Replication factor C (RFC) is a five-subunit
protein complex required for coordinate leading and lagging strand DNA
synthesis during S phase and DNA repair in eukaryotic cells. It
functions to load the proliferating cell nuclear antigen (PCNA), a
processivity factor for polymerases
and
, onto primed DNA
templates. This process, which is ATP-dependent, is carried
out by 1) recognition of the primer terminus by RFC (2) binding to and
disruption of the PCNA trimer, and then 3) topologically linking the
PCNA to the DNA. In this report, we describe the purification and
properties of recombinant human RFC expressed in Sf9 cells from
baculovirus expression vectors. Like native RFC derived from 293 cells,
recombinant RFC was found to support SV40 DNA synthesis and polymerase
DNA synthesis in vitro and to possess an ATPase
activity that was highly stimulated by DNA and further augmented by
PCNA. Assembly of RFC was observed to involve distinct subunit
interactions in which both the 36- and 38-kDa subunits interacted with
the 37-kDa subunit, and the 40-kDa subunit interacted with the 36-kDa
subunit-37-kDa subunit subcomplex. The 140-kDa subunit was found to
require interactions primarily with the 38- and 40-kDa subunits for
incorporation into the complex. In addition, a stable subcomplex
lacking the 140-kDa subunit, although defective for DNA replication,
was found to possess DNA-dependent ATPase activity that was
not responsive to the addition of PCNA.
 |
INTRODUCTION |
Knowledge of the mechanism of mammalian DNA replication is
important not only for understanding how this process is regulated in
normal cells, but also for the design of therapies against cancers in
which this vital function is perturbed. Replication of the mammalian
genome has been investigated in vitro using the SV40 DNA
virus as a model system, because all of the factors required for SV40
DNA replication, except for large T antigen, the initiator protein and
helicase, are host cell-encoded. Recently, all of the factors required
for SV40 DNA replication in vitro have been identified, and
data from numerous biochemical studies suggest the following model for
how these factors function (Refs. 1 and 2, and references therein): 1)
following template preparation (origin recognition and local unwinding,
more extensive DNA unwinding by T antigen, and coating of the single
strands by replication protein A
(RPA)1), primer synthesis and
limited primer extension is carried out by the polymerase
primase
complex to yield an initiator DNA; 2) a switch from usage of polymerase
to polymerase
(pol
) as the primary replicative polymerase
is accomplished by recognition of the initiator DNA primer terminus by
RFC; 3) next, PCNA, the processivity factor for pol
, is loaded onto
the DNA by RFC; 4) leading and lagging strand DNA synthesis is
catalyzed by pol
; and 5) finally, Okazaki fragments are processed
by the coordinate activities of FEN-1, ribonuclease H, and DNA ligase
I. By this model, a potential rate-limiting step for efficient DNA
synthesis is the activity of RFC, which facilitates DNA synthesis by
the highly processive pol
. As implicated by in vitro
studies, the importance of RFC for DNA replication has been confirmed
by in vivo studies that have demonstrated that the
Saccharomyces cerevisiae homologue of human RFC is
essential for viability and functions in DNA replication and
repair (3-8).
In contrast to the function of RFC in DNA replication, its role in DNA
repair is less clear. Nevertheless, both biochemical and genetic
studies have suggested a requirement for RFC in DNA repair (9-13).
Mutations in the S. cerevisiae gene encoding the large
subunit of RFC (cdc44) have been shown to render the cell sensitive to exposure to the alkylating agent methylmethane sulfonate and UV radiation, but not
irradiation, suggesting a role for RFC in
the base excision and nucleotide excision repair pathways (8).
Consistent with this hypothesis, mutations in the pol30 gene
encoding PCNA have been found to suppress the DNA repair defect in
cdc44 mutants (8, 13). In addition, in vitro
reconstitution studies have demonstrated a requirement for RFC for the
pol
(or
)-catalyzed DNA synthesis step during nucleotide
excision repair (14, 15).
RFC has been shown to consist of five subunits, one large subunit of
approximately 100-140 kDa (140 kDa for human, 103 kDa for yeast) and
four small subunits ranging from 40 to 36 kDa (16-20), which have been
found to share considerable sequence identity and similarity. In
addition, a significant degree of sequence identity has been found to
exist between homologous subunits of not only yeast and human RFC but
also between the eukaryotic RFCs and the Escherichia coli
and
' subunits of the
complex, the functional equivalent of
RFC in prokaryotes (21, 22).
All five subunits of RFC have been shown to contain seven conserved
regions that include the nucleotide binding motif found in all known
ATPases, and the "DEAD box" motif found in RNA and DNA helicases
(6). One additional region, conserved only among the large subunits,
has been noted to share a significant degree of similarity with
prokaryotic DNA ligases and has been termed therefore the "ligase
homology box" (6). Biochemical studies have shown that RFC possesses
a structure-specific DNA binding activity, displaying a preference for
substrates with a 5'-overhang (23), and an ATPase activity that is
stimulated by DNA and further augmented in the presence of PCNA (24,
17). To investigate the many interesting structure-function
relationships in human RFC, we have reconstituted in active form a
recombinant human RFC in Sf9 cells and characterized the subunit
requirements for complex assembly and activity.
 |
MATERIALS AND METHODS |
Construction of Recombinant Baculoviruses--
Recombinant
baculoviruses expressing each of the subunits of replication factor C
were created using the Baculogold baculovirus expression system
developed by PharMingen. Baculovirus transfer vectors for each gene
were constructed as follows. pVL1393-140kDa was constructed by
removing the 140-kDa coding sequence from pRFC+ and cloning it into
pVL1393. pVL1393-36kDa was made by removing the 36-kDa subunit coding
sequence from p36NL with NdeI and EcoRI and
placing it into the SmaI and EcoRI sites of
pVL1393. pAcSG2-37kDa was generated by removing the gene for the
37-kDa subunit from pET-37kDa with NdeI and BamHI
and cloning it into the StuI and BglII sites of
pAcSG2. pVL1393-38kDa was created by removing the 38-kDa coding
sequence from pSK-38kDa and placing it into the BamHI and
KpnI sites of pVL1393.
