From the Institute of Veterinary Biochemistry,
University of Zürich-Irchel, Winterthurerstrasse 190,
CH-8057 Zürich, Switzerland, the § Department of
Biology, Department of Biochemical and Biophysical Sciences, Institute
of Molecular Biology, University of Houston, Houston, Texas,
77204-5513, and the ¶ Department of Biology, Texas A&M University,
College Station, Texas 77843
Replication factor C (RF-C) is a heteropentameric
protein essential for DNA replication and repair. It is a molecular
matchmaker required for loading of proliferating cell nuclear antigen
(PCNA) onto double-stranded DNA and, thus, for
PCNA-dependent DNA elongation by DNA polymerases and
. To elucidate the mode of RF-C binding to the PCNA clamp, modified
forms of human PCNA were used that could be 32P-labeled
in vitro either at the C or the N terminus. Using a kinase
protection assay, we show that the heteropentameric calf thymus RF-C
was able to protect the C-terminal region but not the N-terminal region
of human PCNA from phosphorylation, suggesting that RF-C interacts with
the PCNA face at which the C termini are located (C-side). A similar
protection profile was obtained with the recently identified PCNA
binding region (residues 478-712), but not with the DNA binding region
(residues 366-477), of the human RF-C large subunit (Fotedar, R.,
Mossi, R., Fitzgerald, P., Rousselle, T., Maga, G., Brickner, H.,
Messner, H., Khastilba, S., Hübscher, U., and Fotedar, A., (1996)
EMBO J., 15, 4423-4433). Furthermore, we show that the
RF-C 36 kDa subunit of human RF-C could interact independently with the
C-side of PCNA. The RF-C large subunit from a third species, namely
Drosophila melanogaster, interacted similarly with the
modified human PCNA, indicating that the interaction between RF-C and
PCNA is conserved through eukaryotic evolution.
To perform processive, accurate, and rapid DNA synthesis, DNA polymerases (pols)1 require the aid of a set of proteins called DNA replication accessory proteins. The three best known eukaryotic accessory proteins are proliferating cell nuclear antigen (PCNA) (1), replication factor C (RF-C) (2), and replication protein A (3). These proteins are present in all eukaryotic cells examined and are mandatory for the function of replicative pols. Their tasks include the recruitment of pols when needed, the facilitation of pol binding to the primer terminus, an increase in pol processivity, prevention of non-productive binding of pols to single-stranded DNA, the release of pols after DNA synthesis, and communication between the pols and other replication and cell cycle regulating proteins (for a rewiew, see Ref. 4).
RF-C is essential in DNA replication since it is necessary for loading
of PCNA onto DNA and for subsequent DNA synthesis of the leading strand
by pol (5). The gross structure of the RF-C complex appears to be
conserved through eukaryotic evolution since RF-C isolated from either
yeast or human is composed of one large and four small subunits
(Mr = 140, 40, 38, 37, and 36 in human (6, 7)
and Mr = 94.9, 39.7, 38.2, 36.2, and 39.9 in
yeast (8)). RF-C 140 contains the DNA binding activity (9) that has
been further localized between amino acids (aa) 366-477 (10-12) to a
region termed RF-C box I (8). The exact roles of the small subunits
(RF-C 40, RF-C 38, RF-C 37, and RF-C 36) have not been determined;
however, they share seven conserved regions with the large subunit,
termed RF-C boxes II-VIII (8). Since the PCNA binding region within
RF-C 140 (10, 13) contains the conserved RF-C boxes II-IV, all five
subunits could be expected to bind PCNA. To date, only RF-C 40 has been
shown to interact with PCNA (14).
The eukaryotic clamp loader, RF-C, appears to be structurally and
functionally very similar to its prokaryotic and viral counterparts. The Escherichia coli complex consists, in analogy to
human RF-C, of five subunits (
,
,
,
,
) that cooperate
to load the
clamp (the PCNA counterpart) onto the DNA (15). In
bacteriophage T4, the clamp loader consists of two subunits (g44/62p)
that cooperate to load the g45p clamp (16). The amino acid sequence
similarity between some of the subunits of these clamp loaders from
prokaryotic, eukaryotic, and viral systems (8, 17) suggests that their basic mechanism of action may be similar.
