(Received for publication, October 21, 1996, and in revised form, December 22, 1996)
From the Department of Molecular Biology, Vanderbilt University, Nashville, Tennessee 37235
Replication factor C (RF-C), a complex of five
subunits, is an essential eukaryotic protein involved in both DNA
replication and DNA repair. To generate an easily accessible source of
human RF-C for biochemical and genetic studies, we cloned the cDNAs of all five subunits into baculoviruses so that each subunit could be
expressed both as a non-fused polypeptide and as an N-terminal His-tagged fusion (-his). Co-infection of insect cells with five baculoviruses encoding individual RF-C subunits (p140, p40, p38, p37,
and p36) yielded a protein preparation active in two assays characteristic for authentic RF-C: stimulation of DNA polymerase DNA synthesis on singly primed single-stranded DNA template and
formation of a complex of proliferating cell nuclear antigen with
circular double-stranded DNA. Functional recombinant RF-C containing
p40-his, p37-his, or p36-his was isolated using affinity resin. Active
RF-C was reconstituted only by co-expression of all five subunits. A
model for assembly of RF-C from individual subunits was derived from
co-purification experiments performed with various combinations of
His-tagged and non-fused subunits expressed by co-infection of insect
cells with recombinant baculoviruses. p37 and p36 are proposed to form
the first intermediate, which, upon addition of either p40 or p38,
generates stable tertiary complexes: p40·p37·p36 and
p38·p37·p36. The remaining fourth small subunit binds to the
tertiary complex to form a quaternary complex p40·p38·p37·p36.
Large subunit p140 binds last to form the five-subunit protein.
Replication factor C (RF-C)1 was first
isolated from human 293 cells as an essential replication factor in the
in vitro simian virus 40 DNA replication system (1). This
protein was also purified from HeLa cells (2), yeast (3, 4), and calf thymus (5) and assayed by its ability to stimulate pol activity on
a primed ssDNA in the presence of PCNA, RP-A (or Escherichia coli SSB), and ATP. Although discovered as a protein essential for
DNA replication, RF-C itself had little or no effect on DNA synthesis
by polymerases
,
, and
. In contrast, the combination of RF-C
with PCNA, RP-A, and ATP strongly inhibited pol
and stimulated pol
and pol
. Isolation of stable pol
and pol
complexes,
formed with DNA in the presence of RF-C, PCNA, and ATP, indicated that
pol
and pol
directly interact with these auxiliary proteins
(5-8). Biochemical studies on RF-C and PCNA suggested that the
physiological function of RF-C is to catalyze the loading of PCNA onto
a DNA template using the energy of ATP hydrolysis (6, 8, 9). Using
different approaches, it has been shown that the PCNA loaded onto DNA
is able to track along the template (10-14). Preferential binding of
RF-C to the template-primer junction, together with the footprinting
data, suggested that assembly of the RF-C·PCNA complex occurred at
the 3
-OH end of the primer (15). Analysis of loading of
[32P]PCNA onto DNAs of different structure showed that
the assembly of the primary RF-C·PCNA complex does not require 3
-OH
ends and might occur directly on dsDNA, whose topological structure and sequence do not present any restriction to loading. Such an initial salt-sensitive RF-C·PCNA complex was suggested to slide along DNA to
the 3
-OH end of a primer, where the final conversion of the
intermediate complex to the catalytically competent salt-resistant PCNA
clamp takes place (13).
The close cooperation of RF-C and PCNA suggested that these proteins might act together in various DNA transactions. Both RF-C and PCNA were required to reconstitute DNA replication (16) and nucleotide excision repair in vitro (17). Base excision repair has been found to be carried out in two alternative pathways, one of which is PCNA-dependent (18, 19). The PCNA dependence of base excision repair implies the requirement for RF-C in this process as well.
RF-C is a multiprotein complex composed of five subunits, one large subunit and four small subunits, named according their apparent molecular masses on SDS-PAGE: p140, p40, p38, p37, and p36. The cDNAs encoding all five subunits of human RF-C (20-23) and yeast RF-C (24-27) have been cloned. The five subunits are encoded by different genes that show extensive amino acid sequence homology in the middle part of the polypeptides (22, 23, 25, 27). All five genes encoding the subunits of yeast RF-C have been shown to be essential for viability (24-27). Each human RF-C subunit corresponds closely to its yeast counterpart (27; reviewed in Ref. 28), implying a conserved function of this protein in eukaryotic organisms.