Baculoviruses expressing NH2-terminal HA epitope tagged
140- and 37-kDa subunits were generated by cloning each of these
subunits in a CITE vector containing the HA epitope coding sequence
(N-CITE, kindly provided by Drs. Bill Tansey and Winship Herr, Cold
Spring Harbor Laboratory (CSHL)). The 140-kDa subunit sequence was
cloned into N-CITE vector by digesting the plasmid pRFC+ with
HaeII, ligating on the oligonucleotide HaeII
(5'-AATTGCGC-3'; Nucleic Acid Chemistries facility, CSHL), purifying
the DNA by Sephadex G-25 spin column chromatography, and then
incubating the DNA with exonuclease VII. Afterward, the DNA was
extracted with phenol-chloroform and digested with XbaI, and
the 140-kDa gene fragment was gel-purified and ligated to N-CITE
cleaved with SmaI and XbaI. The resulting derivative of N-CITE contained the 140-kDa subunit gene 3' to the HA
epitope coding sequence. The HA epitope-140-kDa subunit cassette was
excised using NcoI and PstI and cloned into the
SmaI and PstI sites of pVL1393.
For construction of an amino-terminal HA epitope 37-kDa subunit gene,
the 37-kDa subunit coding sequence was cloned into the EagI
and PstI of N-CITE by removing from pET-37kDa the gene plus an additional 1024 base pairs (746 base pairs 5' and 278 base pairs 3'
of the 37-kDa subunit gene) with EagI and NheI.
The resulting plasmid was then digested with NdeI and
SmaI and recircularized to remove the additional sequence at
the 5'-end of the gene and to juxtapose the HA epitope and 37-kDa
subunit coding sequences. The HA epitope-37-kDa subunit cassette was
then removed with NcoI and BamHI and cloned into
the SmaI and BglII sites of pVL1393. The N-CITE
derivatives were sequenced with the CITE-1 primer
(5'-GGGGACGTGGTTTTCCTTT-3'; Nucleic Acid Chemistries facility, CSHL) to
determine if in frame fusions were obtained, and the pVL1392 and pAcSG2
derivatives were sequenced with the BVPolH primer
(5'-TCGTAACAGTTTTGTAATAA-3'; Nucleic Acid Chemistries facility, CSHL).
The transfer vectors were used to create recombinant baculoviruses by
homologous recombination in Sf9 cells. Baculovirus clones were
isolated by plaque purification, amplified once, and then screened for
expression of human RFC subunits by SDS-PAGE (Anderson's modified
Laemmli procedure (25)) and immunoblotting. Viruses expressing RFC
subunits were then further amplified until a titer of >1 × 108 plaque-forming units/ml was achieved.
Expression and Purification of Human RFC in Sf9
Cells--
Maintenance and infections of Sf9 cells were
performed in Grace's medium (Life Technologies, Inc.) supplemented
with 10% fetal calf serum (Hyclone). The optimal multiplicity of
infection was empirically determined for each recombinant virus. Each
virus was used at a multiplicity of infection of 10, and expression of
more than one protein at a time was accomplished by the infection of
multiple viruses simultaneously. For purification of the five-subunit RFC complex, cells in 5 T-175-cm2 flask (2 × 107 cells/flask) were infected with each RFC subunit virus
(bvHA-140kDa, bv40kDa, bv38kDa, bv37kDa, and bv36kDa), harvested by
removal with a cell scraper 48 h postinfection, washed once with
phosphate-buffered saline, and then resuspended in buffer PC (50 mM KPO4, pH 7.4, 7 mM CHAPS, 1 mM dithiothreitol, 1 mM EDTA, 1 mM
phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin) plus 300 mM NaCl (10 ml of buffer/2 × 107 cells)
and incubated on ice for 30 min. Next, the lysate (1.2 mg/ml) was
disrupted by Dounce homogenization (10 strokes with a "B" pestle)
and centrifuged at 16,000 × g for 30 min at 4 °C, and the supernatant was incubated for 8 h at 4 °C with 200 µl/50 ml of clarified whole cell lysate of 12CA5-protein A-Sepharose (50% slurry, 500 µg to 1 mg of antibody/ml of bead) prepared as described by Harlow and Lane (26). After binding, the resin was washed
three times for 15 min at 4 °C with 20 ml/wash of buffer PC plus 300 mM NaCl, and RFC was then eluted by incubating the beads at
37 °C for 15 min in 1 ml of buffer PC plus 300 mM NaCl plus 0.5 mg/ml HA peptide (YPYDVPDYA, Protein Chemistries Facility, CSHL). The elution was repeated two more times, and the first and
second eluates were pooled (total of 90-100 µg of protein), concentrated 4-fold on a Centricon 30 (Amicon), and then 200 µl of
the concentrated material was further purified by glycerol gradient
sedimentation (4.8 ml of 15-40% glycerol gradient in buffer PC plus
300 mM NaCl centrifuged in a SW55Ti rotor at 50,000 rpm for
24 h at 4 °C). The gradients (two total) were fractionated from
the top (200 µl/fraction) and assayed for DNA synthesis and ATPase
activity. Active fractions were pooled and stored at
80 °C (total
amount of protein was 50 µg). A subcomplex of RFC lacking the 140-kDa
subunit was purified as described above, except the glycerol gradient
was centrifuged for 19 instead of 24 h.
Immunoprecipitation of RFC Subunits--
Infections and lysate
preparation were performed as described for the RFC purification except
that 1 × 107 cells were lysed with 1 ml of buffer PC
plus 300 mM NaCl, and the lysate was not homogenized but
repeatedly pipetted (10 times) before centrifugation. The clarified
lysate was then incubated with 40 µl of 12CA5-protein A-Sepharose
(50% slurry) for 4 h at 4 °C with rocking, and then the beads
were collected by centrifugation at 1000 × g in an
Eppendorf microcentrifuge, washed for 15 min three times with buffer PC
plus 300 mM NaCl, and finally resuspended in 2 × SDS-PAGE sample buffer. The immunoprecipitates were analyzed by
SDS-PAGE followed by silver staining and/or immunoblotting.
Purification of Other Replication Proteins--
Human RFC was
purified from 293 nuclei as described previously (16). DNA pol
and
topoisomerases I and II were purified from calf thymus as indicated in
Refs. 16 and 27. SV40 large T antigen was purified from
baculovirus-infected Sf9 cells as delineated in Ref. 28. PCNA
and RPA were purified from E. coli as described previously
(28, 29). Fraction IIA, a 0.2 M NaCl, 0.33 M
NaCl phosphocellulose fraction, which supports SV40 DNA replication
when supplemented with topoisomerases I and II, RPA, PCNA, and RFC, was
prepared as described previously (16).