While a high resolution model for the sliding clamp formed by RF-C and
PCNA is not yet available, an increasingly detailed picture of events
occurring at the prokaryotic replication complex is emerging (18). It
has been shown that both the complex and the core pol interact with
the
ring (15). Prior to
clamp assembly on DNA,
shows higher
affinity to the
complex than to the core pol, but once
has been
assembled on DNA, the core develops a stronger affinity for the ring
and outcompetes the
complex. After placing the
ring on the DNA,
the
clamp loader can dissociate from the complex and is ready for
loading other
dimers. Naktinis et al. (19) were able to
assign the
binding function to the
subunit of the
complex.
In the uncharged state,
is buried in the
complex, preventing
its binding to
, but upon ATP binding, the complex undergoes a
conformational change and thus exposing
for interaction with
.
In view of the analogy with the complex, we sought to shed some
light on the structure of the RF-C·PCNA clamp. Specifically, how does
RF-C interact with PCNA in complex formation? The crystal structure of
PCNA shows a homotrimeric, ring-shaped molecule with an overall
negative charge but with a central hole surrounded by positive charges
through which DNA slides (20). This structure is very similar to the
three dimensional structure of the
clamp despite the lack of
significant sequence similarities (21). The C termini of the
subunit, as well as those of PCNA, protrude from structurally
homologous faces (C-side) of the ring and are likely candidates for
interactions with other proteins. The C-terminal amino acids of the
E. coli
ring have been shown to be important for the
interaction with both the clamp loader and the pol (15). Studies by
Fukuda et al. (22) suggested that RF-C binds to the C-side
of human PCNA, specifically to Asp41 and aa 254-257, although the
terminal four amino acids are dispensible. Asp41 is partially exposed
close to the C terminus in the yeast PCNA structure, making the
hypothesis plausible. Care, however, must be taken when interpreting the effects of deletion mutations in PCNA since it has been shown that
deletion of C-terminal residues can have adverse effects on the PCNA
structure (23), and the trimeric structure of PCNA has been shown to be
necessary for the interaction with RF-C (24).
To study the interaction of RF-C with PCNA in more detail, we used modified PCNA carrying artificial phosphorylation sites at either their N or C termini (called nphPCNA and cphPCNA, respectively) (5). In kinase protection assay experiments (KPA) (18), nphPCNA or cphPCNA is incubated with the protein to be assayed for interaction. If the protein binds to PCNA close to either end, the artificial phosphorylation site is expected to become less accessible to the kinase so that a decrease in phosphorylation is observed. In this paper, we show that the pentameric calf thymus RF-C complex protects the C termini, but not the N termini, of PCNA from phosphorylation. We therefore conclude that RF-C interacts with the C-side of the PCNA ring. The RF-C 140 homologue of Drosophila melanogaster is also able to protect the C termini but not the N termini of PCNA, showing, besides its interaction with the C-side of PCNA, that the molecular interaction between these two auxiliary factors is conserved through evolution. More precisely, the interaction between PCNA and RF-C 140 is due to the PCNA binding region, located in human RF-C 140 between aa 478 and 712 (10), which also protects only the C-side of the ring. RF-C 140 is not the only RF-C subunit involved in PCNA binding as we could show that RF-C 36 also interacts with PCNA and is able to protect modified PCNA from phosphorylation.
Radioactive-labeled nucleotides were purchased from Amersham Corp. All other reagents were of analytical grade and were purchased from Merck (Darmstadt, Germany) or Fluka (Buchs, Switzerland).