Some of the human and yeast RF-C subunits have been expressed
individually in E. coli to define their role in RF-C
function. Interaction of RF-C with PCNA was attributed to p40 (29). p40 and p37 interacted directly with pol and pol
, respectively, as
well as with each other (29). Yeast Rfc3p (counterpart of p36) and
Rfc4p (counterpart of p40) yielded a stable complex upon co-expression
in E. coli (25). p37 and its yeast homolog Rfc2p showed
specific primer end binding (26, 29). DNA-dependent ATPase
activity was found to reside on yeast Rfc3p (24). Recombinant yeast
RF-C has been overexpressed in yeast by using a plasmid encoding all
five RF-C subunits (30). Active recombinant human RF-C has been
recently reconstituted using an in vitro coupled transcription/translation system (31) and baculovirus expression system
(32).
In this paper we explored the recombinant baculoviruses encoding individual RF-C subunits to analyze the pathway of assembly of multisubunit RF-C complex. We report here the expression of functionally active recombinant RF-C and its various stable subcomplexes. A model for assembly of individual subunits into the five-subunit complex is proposed.
Proteins
Calf thymus pol (1 unit is defined as incorporation of 1 nmol of dTMP into poly(dA)-oligo(dT) (20:1 base ratio) in the presence of 100 ng of PCNA in 60 min at 37 °C), calf thymus RF-C (Mono Q
fraction), and E. coli SSB have been described (5). Human PCNA was overproduced in E. coli with a plasmid pT7/hPCNA
and purified as described (4). The phosphorylatable derivative of PCNA,
called ph-PCNA, was overproduced in E. coli, purified, and
32P-phosphorylated by cAMP-dependent protein
kinase (catalytic subunit) using [
-32P]ATP (13).
Protein gp II was overproduced in E. coli strain K561
bearing pDG117IIA plasmid (33); the latter was kindly provided by K. Horiuchi (National Institute of Genetics, Mishima, Japan). gp II was
purified from the soluble fraction according to Ref. 34, with some
modifications. Monoclonal antibody raised against RFC140 subunit (mAb
19, Ref. 22) was kindly provided by B. Stillman (Cold Spring Harbor
Laboratory, Cold Spring Harbor, NY). Retrotherm reverse transcriptase
was from Epicentre Technologies; Pwo DNA polymerase was from
Boehringer Mannheim; restriction endonucleases, mung bean nuclease, and
T4 DNA ligase were from Promega. Prestained molecular mass standard
protein mixture (195, 112, 84, 63, 52.5, 35, and 32 kDa) was from
Sigma; the 10-kDa protein ladder kit was from Life Technologies,
Inc.
Nucleic Acids
ssDNA and RF I DNA were prepared from bacteriophage M13(mp19). ssDNA was primed with 30-mer oligonucleotide complementary to nt 6306-6335 of the M13(mp19) genome. A nicked dsDNA substrate was prepared from M13 DNA using a unique cleavage of RF I DNA by gp II protein. The cleavage reaction was carried out in the presence of Ca2+ ions (34). The DNA preparations used for PCNA loading assays contained 75% RF II and 25% RF I DNA.
Baculovirus transfer vectors pVL1393, pBacHisA, and pBacHisC were from Invitrogen. A plasmid pSK/RFC140 encoding cDNA of the large subunit of human RF-C was kindly provided by B. Stillman. The plasmids encoding four small subunits of human RF-C (pET5a/40, pET19b/His38, pET5b/37, and pET19b/His36) were kindly provided by J. Hurwitz (Sloan-Kettering Cancer Center).