DNA Replication Assays--
The in vitro assay for
SV40 DNA replication was performed as described previously (16) using
fraction IIA and purified SV40 large T antigen, topoisomerases I and II
(1 unit of topoisomerase activity is the amount of enzyme that converts
50% of the substrate DNA into relaxed forms in 30 min at 37 °C in a
25-µl reaction containing 500 ng of DNA), RPA, PCNA, and RFC. 50-µl
reactions were performed with 3000 units of topoisomerase I (1000 units/µl), 64 units of topoisomerase II (64 units/µl), 640 ng of
RPA, 1.54 µg of PCNA, 10 µg of SV40 large T antigen, the indicated
amount of RFC (and 0.56 mM CHAPS contributed by
baculovirus-derived RFC), 6.7 µg of fraction IIA, and 500 ng of SV40
origin containing plasmid pSV011. The final NaCl concentration was 25 mM in the experiment described in Fig. 3A and 35 mM NaCl in that described in Fig. 9.
M13 DNA synthesis reactions were carried out as described previously
(30) in 50 mM NaCl using 4 µg/ml single primed M13mp18 DNA (M13mp18 primer 4995; maps to nucleotide 4995), 10 ng of pol
,
20 µg/ml PCNA, 10 µg/ml RPA, and the indicated amount of RFC (0.7 mM CHAPS contributed by baculovirus-derived RFC) in a
30-µl reaction. All reactions for both SV40 and M13 DNA synthesis
were incubated for 1 h at 37 °C and stopped by the addition of
10 mM EDTA, and the dAMP incorporation was determined by
spotting a fraction of the reaction onto DE81 paper (Whatman) that was
then washed in 0.5 M Na2HP04 as
described previously (31). In addition, the replication products from
SV40 DNA replication reactions were purified by proteinase K (200 µg/ml) treatment at 37 °C in 1% SDS, phenol/chloroform
extraction, and ethanol precipitation and then analyzed by neutral
agarose gel electrophoresis and autoradiography.
ATPase Assays--
20-µl reactions were performed in 30 mM HEPES, pH 7.5, 30 mM NaCl, 1 mM
dithiothreitol, 7 mM MgCl2, 100 µg/ml bovine
serum albumin, 100 µM ATP, 3 pmol of
[
-32P]ATP (ICN Pharmaceuticals, Inc.; specific
activity, 800 Ci/mmol), and 0.25 pmol of recombinant RFC (plus 0.7 mM CHAPS contributed by the protein) in the presence or
absence of either 25 µM poly(dT)·oligo(dA) (Pharmacia
Biotech, Inc.; 1:4 molar ratio of poly(dT) (average length of 250 bases) to oligo(dA) (length of 20 bases)) or 25 µM
poly(dT) only. For historical reasons, the five-subunit RFC complex
glycerol gradient fractions were assayed in 50 mM Tris, pH
7.5, 2 mM MgCl2, 30 mM NaCl, 1 mM dithiothreitol, 100 µg/ml bovine serum albumin, 100 µM ATP, and 3 pmol of [
-32P]ATP. The
conditions for these reactions were subsequently discovered to be
suboptimal; therefore, the specific activity of the recombinant RFC and
stimulation by PCNA appears lower than in other experiments. Reactions
were incubated for 1 h (or the indicated time) at 37 °C and
stopped by the addition of EDTA to a final concentration of 10 mM, and the amount of ADP produced was determined by TLC. A small fraction of each reaction (2 µl) was spotted onto a
polyethyleneimine-cellulose plate (pre-developed in 1 M
formic acid), and the plate was then developed in a buffer of 1 M formic acid and 0.5 M LiCl2 for
40 min. The amount of ADP produced was determined by scanning the plates using a FUJI BAS-100 phosphor imager.
Protein Quantitation--
Protein preparations were quantitated
by Bradford assay, using the Bio-Rad Bradford reagent, and by
Coomassie-staining SDS-PAGE gels. Immunoblots were developed using the
SuperSignal Substrate detection system (Pierce).
Other Reagents and Protocols--
The 12CA5 monoclonal antibody
against the HA epitope was obtained from the Monoclonal Antibody
Facility at CSHL. The antibody against the 140-kDa subunit of RFC,
monoclonal antibody 6, was generated as described previously (21).
Rabbit antibodies directed against the 40- and 37-kDa subunits of
RFC were kindly provided by Dr. Jerard Hurwitz, Memorial
Sloane-Kettering Cancer Center. Protein A-Sepharose and CHAPS
were purchased from Pharmacia and Sigma, respectively. Enzymes that
were not purified in the laboratory were purchased from New England
Biolabs. All other protocols that were not outlined were performed as
described by Sambrook et al. (32).
 |
RESULTS |
Reconstitution and Characterization of Baculovirus Human
RFC--
Seven recombinant baculoviruses, each encoding one of the
five RFC subunits were constructed and used to infect Sf9 cells. As shown in Fig. 1A
(lanes 5, 7, 8, and 9), the
40-kDa (lane 5), 37-kDa (both native NH2
terminus (lane 7) and an NH2-terminal HA epitope
variant (lane 8)), and 36-kDa (lane 9) subunits
were expressed well. In contrast, the 140-kDa (both native
NH2 terminus (Fig. 1A, lane 3) and
NH2-terminal HA epitope variant (Fig. 1A, lane 4)) and 38-kDa (Fig. 1A, lane 6)
subunits were consistently not expressed as well. Expression of the
large subunit was confirmed by immunoblotting (data not shown), and
expression of the 38-kDa subunit was confirmed by analysis of protein
synthesis by [35S]methionine labeling of the cells
42 h postinfection, when viral mRNAs were predominantly
translated (Fig. 1B, lane 5).

View larger version (93K):
[in this window]
[in a new window]
|
Fig. 1.
