, DOI 10.1074/jbc.M102423200

 The nucleotide sequences reported in this paper have been submitted to the DDBJ/GenBankTM Data Bank with accesion numbers AB060559, AB060560 and AB060561.

    ABBREVIATIONS

The abbreviations used are: SSB, single-stranded DNA-binding protein; RPA, replication protein A; OB, oligonucleotide/oligosaccharide binding; Pol, DNA polymerase; PCNA, proliferating cell nuclear antigen; Pfu, P. furiosus; DTT, dithiothreitol; PMSF, phenylmethanesulfonyl fluoride; PAGE, polyacrylamide gel electrophoresis; RFC, replication factor C; PI, protein intron (intein).

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Lohman, T. M., and Ferrari, M. E. (1994) Annu. Rev. Biochem. 63, 527-570[CrossRef][Medline] [Order article via Infotrieve]
2. Wold, M. S. (1997) Annu. Rev. Biochem. 66, 61-92[CrossRef][Medline] [Order article via Infotrieve]
3. Iftode, C., Daniely, Y., and Borowiec, J. A. (1999) Crit. Rev. Biochem. Mol. Biol. 34, 141-180[Abstract/Free Full Text]
4. Raghunathan, S., Ricard, C. S., Lohman, T. M., and Waksman, G. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 6652-6657[Abstract/Free Full Text]
5. Bochkarev, A., Pfuetzner, R. A., Edwards, A. M., and Frappier, L. (1997) Nature 385, 176-181[CrossRef][Medline] [Order article via Infotrieve]
6. Bochkarev, A., Bochkareva, E., Frappier, L., and Edwards, A. M. (1999) EMBO J. 18, 4498-4504[Abstract/Free Full Text]
7. Murzin, A. G. (1993) EMBO J. 12, 861-867[Abstract]
8. Bycroft, M., Hubbard, T. J. P., Proctor, M., Freund, S. M. V., and Murzin, A. G. (1997) Cell 88, 235-242[Medline] [Order article via Infotrieve]
9. Gomes, X. V., and Wold, M. S. (1996) Biochemistry 35, 10558-10568[CrossRef][Medline] [Order article via Infotrieve]
10. Pfuetzner, R. A., Bochkarev, A., Frappier, L., and Edwards, A. M. (1997) J. Biol. Chem. 272, 430-434[Abstract/Free Full Text]
11. Kim, D.-K., Stigger, E., and Lee, S.-H. (1996) J. Biol. Chem. 271, 15124-15129[Abstract/Free Full Text]
12. Braun, K. A., Lao, Y., He, Z., Ingles, C. J., and Wold, M. S. (1997) Biochemistry 36, 8443-8454[CrossRef][Medline] [Order article via Infotrieve]
13. Lin, Y. L., Chen, C., Keshav, K. F., Winchester, E., and Dutta, A. (1996) J. Biol. Chem. 271, 17190-17198[Abstract/Free Full Text]
14. Lin, Y.-L., Shivji, M. K. K, Chen, C., Koloddner, R., Wood, R. D., and Dutta, A. (1998) J. Biol. Chem. 273, 1453-1461[Abstract/Free Full Text]
15. Bochkareva, E., Korolev, S., and Bochkarev, A. (2000) J. Biol. Chem. 275, 27332-27338[Abstract/Free Full Text]
16. Bochkareva, E., Frappier, L., Edwards, A. M., and Bochkarev, A. (1998) J. Biol. Chem. 273, 3932-3936[Abstract/Free Full Text]
17. Philipova, D., Mullen, J. R., Maniar, H. S., Lu, J., Gu, C., and Brill, S. J. (1996) Genes Dev. 10, 2222-2233[Abstract]
18. Gomes, X. V., and Wold, M. S. (1995) J. Biol. Chem. 270, 4534-4543[Abstract/Free Full Text]
19. Mer, G., Bochkarev, A., Gupta, R., Bochkareva, E., Frappier, L., Ingles, C. J., Edwards, A. M., and Chazin, W. J. (2000) Cell 103, 449-456[Medline] [Order article via Infotrieve]
20. Dornreiter, I., Erdile, L. F., Gilbert, I. U., von Winkler, D., Kelly, T. J., and Fanning, E. (1992) EMBO J. 11, 769-776[Abstract]
21. Tsurimoto, T., and Stillman, B. (1989) EMBO J. 8, 3883-3889[Abstract]
22. Kenny, M. K., Lee, S. H., and Hurwitz, J. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 9757-9761[Abstract]
23. Radding, C. M. (1991) J. Biol. Chem. 266, 5355-5358[Free Full Text]
24. Kowalczykowski, S. C., Dixon, D. A., Eggleston, A. K., Lauder, S. D., and Rehrauer, W. M. (1994) Microbiol. Rev. 58, 401-465[Abstract]
25. Shinohara, A., and Ogawa, T. (1995) Trends Biochem. Sci. 20, 387-391[CrossRef][Medline] [Order article via Infotrieve]
26. Sugiyama, T., Zaitseva, E. M., and Kowalczykowski, S. C. (1997) J. Biol. Chem. 272, 7940-7945[Abstract/Free Full Text]