Construction of Baculovirus Transfer Vectors
RFC140Plasmid pSK/RFC140 was digested with
HaeII, precipitated with ethanol, and treated with mung bean
nuclease (20 units of nuclease/4.6 µg of digested plasmid, 20-min
incubation at 30 °C). Digested DNA was phenol-extracted and cut with
PstI, resulting in a cDNA fragment containing a
blunt-ended 5-terminus upstream of the ORF and a PstI site
in the untranslated 3
-terminus. In the same way, a plasmid pBacHisC
was digested successively by BamHI, mung bean nuclease, and
PstI. cDNA was ligated into the pBacHisC and the
resulting clones analyzed by sequencing to find clones containing the
RFC140 gene in the reading frame of the His-tag leader with a minimal
number of amino acids derived from 5
-untranslated sequence. Intentional excessive treatment of pSK/RFC140 by mung bean nuclease resulted in partial digestion of the untranslated 5
-terminus of
cDNA fragment. The clone chosen for protein expression,
pBacHis/RFC140, encoded Cys-Gly-Ala-Ala between the His-tag and the
initiation Met of the ORF (Fig. 1). The same preparation of RFC140
cDNA fragment (HaeII, mung bean nuclease, and
PstI digest) was ligated into pVL1393 digested with
SmaI and PstI. The clone chosen for expression contained the ORF of RFC140 starting at position +54 of the mutated polyhedrin gene.
RFC40
The 5-end of RFC40 cDNA was modified by PCR to
generate a BamHI site upstream of the ORF. The reaction was
performed with Pwo pol using the forward primer
dTCAAGGATCCATATGGAGGTGGAGGCCG, the backward primer dGCCCCTGTCATTTGAAGC,
and pET5a/40 (20) as a template. The PCR product was digested by
BamHI and SmaI. The fragment containing the
5
-end of RFC40 cDNA (278 nt) was used to replace the
BamHI/SmaI fragment in the original pET5a/40
plasmid. Next, the whole RFC40 cDNA was excised from the modified
plasmid pET/RFC40 as a BamHI/EcoRI fragment and
cloned into a pSK vector. RFC40 cDNA was then recloned as a
BamHI/HindIII fragment from pSK/RFC40 into
pBacHisC. The BamHI/EcoRI fragment from modified plasmid pET/RFC40 was cloned into plasmid pVL1393.
RFC37 cDNA in the plasmid pET5b/37 lacked the
fragment encoding first 17 amino acids of p37 (21). To complete the
cDNA, we performed reverse transcription followed by PCR. Total RNA from 293S cells was kindly provided by Irene Boche (this laboratory). The 5-terminal 435-nt fragment was cloned by reverse transcription of
1 µg of total RNA by Retrotherm reverse transcriptase with primer
dACACGGCTTCCCATCTGAG, followed by PCR using the forward primer
dTCGCGGATCCATATGCAAGCATTTCTTAAAGG and the same backward primer used for
reverse transcription. The PCR product was digested by BamHI
and BstXI. The 272-nt fragment containing the 5
-end of
RFC37 cDNA was used to replace the
BamHI/BstXI fragment in the original pET5b/37.
The full-length RFC37 cDNA was transfered from modified plasmid
pET/RFC37 as a BamHI/PstI fragment into pBacHisC
and as a BamHI/EcoRI fragment into pVL1393.
The cDNAs of both RFC38 and RFC36 were recloned from the corresponding plasmids pET19b/His38 and pET19b/His36 as BamHI/HindIII fragments into pBacHisA and as BamHI/KpnI fragments into pVL1393.
Growth and maintenance of High Five insect cells (ITC Biotechnology GmbH) in adherent cultures, preparation of recombinant baculoviruses, and infection of the cells were performed according to routine protocols (35). High Five cells were grown in Grace's insect medium (Life Technologies, Inc.) supplemented with 10% fetal calf serum (HyClone). For in vivo recombination, the transfer vectors and BaculoGold DNA (Pharmingen) were co-transfected into insect cells using DOTAP transfection reagent (Boehringer Mannheim). To prepare a control virus, the insect cells were co-transfected with unmodified vector pBacHisC and BaculoGold DNA, thus generating a virus encoding 54 amino acid polypeptide with His-tag at the N terminus. Infected cells were incubated for 48 h prior to harvesting the recombinant proteins.