Expression of the subunits of human RFC in
Sf9 insect cells. Infections of Sf9 cells with
recombinant baculoviruses each encoding a subunit of RFC were carried
out as described under "Materials and Methods." A,
1 × 106 cells were infected with recombinant
baculoviruses expressing the indicated RFC subunit. After 48 h,
they were harvested and lysed in 1 × SDS-PAGE sample buffer and
analyzed by SDS-PAGE. The resulting gel was stained with Coomassie. The
lanes represent cells infected with wild-type AcMNPV
(lane 2), bv140kDa (lane 3), bvHA-140kDa
(lane 4), bv40kDa (lane 5), bv38kDa (lane
6), bv37kDa (lane 7), bvHA-37kDa (lane 8),
and bv36kDa (lane 9). In lane 1, the cells were
mock-infected with media. B, 1 × 106 cells
were infected with the indicated baculoviruses. After 42 h, they
were labeled for 20 min with [35S]methionine. After
harvesting, the cells were lysed in 1 × SDS-PAGE sample buffer
and analyzed by SDS-PAGE and fluorography. Lysates from cells infected
with the following viruses were loaded in the indicated
lanes: wild-type AcMNPV (lane 2), bv140kDa
(lane 3), bv40kDa (lane 4), bv38kDa (lane
5), bv37kDa (lane 6), bv36kDa (lane 7). In
lane 1, the cells were mock-infected with media. The 140-kDa
subunit typically was labeled poorly; therefore, expression of this
subunit was confirmed by immunoblotting (data not shown).
|
|
Using the HA epitope variant of the 140-kDa subunit (HA-140kDa), we
observed co-purification of the 40-, 38-, 37-, and 36-kDa subunits with
the HA-140kDa subunit on a column consisting of the monoclonal antibody
12CA5 linked to protein A-Sepharose (Fig. 2A, lanes labeled
E1 and E2), suggesting
that the RFC complex was reconstituted in the infected cells. A
functional RFC complex was formed as judged by co-sedimentation of both
DNA synthesis and ATPase activities (Fig. 2, C and
D) with all five subunits in a 15-40% glycerol gradient
(Fig. 2B, lanes 14-17). In addition, recombinant
RFC was capable of supporting SV40 DNA synthesis in vitro as
shown in Fig. 3A, with a
specific activity comparable with that of authentic RFC isolated from
human 293 cells as shown in Fig. 3B.

View larger version (64K):
[in this window]
[in a new window]
|
Fig. 2.
Purification of recombinant human RFC from
Sf9 cells. Panel A, infection and lysis of Sf9
cells was as described under "Materials and Methods." A whole cell
lysate from 1 × 108 cells was incubated with
12CA5-protein A-Sepharose, after which the beads were washed and RFC
was eluted with HA peptide (0.5 mg/ml) as described under "Materials
and Methods." A fraction (5 µl) of each stage of the purification
was analyzed by SDS-PAGE, and the resulting gel was silver-stained.
Lane L, the load onto the beads; lane FT, the
material not bound to the beads; lane W1, the
first wash from the beads; lane W4, the fourth
wash from the beads; lanes E1,
E2, and E3, the first,
second, and third elutions, respectively, from the beads; lane
B, the material that remained bound to the beads after the
elutions; lane P, the concentrated pooled elutions.
Panel B, glycerol gradient sedimentation profile of the
concentrated pooled elutions from the 12CA5-agarose beads. The glycerol
gradients (15-40%) were sedimented as described under "Materials
and Methods," and 5 µl of each fraction was analyzed by SDS-PAGE
and silver staining. Lane numbers correspond to fraction numbers (removed from the top of the gradient), and the arrows below denote the positions of the peaks of the protein standards albumin, aldolase, and catalase from an independent gradient
simultaneously sedimented. Panel C, 1 µl of the glycerol
gradient fractions in panel B were assayed for the ability
to support pol DNA synthesis on singly primed M13 DNA as described
under "Materials and Methods." Panel D, 2.5 µl of the
glycerol gradient fractions in panel B were assayed for
ATPase activity as described under "Materials and Methods." Each of
the indicated fractions were tested in the absence (squares)
and presence (diamonds) of 20 µg/ml PCNA. All reactions
contained 25 µM poly(dT)·oligo(dA).
|
|

View larger version (35K):
[in this window]
[in a new window]
|
Fig. 3.
SV40 DNA synthesis in vitro by
recombinant RFC and comparison of the pol -stimulatory activities of
recombinant and authentic RFC. A, reconstitution of SV40 DNA
synthesis in vitro was performed as described under
"Materials and Methods." The recombinant RFC was titrated in this
assay, and the amount of DNA synthesis was quantitated and graphed. The
DNA synthesis product from the reactions containing 0.03, 0.13, 0.26, and 0.51 pmol of RFC was analyzed by neutral agarose gel
electrophoresis and autoradiography. The graph shows incorporation of
dAMP in the reconstituted replication reaction. B, DNA
synthesis by pol on singly primed M13 DNA was performed as
described under "Materials and Methods." Both the recombinant RFC
(diamonds) and authentic RFC derived from 293 cells
(squares) were titrated in this assay, and the amount of DNA
synthesis was graphed as a function of protein concentration.
|
|
Similar to native RFC, the recombinant RFC was observed to hydrolyze
ATP in a DNA-dependent manner, displaying a preference for
primer-template substrates compared with unprimed single-stranded DNA
(Fig. 4A). The approximate
rate of ATP hydrolysis as determined from a time course of the reaction
(Fig. 4B) was 0.95 pmol/min, indicating a specific activity
of 3.7 mol of ADP produced/mol of RFC/min. The ATPase activity of
recombinant RFC in the presence of DNA was found to be stimulated
4-fold by PCNA (Fig. 4C), and no stimulation by PCNA was
observed in the absence of DNA or in the absence of primer (data not
shown).

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 4.
Characterization of the ATPase activity of
recombinant human RFC. ATPase assays were performed with 0.25 pmol
of RFC and the indicated amount of either poly(dT)·oligo(dA) or
poly(dT) only as described under "Materials and Methods."
A, ATPase assays were performed in the presence of
increasing concentrations (0.25, 1, 5, 10, 25, 50, and 75 µM) of either poly(dT)·oligo(dA) (squares) or poly(dT) only (diamonds). The amount of ADP produced by
RFC in the absence of DNA in this experiment was 45 pmol. B
shows the time course of ATP hydrolysis in the presence of either
poly(dT)·oligo(dA) (squares) or poly(dT) only
(diamonds). C shows an analysis of RFC ATP
hydrolysis in the presence of increasing concentrations (5, 15, 45, and
90 µg/ml) of PCNA with either 25 µM
poly(dT)·oligo(dA) (squares) or 1 µM
poly(dT)·oligo(dA) (diamonds). The amount of ADP produced
in the presence of 90 µg/ml PCNA with 25 µM
poly(dT)·oligo(dA) but without RFC was 0.39 pmol.
|
|
Analysis of the Subunit Interactions Necessary for Complex
Assembly--
The arrangement within the RFC complex of the five
subunits was investigated by performing immunoprecipitation experiments from extracts prepared from cells infected with subsets of viruses. Using an HA epitope-tagged variant of the 37-kDa subunit, we tested which of the other RFC subunits could associate with the 37-kDa subunit. When Sf9 cells were co-infected with all five RFC
subunit viruses, bvHA-37kDa, bv140kDa, bv40kDa, bv38kDa, and bv36kDa, all of the untagged RFC subunits were observed to co-purify with the HA
epitope-tagged 37-kDa subunit (Fig.