27. Sung, P. (1997) J. Biol. Chem. 272, 28194-28197[Abstract/Free Full Text]
28. Gupta, R. C., Golub, E. I., Wold, M. S., and Radding, C. M. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 9843-9848[Abstract/Free Full Text]
29. Benson, F. E., Baumann, P., and West, S. C. (1998) Nature 391, 401-404[CrossRef][Medline] [Order article via Infotrieve]
30. Shinohara, A., and Ogawa, T. (1998) Nature 391, 404-407[CrossRef][Medline] [Order article via Infotrieve]
31. New, J. H., Sugiyama, T., Zaitseva, E., and Kowalczykowski, S. C. (1998) Nature 391, 407-410[CrossRef][Medline] [Order article via Infotrieve]
32. Sung, P. (1997) Genes Dev. 11, 1111-1121[Abstract]
33. Petukhova, G., Stratton, S., and Sung, P. (1998) Nature 393, 91-94[CrossRef][Medline] [Order article via Infotrieve]
34. Petukhova, G., Van Komen, S., Vergano, S., Klein, H., and Sung, P. (1999) J. Biol. Chem. 274, 29453-29462[Abstract/Free Full Text]
35. Seitz, E. M., Brockman, J. P., Sandler, S. J., Clark, A. J., and Kowalczykowski, S. C. (1998) Genes Dev. 12, 1248-1253[Abstract/Free Full Text]
36. Kil, Y. V., Baitin, D. M., Masui, R., Bonch-Osmolovskaya, E. A., Kuramitsu, S., and Lanzov, V. A. (2000) J. Bacteriol. 182, 130-134[Abstract/Free Full Text]
37. Spies, M., Kil, Y., Masui, R., Kato, R., Kujo, C., Ohshima, T., Kuramitsu, S., and Lanzov, V. (2000) Eur. J. Biochem. 267, 1125-1137[Abstract/Free Full Text]
38. Komori, K., Miyata, T., DiRuggiero, J., Holley-Shanks, R., Hayashi, I., Cann, I. K., Mayanagi, K., Shinagawa, H., and Ishino, Y. (2000) J. Biol. Chem. 275, 33782-33790[Abstract/Free Full Text]
39. Chedin, F., Seitz, E. M., and Kowalczykowski, S. C. (1998) Trends Biol. Sci. 23, 273-277[CrossRef]
40. Kelly, T. J., Simancek, P., and Brush, G. S. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 14634-14639[Abstract/Free Full Text]
41. Kelman, Z., Pietrokovski, S., and Hurwitz, J. (1999) J. Biol. Chem. 274, 28751-28761[Abstract/Free Full Text]
42. Jarvis, T. C., Paul, L. S., and von Hippel, P. H. (1989) J. Biol. Chem. 264, 12709-12716[Abstract/Free Full Text]
43. Komori, K., Sakae, S., Shinagawa, H., Morikawa, K., and Ishino, Y. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 8873-8878[Abstract/Free Full Text]
44. Fiala, G., and Stetter, K. O. (1986) Arch. Microbiol. 145, 56-61
45. Uemori, T., Ishino, Y., Toh, H., Asada, K., and Kato, I. (1993) Nucleic Acids Res. 21, 259-265[Abstract]
46. Cann, I. K. O., Komori, K., Toh, H., Kanai, S., and Ishino, Y. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 14250-14255[Abstract/Free Full Text]
47. Hayashi, I., Morikawa, K., and Ishino, Y. (1999) Nucleic Acids Res. 27, 4695-4702[Abstract/Free Full Text]
48. Cann, I. K. O., Ishino, S., Yuasa, M., Daiyasu, H., Toh, H., and Ishino, Y. (2001) J. Bacteriol. 183, 2614-2623[Abstract/Free Full Text]
49. Cann, I. K. O., Ishino, S., Hayashi, I., Komori, K., Toh, H., Morikawa, K., and Ishino, Y. (1999) J. Bacteriol. 181, 6591-6599[Abstract/Free Full Text]
50. Bocquier, A. A., Liu, L., Cann, I. K. O., Komori, K., Kohda, D., and Ishino, Y. (2001) Curr. Biol. 11, 452-456[CrossRef][Medline] [Order article via Infotrieve]
51. Komori, K., Fujita, N., Ichiyanagi, K., Morikawa, K., and Ishino, Y. (1999) Nucleic Acids Res. 27, 4167-4174[Abstract/Free Full Text]
52. Golub, E. I., Gupta, R. C., Haaf, T., Wold, M. S., and Radding, C. M. (1998) Nucleic Acids Res. 26, 5388-5393[Abstract/Free Full Text]
53. Wadsworth, R. I., and White, M. F. (2001) Nucleic Acids Res. 29, 914-920[Abstract/Free Full Text]
54. Bullock, P. A. (1997) Crit. Rev. Biochem. Mol. Biol. 32, 503-568[Abstract]
55. Yuzhakov, A., Kelman, Z., Hurwitz, J., and O'Donnell, M. (1999) EMBO J. 18, 6189-6199[Abstract/Free Full Text]


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.