Purification of Non-fused Recombinant RF-C
Analytical Purification on HAP3.8 × 107 cells infected either with five viruses encoding all RF-C subunits or with control virus were lysed in 1.5 ml of buffer A (20 mM Tris-HCl (pH 7.5), 1 mM DTT, 1 mM EDTA, 0.2% (v/v) Nonidet P-40, 10 mM NaHSO3, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml each of aprotinin, leupeptin, and pepstatin) containing 0.1 M NaCl. Cell debris was removed by centrifugation and supernatant loaded onto a 0.2-ml HAP column, equilibrated in buffer A, 0.1 M NaCl. The column was washed with 0.5 ml of buffer A, 0.1 M NaCl, and 1 ml of 0.2 M potassium phosphate in buffer B (potassium phosphate (pH 7.5) at the indicated concentration, 10% (v/v) glycerol, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml each of aprotinin, leupeptin, and pepstatin). Proteins were eluted with 0.4 ml of buffer B, 0.4 M potassium phosphate.
Preparative Purification2.7 × 108 cells
infected with five viruses encoding all RF-C subunits were lysed in 10 ml of buffer A, 0.1 M NaCl. Cell debris was removed by
centrifugation and supernatant loaded onto 2-ml HAP column,
equilibrated in buffer A, 0.1 M NaCl. The column was washed
with 10 ml of buffer B, 0.1 M potassium phosphate. Proteins were eluted with a 25-ml linear gradient of potassium phosphate from
0.1 M to 0.6 M in buffer B. Fractions active in
the pol holoenzyme assay were pooled, diluted 4.3-fold with buffer
C (20 mM Tris-HCl (pH 8.0), 10% (v/v) glycerol, 1 mM DTT, 1 mM EDTA, 0.01% Nonidet P-40, 1 µg/ml each of aprotinin, leupeptin, and pepstatin), and loaded onto a
1-ml Mono Q column equilibrated in buffer C, 0.1 M NaCl.
Proteins were eluted with a 15-ml gradient of NaCl from 0.1 to 0.4 M in buffer C.
3.8 × 107 cells infected by various combinations of viruses were lysed in 1.5 ml of buffer A, 0.1 M NaCl, omitting DTT and EDTA. Cell debris was removed by centrifugation and the supernatant mixed with 30 µl of Ni-NTA resin (QIAGEN). The suspension was gently mixed for 1 h at 4 °C, and the resin was pelleted by centrifugation and washed three times in batch with 0.5 ml of buffer D (20 mM Tris-HCl (pH 7.5), 5 mM imidazole-HCl, 300 mM NaCl, 0.02% (v/v) Nonidet P-40). The proteins were eluted from the resin with 100 µl of buffer E (200 mM imidazole-HCl (pH 7.2), 10% (v/v) glycerol, 0.01% (v/v) Nonidet P-40).
PCNA Loading Assay
Reaction mixtures (final volume of 20 µl) contained 40 mM triethanolamine-HCl (pH 7.5), 1 mM DTT, 10 mM MgCl2, 1 mM ATP, 25 ng of [32P]ph-PCNA, 100 ng of nicked DNA, and RF-C as indicated. Samples were incubated for 4 min at 37 °C, then glutaraldehyde was added to a final concentration of 0.1% (w/v), and the mixtures incubated for another 10 min at 37 °C. The samples were loaded and analyzed on a neutral 0.8% agarose gel in the presence of 0.1% SDS as described (13).
Polymerase Holoenzyme Assay
Reaction mixtures (final volume of 25 µl) contained 40 mM Tris-HCl (pH 7.5), 0.2 mg/ml bovine serum albumin, 1 mM DTT, 10 mM MgCl2, 1 mM ATP, 50 µM each of dATP, dGTP, dCTP, 20 µM [-32P]dTTP (500 cpm/pmol), 100 ng of
primed ssDNA, 100 ng of PCNA, 1.2 µg of E. coli SSB, 0.25 unit of pol
, and RF-C as indicated. Samples were incubated for 30 min at 37 °C, the reactions were terminated by adding 1 ml of
ice-cold 10% (w/v) trichloroacetic acid, and acid-insoluble material
was analyzed by scintillation counting. For product length analysis,
the reactions were terminated by treating them with proteinase K (60 µg/ml) for 30 min at 37 °C in the presence of 1% (w/v) SDS and 20 mM EDTA (pH 8.0). The DNA was then precipitated with
ethanol and the products analyzed on an alkaline 1.5% agarose gel as
described (36).
Other Methods
Protein concentration was determined according to Ref. 37 using Bio-Rad reagents and IgG as a standard.
For Western blot analysis, the proteins separated by SDS-PAGE were blotted to Optitran nitrocellulose membrane (Schleicher & Schuell) and probed with mAb 19 (22). The bands were visualized by using secondary anti-mouse antibody conjugated to alkaline phosphatase (Promega).