5A, silver-stained gels,
lane 5; Fig. 5B, immunoblots of the samples in
Fig. 5A probed with either an
-140kDa monoclonal antibody
or
-40kDa rabbit antibody, lane 5). The samples in Fig. 5
were subjected to electrophoresis four separate times, and the
silver-stained gels (Fig. 5A, top and
bottom) were developed either completely (top) to
visualize the 140-kDa subunit or less extensively (bottom)
to view the small subunits, which, due to their similar molecular
weights, run close together on the gel. Subsequent purification of the
proteins shown in Fig. 5A, lane 5, by
phosphocellulose chromatography, single-stranded DNA cellulose column
chromatography, and glycerol gradient sedimentation confirmed the
presence of a functional five-subunit RFC complex (data not shown).
Thus, a functional five-subunit RFC complex could be purified using two
different epitope-tagged versions of the complex.

View larger version (62K):
[in this window]
[in a new window]
|
Fig. 5.
Immunoprecipitation of the 140-, 40-, 38-, and 36-kDa subunits with the HA-37kDa subunit. Immunoprecipitation
of the HA epitope variants of RFC using the -HA monoclonal antibody 12CA5 was carried out as described under "Materials and Methods." The proteins bound to the beads were analyzed by SDS-PAGE, and the gels
were subsequently silver-stained (A) or subjected to immunoblot analysis using antibodies against either the 140- or 40-kDa
subunits (B). Lysates from cells infected with the pertinent viruses (denoted as plus signs) were analyzed and loaded in
the numbered lanes as indicated. Loaded in lane 1 is the immunoprecipitate from cells mock-infected with media, and in
lane 2 is the immunoprecipitate from cells infected with
wild-type AcMNPV. A, two silver-stained gels, one developed
thoroughly (top) and the other moderately (bottom), were prepared with the samples from the
immunoprecipitation reactions described above to allow detection of the
140-kDa subunit (top gel) and 40-kDa subunit (bottom
gel), respectively. B shows immunoblots of the lysates
used for the immunoprecipitation reactions described above before the
addition of 12CA5 (left) as well as the immunoprecipitation
reactions (IP) (right) using antibodies against
the 140-kDa subunit (top) and 40-kDa subunit
(bottom).
|
|
When the 140-kDa subunit was omitted from the co-infection, a
quaternary complex consisting of the 40-, 38-, 37-, and 36-kDa subunits
was observed (Fig. 5A, lane 6, and Fig.
5B,
40kDa immunoblot, lane 6), indicating no
dependence on the 140-kDa subunit for the small subunits to assemble
into a complex. However, assembly of the 140-kDa subunit into the
complex was observed to be dependent upon the 38- and 40-kDa subunits,
because omission of the 38-kDa subunit from the complex resulted in
complete loss of the 140-kDa subunit from the complex (Fig.
5A, lane 8, and Fig. 5B,
140kDa immunoblot, lane 8), and omission of the 40-kDa subunit
resulted in significantly reduced assembly of the 140-kDa subunit into the complex (Fig. 5A, lane 7, and Fig.
5B,
140kDa immunoblot, lane 7). The omission
of the 36-kDa subunit was observed to diminish dramatically the
incorporation of the 40-kDa subunit into the complex (Fig.
5A, lane 9, and
40kDa immunoblot in Fig.
5B, lane 9). In addition, a small reduction in
the incorporation of the 140-kDa subunit (Fig. 5A,
lane 9, and Fig. 5B
140kDa immunoblot, lane 9), presumably due to loss of the 40-kDa subunit, was
detected. Neither the 38-kDa subunit nor the 36-kDa subunit was found
to manifest a dependence on the other RFC subunits for assembly into a
complex with the 37-kDa subunit, since omission of each of the other
RFC subunits had no effect on the ability of the 36- and 38-kDa
subunits to co-immunoprecipitate with the 37-kDa subunit (Fig.
5A, lanes 5, 6, 7, and
9 (36-kDa subunit) and lanes 5, 6, 7, and 8 (38-kDa subunit)).
To further characterize the subunit interactions, we tested pairwise
interactions between the untagged RFC subunits and either the HA-37kDa
subunit (Fig. 6) or the HA-140kDa subunit
(Fig. 7). The 36- and 38-kDa subunits
were found to interact directly with the 37-kDa subunit (Fig. 6,
lanes 5 and 6), whereas the 140- and 40-kDa
subunits were not (Fig. 5, lane 10, and Fig. 6, lane
4). However, the 40-kDa subunit was discovered to interact with
the 37-kDa subunit when the 36-kDa subunit was co-expressed with these two subunits (Fig. 6, lane 9). The interaction was found to
be facilitated specifically by the 36-kDa subunit, since neither the
38-kDa subunit (Fig. 6, lane 8, and immunoblot analysis not shown) nor the 140-kDa subunit (data not shown) was capable of promoting this interaction. Thus, the 40-kDa subunit appears to interact either directly with the 36-kDa subunit, or with a unique interface created by the 36-kDa subunit-37-kDa subunit interaction.

View larger version (41K):
[in this window]
[in a new window]
|
Fig. 6.
Immunoprecipitation of the 40-, 38-, and
36-kDa subunits with the HA-37kDa subunit of RFC. The 40-, 38-, and 36-kDa subunits were examined for the ability to interact with the
37-kDa subunit. Infections with the indicated viruses, preparation of cell lysates, and immunoprecipitations were carried out as described under "Materials and Methods." 5-µl aliquots of the cell lysates were removed before the addition of 12CA5 and analyzed by SDS-PAGE and
Coomassie staining (B). The immunoprecipitates were analyzed by SDS-PAGE and silver staining (A). The numbered
lanes contain immunoprecipitates from the indicated
infections, with viruses used for infections denoted as plus
signs. The immunoprecipitate loaded in lane 1 was from
cells infected wild-type AcMNPV. The 38-kDa subunit was not visible in
these whole cell lysates.
|
|

View larger version (47K):
[in this window]
[in a new window]
|
Fig. 7.