BuffersThe following buffers were used. Buffer A contained
25 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.01%
(v/v) Nonidet P-40, 1 mM DTT, 10 mM
NaHSO3, 1 mM PMSF, and 1 µg/ml each
pepstatin, leupeptin, and aprotinin. Buffer B contained buffer A plus
10% (v/v) glycerol. Buffer C contained 20 mM Tris-HCl (pH
7.5), 300 mM NaCl, 0.4 mM PMSF, and 1 µg/ml
each aprotinin, pepstatin, and leupeptin. Buffer D contained 20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.4 mM PMSF, 1 mM DTT, 1 mM EDTA, and
0.5% Nonidet P-40. Buffer G contained 40 mM Tris-HCl (pH
7.5) and 10 mM MgCl2. Buffer H contained 40 mM Tris-HCl (pH 7.5), 30 mM imidazole-HCl, 10 mM MgCl2, 50 mM NaCl, and 0.1%
Nonidet P-40. Buffer I contained 40 mM Tris-HCl (pH 7.5),
10 mM MgCl2, and 0.2 mg/ml bovine serum
albumin. Buffer L contained 10 mM Tris-HCl (pH 8.0), 200 mM NaCl, 1 mM EDTA, 10% (v/v) glycerol, 1 mM PMSF, and 1 mM DTT. KPA buffer contained 20 mM Tris-HCl (pH 7.5), 12 mM MgCl2,
200 µg/ml bovine serum albumin, and 1 mM DTT. SDS-gel
loading buffer contained 60 mM Tris-HCl (pH 6.8), 2% (w/v)
SDS, 2% (v/v) glycerol, 0.005% (w/v) bromphenol blue, and 2%
(v/v) -mercaptoethanol.
Poly(dA)1000-1500 and oligo(dT)12-18 were purchased from Sigma and Pharmacia, respectively. Poly(dA)/oligo(dT) (base ratio 10:1) was prepared as described (25). Singly primed M13 single-stranded DNA was prepared according to (26). Oligonucleotides for modification and sequencing of the PCNA gene were from Microsynth (Balgach, Switzerland). The plasmid pT7/hPCNA carrying the cDNA of human PCNA was kindly provided by B. Stillman (Cold Spring Harbor Laboratory, NY). The pET19b/p36His, pET5a/40K, and pET16/p128 expression vectors containing the cDNA of RF-C 36, RF-C 40, and RF-C 128, respectively, of human RF-C were a gift of J. Hurwitz (New York). The cDNAs encoding the fragments of the human RF-C 140 were from A. Fotedar (La Jolla, CA).
Enzymes and ProteinsRestriction and modifying enzymes were
purchased from U. S. Biochemical Corp., New England Biolabs (NEB), and
Boehringer Mannheim. The catalytic subunit of
cAMP-dependent protein kinase from bovine heart muscle was
from Sigma. Human wtPCNA was produced in E. coli using the plasmid pT7/hPCNA and purified as described
previously (27). Calf thymus pol and pentameric RF-C were isolated
as described (25, 28). The three fragments of human RF-C 140, A, B, and
A + B were purified according to (10). The N-terminal phosphorylatable
PCNA (nphPCNA) was produced and purified as described in Podust
et al. (5). Cloning and isolation of RF-C 140 of D. melanogaster will be described
elsewhere.2 Polyclonal antibodies against
PCNA were raised in chicken and affinity purified with protein
A-Sepharose. The alkaline phosphatase-conjugated anti-chicken antibody
was from Sigma.
The poly(dA)/oligo(dT) assay (RF-C-independent assay) was carried out according to (25). DNA replication on singly primed M13 DNA (RF-C-dependent assay) was performed as described previously (26).
Construction of PCNA with a C-terminal Phosphorylation SiteA construct encoding PCNA with the C-terminal consensus
phosphorylation sequence for cAMP-dependent protein kinase
(Arg-Ala-Ser-Val-Ala) was generated by PCR in analogy to a similar
strategy on the N terminus (5). The original pT7/hPCNA expression
vector (27) was used as a template for PCR with a 28-mer T7-promoter
primer sequence and a 49-mer lower primer (5-GCG CGG ATC CTA TGC AAC ACT TGC TCT TCT AGA TCC TTC TTC ATC CTC G-3
) designed to introduce the
phosphorylation site at the C terminus. The PCR product was digested
with NdeI and BamHI and recloned into the pT7
expression vector. The resulting clone (called pT7/cphPCNA) was
sequenced to confirm the absence of mutations.
E. coli BL21(DE3)pLysS
cells transformed with the pT7/cphPCNA expression construct were grown
at 37 °C in 200 ml of LB medium to A600 = 0.4 and induced with 0.4 mM isopropyl thiogalactoside. After
3 h, cells were harvested by centrifugation and lysed by freezing/thawing in 10 ml of buffer A containing 25 mM
NaCl. The mix was sonicated and centrifuged for 30 min at 25,000 × g. The supernatant was loaded onto a 4-ml Q-Sepharose
column and equilibrated in buffer B containing 100 mM NaCl.