Radioactivity in the agarose gels was quantified using a PhosphorImager (Molecular Dynamics). Ethidium bromide-stained agarose gels and Coomassie-stained polyacrylamide gels were photographed using Image Store 7500 (UVP) and analyzed with NIH Image 1.57 software.
The cDNAs of
all five RF-C subunits were cloned into baculovirus vectors as
described under "Materials and Methods," to produce each subunit
both as a non-fused polypeptide and as an N-terminal His-tagged fusion.
The 5-termini of cDNA in all 10 transfer vectors were re-sequenced
using a primer complementary to the polyhedrin promoter. The sequences
around each start codon are shown on Fig. 1. The
recombinant baculoviruses were named according to the encoded protein
using the prefix v- (e.g. v140 for the virus encoding p140
protein, etc.).
Two assays were used to address the question
whether the functionally active RF-C can be produced in the insect
cells by co-infection with five recombinant baculoviruses encoding
individual RF-C subunits. The first step of pol holoenzyme
assembly, the loading of the sliding clamp, was analyzed by assaying
the RF-C-dependent formation of the complex between
[32P]PCNA and circular dsDNA (13). The
RF-C-dependent assembly of pol
holoenzyme was analyzed
by measuring DNA synthesis by pol
on singly primed ssDNA template
in the presence of RF-C, PCNA, RP-A (or E. coli SSB), and
ATP (2, 3, 38). Extracts from insect cells, infected either with five
RF-C viruses or control virus, were chromatographed on HAP (Fig.
2, A and B). Although most of the
DNA synthesis activity in the extracts was not dependent on exogenous
pol
and was found at about equal level in both extracts, a new pol
-dependent activity was evident upon co-infection of the
cells with the RF-C viruses. This pol
stimulating activity copurified with PCNA loading activity (Fig. 2B), confirming
that these two novel activities correspond to the recombinant RF-C. The
PCNA loading assay proved to be a quite specific test for the
expression of recombinant human RF-C in insect cells, as host polymerases did not interfere with it and insect RF-C appeared not to
interact efficiently with human PCNA. The small scale analytical HAP
chromatography with step elution was sufficient to reveal recombinant
RF-C activity poorly detectable in crude extracts. PCNA loading
activity of recombinant RF-C was about 40-fold higher than the
background in the control experiment (Fig. 2C). The
recombinant RF-C was further purified on a Mono Q column. The pol
stimulating and PCNA loading activities coeluted at 0.25-0.3
M NaCl and the active fractions were essentially free of
host pol activity (data not shown). The chromatographic behavior of the
recombinant protein on HAP and Mono Q was similar to that of natural
mammalian RF-C (1, 2, 5).
Polymerase holoenzyme DNA synthesis was linearly dependent on low
concentrations of calf thymus RF-C, thus allowing to quantify the
amounts of RF-C in terms of activity units (1 unit corresponded to the
incorporation of 1 nmol of total dNMP into singly primed ssDNA in the
presence of pol
, PCNA, and E. coli SSB in 30 min at
37 °C, Ref. 5). Additional RF-C in the reaction mixture led to
saturation of DNA synthesis (Fig. 3A). These
saturation conditions apparently corresponded to the loading of at
least one PCNA clamp per available template-primer junction and no
additional RF-C was required once pol
holoenzyme DNA synthesis
started (36). Under our experimental conditions, pol
holoenzyme DNA synthesis in the presence of saturating amounts of calf thymus RF-C was
40 pmol of dNMP in 30 min. Polymerase
holoenzyme DNA synthesis in
the presence of recombinant RF-C also reached saturation, with maximal
dNMP incorporation of 14 pmol in 30 min, corresponding to 35% of the
value obtained for calf thymus RF-C (Fig. 3B). Analysis of
DNA synthesis products on alkaline agarose gel showed that the average
size of the fragments synthesized in the presence of recombinant RF-C
was about 400-1000 nt in comparison with products of 600-5000 nt
synthesized by pol
in the presence of calf thymus RF-C (Fig.