Immunoprecipitation of the small subunits of
RFC with the HA-140kDa subunit. The small subunits of RFC were
tested for the ability to interact with the 140-kDa subunit. Lysates from cells co-infected with the viruses noted below were used in
immunoprecipitation reactions (procedure described under "Materials and Methods"). The immunoprecipitates were analyzed by SDS-PAGE, and
the gels were either silver-stained as shown in panel A or subjected to immunoblot analysis using an antibody against the 40-kDa
subunit as shown in panel B. The numbered lanes
were loaded with the immunoprecipitates from the indicated infections
with the viruses used denoted by plus signs. In lanes
1 and 2, the cells were either mock-infected with media
(lane 1) or infected with wild-type AcMNPV (lane
2). In panel C is a Coomassie-stained gel of an aliquot
(5 µl) of the lysates used for the immunoprecipitations in
panels A and B plus lysates from additional
control infections with single subunits, which were used for
immunoprecipitation reactions but not loaded on the gels in
panels A and B because they were redundant. The
aliquots were removed before the addition of antibody. The numbered
lanes correspond to cells infected with media only
(lane 1); wild-type AcMNPV (lane 2); bv40kDa,
bv38kDa, bv37kDa, and bv36kDa (lane 3); bvHA-140kDa
(lane 4); bv40kDa (lane 5); bv38kDa (lane
6); bv37kDa (lane 7); bv36kDa (lane 8);
bvHA-140kDa, bv40kDa, bv38kDa, bv37kDa, and bv36kDa (lane
9); bvHA-140kDa, bv38kDa, bv37kDa, and bv36kDa (lane
10); bvHA-140kDa, bv40kDa, bv37kDa, and bv36kDa (lane
11); bvHA-140kDa, bv40kDa, bv38kDa, and bv36kDa (lane
12); bvHA-140kDa, bv40kDa, bv38kDa, and bv37kDa (lane
13). In panel D is a model for the organization of the
subunits within the RFC complex. The complex is illustrated as two
"domains," one consisting of the 40-, 36-, and 37-kDa subunits and
the other consisting of the 140- and 38-kDa subunits (depicted in
gray). These two subcomplexes are connected by the 40-kDa
subunit-140-kDa subunit and 38-kDa subunit-37-kDa subunit interactions,
the abolition of which results in the loss of complex formation.
|
|
Analyses of pairwise subunit interactions between untagged RFC subunits
and the HA epitope-tagged 140-kDa subunit were difficult because of the
substantial degradation of this subunit in the absence of more than one
small subunit (Fig. 7A and data not shown). However, weak
interactions between some of the small subunits and the HA-140kDa
subunit were detectable when a single subunit was omitted from the
co-infections. When either the 40- or 38-kDa subunit was omitted, only
the 36- and 37-kDa subunits were found to co-immunoprecipitate with the
HA-140kDa subunit (Fig. 7A, silver-stained gel, lanes
6 and 7, and data not shown). Upon omission of the 37-kDa subunit, unexpectedly, only the 36-kDa subunit was found to
interact with the 140-kDa subunit; neither the 40- nor the 38-kDa
subunit was detected in the immunoprecipitate (Fig. 7A, lane 8; Fig. 7B,
40kDa immunoblot of the
samples in Fig. 7A). When the 36-kDa subunit was excluded
from the co-infection, an interaction between the HA-140kDa subunit and
the 38-, 37-, and 40-kDa subunits was observed (Fig. 7A,
lane 9; Fig. 7B,
40kDa immunoblot, lane
9). The detection of this complex is consistent with the results
in Fig. 5 that show that a complex consisting of the 140-kDa, 40-kDa,
38-kDa, and HA-37kDa subunits can be formed in the absence of the
36-kDa subunit (Fig. 5, lane 9). The significance for RFC
complex assembly of the interaction between the 140-kDa subunit and 36- and 37-kDa subunits is questionable for two reasons. 1) the 140-kDa
subunit manifested no requirement for the 36-kDa subunit for
incorporation into the RFC complex. As shown in Fig. 5, lanes
7 and 9, omission of the 40-kDa subunit (lane
7) had a greater effect on the assembly of the 140-kDa subunit
into complex than omission of the 36-kDa subunit (lane 9),
which instead hindered assembly of the 40-kDa subunit. 2) No direct
interaction between the 140- and 37-kDa subunits was observed in the
experiment in Fig. 5, lane 10. Therefore, the interaction
between the 140-kDa subunit and 37-kDa subunit most likely is indirect
and is mediated probably through the 36-kDa subunit, which was found to
interact directly with the 37-kDa subunit (Fig. 6, lane 6).
Thus, it appears that for RFC complex assembly (summarized in Fig.
7D), both the 38- and 40-kDa subunits are required for the
140-kDa subunit to assemble into the complex. These two subunits in
turn interact with the 37- and 36-kDa subunits, the 38 kDa subunit
directly with the 37-kDa subunit and the 40-kDa subunit either directly with the 36-kDa subunit or with a unique protein surface created by the
interaction of the 36- and 37-kDa subunits.
A Subcomplex of RFC Lacking the 140-kDa Subunit Is Inactive for DNA
Replication but Possesses ATPase Activity--
Given the previous
observations suggesting that a stable complex between the four small
subunits of RFC can be formed in the absence of the large subunit, we
next attempted to isolate the complex of four small subunits to analyze
its biochemical properties. Similar to the entire RFC complex, the 40-, 38-, and 36-kDa subunits were observed to co-purify with the HA
epitope-tagged 37-kDa subunits through 12CA5-protein A-Sepharose
chromatography (Fig. 8A), and all four small subunits appeared to co-sediment on a 15-40% glycerol gradient (Fig. 8B, lanes corresponding to
fractions 10 and 12). Fractions 10-12 from the glycerol gradient were
pooled, and the purified small subunit complex was tested for the
ability to support SV40 DNA replication, pol
DNA synthesis on
singly primed M13, and ATPase activity (Fig.
9 and Table
I). Unlike the entire RFC complex, the
small subunit complex appeared to be unable to support full SV40 DNA
synthesis in vitro, with only aborted initiation events
detected (Fig. 9 lanes labeled BVRFC-140) (16, 33). In the
M13 DNA synthesis assay, no stimulation of pol
DNA synthesis was
observed with the amounts of small subunit complex used in the SV40 DNA
synthesis experiment (data not shown). Collectively, these results
indicate a requirement for the 140-kDa subunit for DNA synthesis.

