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
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ABSTRACT |
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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.
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 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.
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- 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 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.
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.
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.
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.
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).
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.
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 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.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-primase complex, resulting in the
formation of an initiation complex at the replication origin (20). The
RPA also stimulates Pol
-primase activity and
PCNA-dependent Pol
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).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-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-
-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-
-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.
1 cm
1 for RPA41, RPA32, and
RPA14, respectively.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (58K):
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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 helix and the
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.
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[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.
View larger version (25K):
[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.
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[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.
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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 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.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-primase complex
is recruited to synthesize primers. The interaction between RPA and the
DNA polymerase
-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
-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
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.
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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.
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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.
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.
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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).
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