3C). The length of the products synthesized by pol
holoenzyme is known to be dependent on incubation time (8, 36) and
concentration of pol
core enzyme (39), but not on concentration of
RF-C (2). To assess whether the slightly decreased activity of
recombinant RF-C could be caused by a substoichiometric amount of the
large subunit (see Fig. 4 below), we analyzed whether a
complex of the four small RF-C subunits could inhibit pol
holoenzyme DNA synthesis. Recombinant four-subunit complex
p40-his·p38·p37·p36 (see below in the text and Fig. 6) in amounts
up to 1 µg, corresponding to about 300-fold molar excess over
five-subunit RF-C, did not significantly decrease bovine
RF-C-dependent DNA synthesis (data not shown). Therefore
the decreased DNA synthesis performed in the presence of recombinant
five-subunit RF-C was not due to non-stoichiometric expression of RF-C
subunits. Another possible explanation for the apparently lower
activity of human recombinant RF-C was that we used bovine pol
for
the DNA synthesis assay. Some species specificity in the interaction of
human RF-C and bovine pol
cannot be ruled out. Further comparison
of natural human and bovine RF-C with recombinant human protein will be
required to determine which function or property of RF-C affects the
length of DNA products.
Expression of Functional Recombinant RF-C Containing His-tagged Subunits
The recombinant baculoviruses were constructed to
produce each RF-C subunit both as non-fused and His-tag-fused proteins, the latter designed for purification using affinity chromatography. Ni-NTA resin chromatography of the extracts from cells infected with
any recombinant virus encoding non-fused RF-C subunit or with control
virus reproducibly contained the same background polypeptides, most of
them over 60 kDa. Only four minor host cell polypeptides were detected
in the region of 35-50 kDa, and these were independent on the viruses
used for infection (see, e.g., Figs. 4C,
5C, and 6A). Upon
infection of insect cells with baculoviruses encoding individual
His-tagged RF-C subunits, followed by Ni-NTA resin chromatography, the
purified p40-his, p38-his, p37-his, and p36-his could be clearly
detected on Coomassie-stained denaturing polyacrylamide gels. The
amounts of soluble individual RF-C subunits purified using Ni-NTA resin
were estimated using the bovine serum albumin standards loaded on the
same denaturing gel. 3.8 × 107 cells (one
150-cm2 flask) infected with the corresponding baculovirus
yielded 16.5 µg of p37-his, 11 µg of p40-his, 8.5 µg of p36-his,
and 1.2 µg of p38-his (data not shown).
We investigated whether any of the His-tag fusions can be used to purify the whole five-subunit complex by affinity chromatography on Ni-NTA resin. Cells were infected with five viruses, one of them encoding a His-tagged subunit. The proteins from the cell extracts were purified on Ni-NTA resin as described under "Materials and Methods." When p40, p37, or p36 was His-tag-fused, the purified fractions displayed PCNA loading activity (Fig. 4B), and this activity was reproducibly highest in preparations containing the p36-his subunit. SDS-PAGE analysis followed by Coomassie staining showed that the fractions active in the PCNA loading assay contained polypeptides with molecular masses characteristic for small human RF-C subunits (Fig. 4C; compare the protein bands marked with dots in lanes 4, 6, and 7 with the background in lane 2).
Although the small polypeptides were directly detectable by Coomassie staining, the large RF-C subunit could not be seen, suggesting that this subunit might be underexpressed in comparison with other subunits. To pursue this question, the expression and co-purification of p140 with other subunits was analyzed by Western blot with mAb 19 against RFC140 (22). A 140-kDa polypeptide reacting with mAb was observed in the preparations containing p40-his, p37-his and p36-his (Fig. 4D), confirming that all five subunits copurify with the PCNA loading activity by affinity chromatography.
The results presented in Fig. 4 allowed us to address the question whether all five RF-C subunits are required to reconstitute active human RF-C in insect cells. Affinity-purified fractions were prepared from insect cells co-infected with combinations of viruses as shown at the top of Fig. 5. Omission of any of the five subunits resulted in the loss of PCNA loading activity (Fig. 5B) and the complete absence of p140 in the purified fractions (Fig. 5D), while the subcomplexes of small subunits could be seen (Fig. 5C). This result indicated that all five subunits are required to reconstitute functional RF-C and that p140 binds stably to the small subunits only when all four of them are co-expressed. The conclusion that all of the subunits are required for biochemical activity of RF-C agrees with the findings that all five genes of yeast RF-C are essential for viability (24-27).