View larger version (55K):
[in this window]
[in a new window]
|
Fig. 8.
Purification of a subcomplex of RFC
consisting of the four small subunits. The infections, lysate
preparation, immunoaffinity chromatography on 12CA5-protein
A-Sepharose, and glycerol gradient sedimentation were accomplished as
described under "Materials and Methods." 5-µl aliquots from each
stage of the purification were removed and examined by SDS-PAGE and
silver staining as shown in panel A. Lane L, the
lysate before incubation with the beads; lane FT, the lysate
after incubation with the beads; lane W1, the
first wash from the beads; lanes E1 and
E2, the first and second elutions from the
beads. Panel B shows fractions from the glycerol gradient
(15-40%; fractionated from the top) that were analyzed by SDS-PAGE
and silver staining. The lane numbers correspond to the
fraction numbers, and the arrows denote the positions of the
peaks of the protein standards from an independent gradient sedimented
simultaneously.
|
|

View larger version (89K):
[in this window]
[in a new window]
|
Fig. 9.
Impaired ability to support SV40 DNA
replication by a subcomplex of RFC lacking the 140-kDa subunit.
In vitro SV40 DNA replication reactions were performed as
described under "Materials and Methods" with increasing amounts of
either the entire RFC complex (labeled BVRFC; 0.013, 0.03, 0.13, and 0.32 pmol used) or the 40-, 38-, 37-, and 36-kDa subunits
(labeled BVRFC-140; 0.05, 0.13, and 1.3 pmol used). Graphed
below is the amount of dAMP incorporated.
|
|
View this table:
[in this window]
[in a new window]
|
Table I
ATPase activity of the four small subunit complex of RFC
Reactions were carried out as described under "Materials and
Methods" in the presence or absence of 25 µM
poly(dT) · oligo(dA) using 0.25 pmol of the entire RFC complex
or 0.5 pmol of the four small subunit complex.
|
|
When the small subunit complex was assayed for ATPase activity in the
presence or absence of poly(dT)·oligo(A) (Table I), an ATPase
activity that was highly stimulated (approximately 20-fold) by DNA was
detected. Attempts to purify further the glycerol gradient pool by ion
exchange chromatography on phosphocellulose, Affi-Gel blue Sepharose,
and Q-Sepharose were futile, because under the conditions used, neither
the RFC subunits nor this DNA-dependent ATPase activity
interacted with any of these resins; both the activity and the RFC
proteins were recovered in the flow-through (data not shown). Hence,
this DNA-dependent ATPase activity appears to be intrinsic
to the small subunits of RFC. In addition, the activity of the small
subunit ATPase was examined in the presence of PCNA (Fig.
10). In contrast to the entire
five-subunit complex, this small subunit complex appeared to be
refractory to PCNA, indicating a requirement for the 140-kDa subunit
not only for DNA synthesis but also for PCNA-dependent
ATPase activity.

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 10.
The four small subunit complex ATPase
activity is not responsive to PCNA. ATPase reactions were
performed in the presence of 25 µM poly(dT)·oligo(dA)
as described under "Materials and Methods." The amount of ATP
hydrolysis by 0.38 pmol of the entire RFC complex (squares)
and the four small subunit complex (diamonds) were measured
in the presence of increasing concentrations of PCNA. The amount of ADP
formed in the absence of RFC is represented by the
circles.
|
|
 |
DISCUSSION |
Biochemical characterization of mammalian DNA replication has been
hampered due to lack of sufficient quantities of the replication factors necessary for such studies. Here we report the reconstitution of human replication factor C in Sf9 cells, characterization of its activities in vitro, and analysis of the subunit
requirements for complex assembly and activity. Similar expression of
RFC has been reported elsewhere (34-36). The five subunits of RFC
share a significant degree of homology, which is manifested as seven conserved sequence motifs. These include the P-loop and DEAD-box motifs
found in other ATP/GTPases. Despite the apparent functional similarity,
in vivo the function of each subunit is not redundant. This
raises the question of why so many similar subunits are necessary. The
studies in this report suggest that all five RFC subunits are essential
to form a stable complex competent for DNA synthesis. Central to the
complex is the 37-kDa subunit, which functions as a scaffold on which
the other subunits can assemble (Fig. 7D). The direct
interaction of the 36- and 38-kDa subunits with the 37-kDa subunit are
required for incorporation of the 40- and 140-kDa subunits,
respectively, into the complex. The stability of the 140-kDa subunit in
the complex depends on the presence of both the 38- and 40-kDa
subunits. In the absence of the 140-kDa subunit, the 40-kDa subunit
displays an absolute requirement for the 36-kDa subunit for
incorporation into a subcomplex. However, a subcomplex consisting of
the 140-, 40-, 38-, and 37-kDa subunits can form, albeit inefficiently,
in the absence of the 36-kDa subunit, implying that other
protein-protein interactions, most notably with the 140-kDa subunit,
may stabilize the 40-kDa subunit in the complex.
The subunit interactions required for the assembly of RFC have also
been investigated by Uhlmann et al. (34) and Podust and
Fanning (35). In each report (this one and the aforementioned ones), a
stable interaction between the 40-, 37-, and 36-kDa subunits was
reported. Moreover, this report and that of Podust and Fanning (35)
have described the assembly of this subcomplex, which was found to
involve a direct interaction between the 37- and 36-kDa subunits and
the interaction of the 40-kDa subunit with the 37-kDa subunit-36-kDa
subunit complex. A requirement for the 38-kDa subunit for incorporation
of the 140-kDa subunit into the RFC complex has been shown here and in
Uhlmann et al. (34). Unique findings regarding RFC assembly
reported here are 1) a stable interaction of the 38-kDa subunit with
the 37-kDa subunit that was not found to be dependent on any other
subunit (Figs. 5A and 6), 2) the requirement for the 40-kDa
subunit for efficient incorporation of the 140-kDa subunit (Fig.