The column was washed with the same buffer, and proteins were eluted
with a 50-ml linear gradient from 0.1 to 0.7 M NaCl in
buffer B. The fractions containing cphPCNA were pooled and loaded on a
1.5-ml phenyl-Sepharose column equilibrated with buffer A containing
1.2 M NaCl. The column was washed with buffer A without
Nonidet P-40, and cphPCNA was eluted with a 20-ml linear gradient from
1.2 to 0 M NaCl in buffer A without Nonidet P-40. cphPCNA
containing fractions were pooled, brought to 50% (v/v) glycerol in
buffer A and stored at 20 °C until use.
E. coli BL21(DE3) cells were transformed with the pET19b plasmid containing RF-C 36 sequence linked to a His tag at its N terminus. The cells were grown in 1 liter of LB medium to A600 = 0.6, and isopropyl thiogalactoside was added to a final concentration of 0.4 mM. The cells were further grown for 5 h at 37 °C, harvested by centrifugation at 5,000 × g for 20 min, and then lysed in buffer C by three passages through a French press. The lysate was centrifuged for 20 min at 18,000 × g, and the pellet was solubilized in 10 ml of buffer C containing 6 M guanidinium-HCl. After ultracentrifugation at 35,000 × g for 40 min, the supernatant was dialyzed sequentially against buffer C containing urea (8, 4, 2, and finally 0 M). Most of the protein precipitated during the last dialysis step but could be solubilized by resuspending it in buffer C containing 0.05% (v/v) Tween 20 and 0.01% (v/v) Nonidet P-40.
Subcloning, Expression, and Purification of the His-tagged RF-C 40The gene encoding RF-C 40 was cut out of the original pET5a vector with EcoRI and cloned into the pET28a+ vector, to gain RF-C 40 with a His tag at its N terminus. The resulting clone was sequenced to confirm the reading frame and the presence of the His tag. RF-C 40 was expressed and purified essentially as RF-C 36 except that buffer D was used (instead of buffer C). Soluble protein was obtained by serial dialysis against buffer D containing urea (4, 3, 2, 1.5, 1, 0.5, 0.25, and finally 0 M), 10% glycerol, and 1 µg/ml each of aprotinin, pepstatin, and leupeptin.
Subcloning, Expression, and Purification of the His-tagged Human RF-C 140A linker encoding a 6-His tag was cloned into the single BstBI restriction site at the end of the gene encoding RF-C 140 in pET16b. This resulted in the expression of the human RF-C 140 His-tagged at its C terminus. RF-C 140 was expressed and purified as described above for RF-C 40.
KPA250 ng (3 pmol) of cphPCNA were incubated with an
excess of the protein to be tested and with 3 µCi
[-32P]ATP in a 10-µl total volume of KPA buffer.
After 2 min at 37 °C, the phosphorylation reaction was started by
the addition of 0.75 units of cAMP-dependent protein
kinase. The reaction mix was further incubated at 37 °C. After 1, 2, 5, and 10 min, 2-µl aliquots were withdrawn, and the reactions were
stopped with 8 µl of 2% SDS. The samples were then analyzed by
electrophoresis on a 12% SDS-polyacrylamide gel. Gels were fixed in
10% trichloroacetic acid, soaked in 10% acetic acid/12% methanol,
dried, and autoradiographed. Gels with labeled products were exposed on
a special screen for PhosphorImager (Molecular Dynamics, Inc.). After
exposure, the screen was scanned into the PhosphorImager and
quantitation was performed with the Image Quant program of Molecular
Dynamics. To exclude false-positive results due to intrinsic ATPase
activity, samples were tested for the presence of free phosphate by
thin layer chromatography.
80 pmol of the His-tagged proteins to be assayed were mixed with 20 µl of Ni-NTA resin in buffer G. The mixture was incubated by rocking for 1 h at 4 °C. After 3 min of centrifugation at 2,000 × g, the beads were washed 3 times with 1 ml of buffer G. Then 80 pmol of human wtPCNA were added to the beads in a 100-µl total volume of buffer I. In order to allow binding of wtPCNA to the Ni-NTA coupled proteins, the suspension was incubated at room temperature for 1 h. After centrifugation as above, the resin was washed 4 times with buffer H, and the proteins were eluted by boiling the beads in 30 µl of SDS-gel loading buffer. The proteins were separated by electrophoresis on a 12% SDS-polyacrylamide gel, and PCNA was detected by immunoblotting with polyclonal antibodies.