Subunit Interactions in Recombinant RF-CWhile the presence of p140 in the purified fractions could be clearly detected with mAb, this subunit appeared to be stoichiometrically underrepresented relative to the small subunits. Thus p140 might bind last to a preformed subcomplex of small subunits. Furthermore, all four small subunits must comprise this subcomplex, since omission of any of the small subunits resulted in the loss of p140 from the purified fractions (Fig. 5). To assess this possibility, we analyzed the co-purification of proteins upon co-infection of the cells with various combinations of the viruses encoding small subunits, one of them as a His-tag fusion.
Co-infection of the cells with v40-his, v38, v37, and v36 resulted in a protein preparation containing the four corresponding subunits: p40-his, p37, and p36 in stoichiometric amounts and p38 in about half the amount of the other subunits (Fig. 6A, lane 3). Upon infection of the cells with corresponding pairs of viruses: v40-his·v40, v40-his·v38, v40-his·v37, and v40-his·v36, no protein co-purified with p40-his, indicating that p40 does not form stable complexes with other any single subunit and does not tend to oligomerize (data not shown). Next, various combinations of three viruses were analyzed. Only p40-his was found in the purified fraction upon co-infection of the cells with v40-his, v38, and v37, indicating that these three subunits do not form a stable subcomplex (Fig. 6A, lane 4). Co-infection of the cells with v40-his, v38, and v36 showed only very weak binding of p36 to p40-his (Fig. 6A, lane 5). In contrast, a stoichiometric complex of p40-his, p37, and p36 could be isolated upon co-infection of the cells with the corresponding viruses (Fig. 6A, lane 6), indicating that these three subunits form a stable intermediate. Since p38 is not required to form p40-his·p37·p36 complexes, the protein preparation in lane 3 is apparently a mixture of trimer p40-his·p37·p36 and tetramer p40-his·p38·p37·p36, the amount of the latter being limited by the expression of p38.
Assembly of subcomplexes was further investigated in co-infections
using the v37-his virus in place of v40-his. When the cells were
co-infected with v37-his, v40, v38, and v36, the four corresponding subunits could be seen in the purified fraction, with p37-his migrating
most slowly (Fig. 7A, lane 2). In
contrast to the v40-his results, the co-infections with pairs of
viruses showed that p38, p37, and p36, but not p40, co-purify to some
extent with p37-his (Fig. 7A, lanes 3-6). Upon
co-infection of the cells with v37-his, v40, and v38, no proteins
co-purified with p37-his and even the weak interaction with p38 was
inhibited (Fig. 7A, lane 7; see also Fig.
5C, lane 8). This result confirms the above
observation that the subunits p40, p38, and p37 do not form a stable
subcomplex. Upon co-infection of the cells with v37-his·v40·v36 and
v37-his·v38·v36, the corresponding tertiary complexes of subunits
could be observed, although not with stoichiometric ratios of subunits
as for p40-his·p37·p36 (see Fig. 6A, lane
6).
To characterize these two complexes more carefully, 6-fold larger amounts of the cells were infected with the corresponding viruses, proteins were purified using Ni-NTA resin, and the eluted fractions were chromatographed on a Mono Q column (Fig. 7, B and C). Analysis of the extract from the cells infected with v37-his·v38·v36 showed that the complex containing p37-his and p36 eluted from the Mono Q column first, followed by the complex of p37-his, p38, and p36 (Fig. 7B). We supposed that p38 was underexpressed in comparison with p37-his and p36, resulting in the appearance of the excess of p37-his·p36 complex. The ratio of the band intensities for p37-his·p36 complex was 1:0.55 (determined in fraction 9), and for p37-his·p38·p36 complex it was 1:0.62:0.64 (fraction 13). In the case of infection with v37-his, v40, and v36, no p37-his·p36 complex could be found, while the complex p37-his·p40·p36 eluted as a sharp symmetrical peak with a band ratio 1:0.66:0.70 for p37-his, p40, and p36, respectively (Fig. 7C).