5A), and 3) formation of a stable complex between the 140-, 40-, 38-, and 37-kDa subunits in the absence of the 36-kDa subunit
(Figs. 5A, 7A, and 7B). Discordant with the results in Figs. 5A and 6A are those
presented in Uhlmann et al. (34), which 1) implied that the
40-kDa subunit is dispensable for complex assembly and 2) did not
address the dependence on the 36-kDa subunit for assembly of the 40-kDa
subunit into the RFC complex but instead advocated a direct interaction
between the 40- and 37-kDa subunits. The requirement for the 36-kDa
subunit for a 37-kDa subunit-38-kDa subunit interaction described by
Podust and Fanning (35) is contradictory to the results shown in Fig. 6, which demonstrated a direct interaction between the 38- and 37-kDa
subunits in the absence of any other RFC subunit. In summary, we
believe the RFC complex is probably organized as two "domains," one
consisting of the 40-, 36-, and 37-kDa subunits and the other consisting of the 140- and 38-kDa subunits. These two subcomplexes are
juxtaposed by the 40-kDa subunit-140-kDa subunit and 38-kDa subunit-37-kDa subunit interactions, which, if eliminated, are predicted to result in the loss of complex formation (see Fig. 7D). This model provides an explanation for why omission of
the 140- and 36-kDa subunits from the complex permits the formation of
stable quaternary complexes of the 140-, 40-, 38-, and 37-kDa subunits
(Fig. 5A, lane 9; Fig. 7A, lane
9) and 40-, 38-, 37-, and 36-kDa subunits (Fig. 5A,
lane 6; Fig. 6, lane 7), respectively.
The primary role of RFC during DNA synthesis is to load PCNA, the
processivity factor for pol
, onto the DNA, thus facilitating highly
efficient leading and lagging strand DNA synthesis. This process
entails recognition of the primer terminus by RFC, binding and
disruption of the PCNA trimer, and topologically linking PCNA to the
DNA. How these activities are distributed among the five subunits is
unknown. In vivo experiments in yeast have shown a requirement for all five RFC subunits for viability (4-7, 37) and the
involvement of RFC in DNA replication, DNA repair, and cell cycle
checkpoint pathways (8, 14, 15, 38). However, the function(s) of each
subunit in these cellular processes remain(s) unclear. In
vitro experiments presented in this report and elsewhere have
shown biochemically a requirement for the large subunit of RFC for DNA
replication, because a subcomplex lacking the large subunit was found
to be incompetent for DNA synthesis. RFC has been shown to be a
structure-specific DNA-binding protein, displaying a preference for DNA
molecules mimicking DNA replication substrates (23). Deletion analyses
of the 140-kDa subunit have defined the presence of at least two
"domains" involved in DNA binding, one at the NH2
terminus that includes the ligase homology domain (39, 40) and one
located near the COOH terminus (40). However, the specificity of these
putative large subunit DNA binding domains for primer termini is
inconclusive.
Present in all five RFC subunits are motifs termed RFC box III and RFC
box V that are similar to the phosphate binding loop (P-loop) and DEAD
box consensus sequences, respectively, found in proteins that hydrolyze
ATP or other nucleotide triphosphates. The RFC complex possesses ATPase
activity, and stable binding of RFC to DNA has been shown to require
ATP binding and/or hydrolysis. Moreover, DNA appears to be a co-factor
for the ATPase of RFC, stimulating this activity approximately 20-fold.
The subunit(s) responsible for this activity is unknown. In this
report, evidence is presented indicating that a complex of the small
subunits, although inactive for DNA replication, is an efficient
ATPase. Similar to the entire RFC complex, the ATPase activity of the small subunit complex was found to be highly stimulated by DNA, but,
unlike the entire RFC complex, its ATPase activity was not responsive
to PCNA. This observation is consistent with the findings of McAlear
et al. (8), which indicated that a direct interaction between the large subunit of yeast RFC (encoded by cdc44)
and PCNA (encoded by pol30) is required for RFC function.
The apparent inability of PCNA to stimulate the activity of the small
subunit ATPase is contradictory to the observations recently reported by Cai et al. (41). Further investigation of the interaction between the small subunit complex and PCNA may shed some light on this
issue.
As noted earlier, the 140-kDa subunit has been reported to contain two
distinct DNA binding domains; but which, if either, of these regions is
responsible for the DNA binding properties of RFC is unclear.
Experiments presented in this report suggest that a complex of the
small subunits of RFC is a DNA-stimulated ATPase, implying that at
least one of these subunits has DNA binding activity. In the report of
Uhlmann et al. (40), the four small subunits of RFC were
tested individually for the ability to bind DNA, and none exhibited any
DNA binding activity. Perhaps these subunits bind DNA cooperatively,
and consequently, a complex of these subunits is required for stable
DNA binding. Alternatively, a complex of the small subunits is
necessary to form a functional ATPase that is competent to stably bind
DNA. Indeed, although each subunit of RFC has the primary features of
an ATPase, reconstitution experiments have revealed that a minimum of
three subunits (the 40-, 37-, and 36-kDa subunits) are required for
DNA-dependent ATPase activity in
vitro.2 How the DNA
binding, ATP binding, and ATPase activities of RFC are distributed
among the subunits remains to be determined.
In contrast to RFC, much more is known about the prokaryotic equivalent
of RFC, the
complex of E. coli. It consists of five subunits (

'
) and like RFC possesses a
DNA-dependent ATPase activity that is required for loading
the
clamp, the E. coli equivalent of PCNA, onto DNA
(42). Although all five subunits of the
complex are necessary for
optimal activity in vitro, the minimum complex active for
DNA synthesis consists of
,
, and
' (43). The
and
'
subunits are homologous to the small subunits of RFC and contain
nucleotide binding motifs (6, 22). However, the
subunit has been
demonstrated to be the sole functional ATPase in the complex, and it is
optimally active only when present in a complex with the
and
'
subunits (43, 44). The ATPase activity of the 

' complex was
found to be stimulated by a complex of the
and
subunits, as
well as by the
clamp, which has been shown to interact with the
subunit (45, 46). Given the similarity among all five RFC subunits and,
consequently, the greater complexity of functional analyses, it will be
interesting to see if a similar distribution of the functions of the
complex among the five subunits is achieved in RFC. Further
characterization of the conserved regions shared among the subunits
will undoubtedly yield interesting insights into how multisubunit
protein complexes coordinate multiple activities.
We thank J. Hurwitz at the Memorial
Sloane-Kettering Cancer Center for providing the RFC small subunit
plasmids and antibodies; Fred Bunz and Nancy Bei for construction of
the native NH2 terminus 140-kDa subunit virus; and Gerhard
Cullmann and Shou Waga for valuable discussions.