The C termini of PCNA, as well as those of the clamp in prokaryotes, protrude from one face of the ring, making them
candidates for interaction with other proteins. The C termini of the
clamp in E. coli have been shown to be involved both in
the recognition of
by the
clamp loader and in the binding of
the pol (15). Considering the structural and functional similarities of
the prokaryotic and eukaryotic pol sliding clamps, we designed
experiments to test the hypothesis that PCNA and RF-C interact in a way
similar to that of the
clamp and its clamp loader. For this
purpose, we used plasmids containing the human PCNA open reading frame with an artificial phosphorylation site for cAMP-dependent
protein kinase fused either to its 3
- or 5
-end (5). Predictably, these modified forms of PCNA (called cphPCNA and nphPCNA, respectively) run slightly slower than unmodified PCNA on an SDS-polyacrylamide gel
due to the presence of the phosphorylation sites (Fig.
1A, cphPCNA). Unlike the unmodified PCNA,
cphPCNA and nphPCNA (see Ref. 5) can be efficiently phosphorylated
in vitro (Fig. 1B, cphPCNA). Moreover, these PCNA
forms have full activity in replication assays on primed
single-stranded M13 DNA (RF-C-dependent assay; Fig.
1C, cphPCNA) and on poly(dA)/oligo(dT) (RF-C-independent assay, data not shown) (see also Ref. 5).
The Calf Thymus RF-C Complex Protects the C Termini, But Not the N Termini, of PCNA from Phosphorylation
First, we studied with KPA
the interaction of the calf thymus RF-C complex with human PCNA. For
this purpose, cphPCNA or nphPCNA and a 2-fold molar excess of the calf
thymus RF-C complex were preincubated with [-32P]ATP
for 2 min at 37 °C to allow interaction between the proteins. After
addition of the cAMP-dependent protein kinase, the kinetics of the phosphorylation reaction were assayed. If RF-C interacts with
PCNA close to either terminus, the accessibility of the respective artificial phosphorylation sites to the kinase is expected to be
reduced. This results in a reduction of the amount of radioactivity incorporated in the PCNA ring. In Fig. 2, we show that a
2-fold molar excess of RF-C inhibited phosphorylation of cphPCNA by
approximately 50% after 10 min but did not inhibit the phosphorylation
of nphPCNA. This experiment provided evidence that RF-C interacts with
PCNA close enough to the C termini to protect them from
phosphorylation.
The PCNA Binding Region of Human RF-C 140 Is Able to Protect PCNA from Phosphorylation, the DNA Binding Region Is Not
Having shown
that the pentameric calf thymus RF-C complex interacts with the C-side
of human PCNA, we next wanted to investigate the role of RF-C 140 in
this interaction. Two functional regions of human RF-C 140 have
recently been identified (10), the DNA binding (fragment A) and the
PCNA binding regions (fragment B). The DNA binding activity was
localized between aa 366 and 477, where the conserved box I (restricted
to the large subunit) is situated (8). This region shows striking
similarity to the N-terminal regions of all bacterial DNA ligases
sequenced to date (11, 29-31) and somewhat less, but still
significant, similarity to the automodification domain of eukaryotic
poly(ADP-ribose) pols. The PCNA binding region, which is adjacent to
but does not overlap with the DNA binding region, includes aa 478-712.
This region of the protein has been shown to inhibit SV40 DNA
replication in vitro, as well as RF-C-dependent
loading of PCNA onto DNA and RF-C-dependent DNA synthesis.