To confirm these results, the same series of experiments were carried
out using v36-his co-infections. Though p36-his appears to be the most
useful for the purification of biologically active RF-C, its subunit
interaction analysis was most difficult to interpret, since p36-his
overlapped with p40 in SDS-PAGE. Co-infection of the cells with
v36-his, v40, v38, and v37 resulted in a poorly resolved complex, and
only p37 could be clearly identified (Fig. 8A, lane 2). Co-infections with
pairs of viruses showed that p36-his binds to p37 (lane 7),
but does not interact detectably with p38 (lane 6); no
conclusion could be made for p40 (lane 5). Co-infection of
the cells with v36-his, v40, and v38 yielded no polypeptide band
co-purifying with p36-his (lane 3). Although
p36-his·p40·p37 complex could not be resolved into three
components, it was evident that the amount of p37 bound to p36-his
complexes was higher for v36-his·v40·v37 than for v36-his·v37
infections (compare lanes 4 and 7). This
cooperative interaction of p40 and p37 further validated the data
obtained using p40-his and p37-his (Figs. 6 and 7). The protein
complex(es) resulting from co-infection of the cells with
v36-his·v38·v37 were purified using Ni-NTA resin followed by
chromatography on the Mono Q column (Fig. 8B). The complex
p36-his·p37 eluted first, followed by the complex p36-his·p38·p37 (Fig. 8B). The ratio of the band intensities was 1:0.68 for
p36-his·p37 (fraction 10) and 1:0.56:0.69 for
p36-his·p38·p37 (fraction 15).
Model of Assembly of Five-subunit RF-C Complex (Fig. 9)
The RF-C intersubunit interactions described
above (Figs. 4, 5, 6, 7, 8) allow to propose a model for assembly of RF-C
subunits into the five-subunit complex. p37 and p36 form a dimeric
complex stable enough to copurify on affinity resin and
anion-exchanger. We found only two stable tertiary subcomplexes,
p40·p37·p36 and p38·p37·p36, which might be intermediates in
the RF-C assembly. Both subcomplexes could be derived from the common
precursor p37·p36. Therefore, we postulate a bifurcated pathway for
the RF-C assembly; either p40 or p38 binds to p37·p36 dimer to form
tertiary complex. The missing fourth small subunit binds to the
tertiary complex, forming a quaternary complex of small subunits. The
latter binds the large subunit to form a catalytically competent
five-subunit complex (Fig. 9).
A more limited model for assembly of RF-C subunits co-expressed using an in vitro coupled transcription/translation system has also been proposed recently (31). Data from that system indicated the following. (i) No stable complex with two subunits was formed, although weak interactions could be observed for several pairs of subunits; (ii) the only stable three-subunit core complex was formed with p40, p37, and p36; (iii) p140 and p38 bound only cooperatively to the core complex p40·p37·p36 (31). However, co-expression of RF-C subunits using baculoviruses revealed additional intersubunit interactions, resulting in a significantly expanded model (Fig. 9). Thus the model based on the in vitro coupled transcription/translation system appears to represent one of the alternative assembly pathways observed in the baculovirus expression system. The inability to detect some of the subcomplexes in the in vitro expression system (31) that were detected using the baculovirus system could derive from differences between the expression systems, for example in protein modification. Although baculovirus expression does not guarantee the correct processing of mammalian polypeptides, the posttranslational modification of proteins in insect cells might be very close to those occurring in mammalian cells (see Ref. 40, and references therein). Two closely migrating bands of p140 were detected in Western blots of recombinant human RF-C (Fig. 4D). Two closely migrating bands of the large subunit of mouse RF-C were also visible in Western blots of mouse whole cell extracts (41). These results suggest that the baculovirus expression system might allow proper posttranslational modification of RF-C subunits.
The possibility to produce soluble individual RF-C subunits, several subcomplexes, as well as functional five-subunit RF-C using recombinant baculoviruses provides a useful approach to the detailed biochemical analysis of the structure and mechanism of action of this protein, as well as its role in DNA replication and DNA repair machineries as a whole. This will allow the in vitro characterization of functional subcomplexes of the subunits and various mutant polypeptides and will shed light on the biochemical role of each subunit in the overall function of RF-C.
We thank J. Hurwitz for plasmids; B. Stillman for plasmids and antibody preparation; K. Horiuchi for the E. coli gp II overproducer strain; D. Barnard for help with the modification of RFC40 cDNA; and I. Boche, C. Rehfuess, and C. Voitenleitner for sharing experimental advice, cells, and materials.