It also showed a dominant negative phenotype when expressed in
mammalian cells (10). Since the pentameric RF-C complex could protect
cphPCNA from phosphorylation (see Fig. 2), the effect of the PCNA
binding region of RF-C 140 (fragment B) alone was investigated in the
same assay. Using a 6-fold molar excess of RF-C 140 fragment B, 80%
less phosphorylation of cphPCNA was observed after 10 min of incubation
(Fig. 3A). The RF-C 140 fragment B, however,
did not significantly protect human PCNA containing an artificial
phosphorylation site at its N terminus (data not shown). This result
further supported the notion that RF-C binds to PCNA on the side where
its C termini are located rather than to the other face of the ring. To
exclude the possibility that the protection seen is an artifact caused by the intrinsic ATPase activity of RF-C 140 fragment B, we separated 2 µl of the KPA samples on polyethyleneimine-thin layer chromatography after different incubation times at 37 °C. In all cases tested, the
amount of free [32P]phosphate produced was negligible
(data not shown). Consistent with its lack of interaction with PCNA, a
6-fold molar excess of fragment A produced no inhibition of
phosphorylation (Fig. 3B). RF-C 140 fragment A + B, a
fragment containing both regions A and B, produced 60% inhibition of
phosphorylation. In summary, these results supported the hypothesis
that the PCNA binding region of RF-C 140 interacts with sites close to
the C termini of PCNA.
RF-C 140 Homologue of D. melanogaster Protects the C Termini of PCNA from Phosphorylation
Since it has been observed that PCNA
from distantly related species can be interchanged in
vitro and in vivo, we next tested RF-C 140 of
D. melanogaster. Although human and Schizosaccharomyces pombe PCNA are only 51% identical, the human protein can
complement a Schizosaccharomyces PCNA deletion mutation
(32). Similarly, D. melanogaster PCNA could substitute for
mammalian PCNA in SV40 in vitro replication although the
proteins are only 71% identical (33). We used the cloned RF-C 140 of
D. melanogaster fused with maltose binding
protein2 and tested the purified fusion protein in KPA. A
3-fold molar excess of D. melanogaster RF-C 140 led to a
45% inhibition of cphPCNA phosphorylation by
cAMP-dependent kinase (Fig. 4). A 7-fold excess of the D. melanogaster RF-C 140 did not cause any
protection of the N-terminally phosphorylatable PCNA (data not shown),
further supporting the involvement of the PCNA region close to the C
termini in the interaction with RF-C. The DNA binding region of
D. melanogaster RF-C 140 did not protect cphPCNA from
phosphorylation (data not shown), consistent with our results with
human RF-C 140 fragment A (see above). These results confirmed that
RF-C interacts with PCNA at the C-side, and not at the N-side, and that
the large subunit is involved in this interaction. Moreover, the
molecular interactions between PCNA and RF-C 140 have been conserved
through eukaryotic evolution.
Human RF-C 36 Interacts Independently with PCNA Close to the C Termini, whereas RF-C 40 Is Not Able to Protect PCNA in KPA or to Inhibit DNA Replication in Vitro
An interaction between the four
small RF-C subunits and PCNA is plausible because all of them show
sequence similarity to the PCNA binding region (10) of RF-C 140 (Fig.
5). Of the four individual small subunits, only RF-C 36 and RF-C 40 could be produced as soluble single subunits. This is not
surprising in view of the work published by Uhlmann et al.
(34) where the coexpression of at least a subset of small subunits was
required to get soluble proteins. Nevertheless, we managed to
solubilize RF-C 36 and RF-C 40, and tested their ability to protect
cphPCNA and nphPCNA in the KPA. Incubation of RF-C 36 with cphPCNA for
10 min resulted in a 60% inhibition of phosphorylation of PCNA (Fig.
6, A and B), providing evidence
that RF-C 36 interacts with PCNA at the C-side. On the other hand, no
protection was observed when RF-C 36 was incubated with nphPCNA (data
not shown). Further evidence for an interaction between PCNA and RF-C
36 was obtained with RF-C-dependent replication assays on
singly primed M13 DNA. RF-C 36 inhibited DNA replication (Fig.
6C), most likely by interfering with the function of RF-C.
These results were confirmed by the inability of RF-C 36 to inhibit
RF-C-independent DNA synthesis (Fig. 6D). On the other hand,
we were unable to show any inhibition of DNA synthesis by RF-C 40 (both
RF-C-dependent and -independent) (Fig. 6, C and
D) even by using very low amounts (0.1 and 0.05 units) of
pol and PCNA (data not shown), nor could we detect protection of
either cph- or nph-PCNA from phosphorylation in the KPA (data not
shown). However, we found weak ATP-dependent PCNA binding
activity in RF-C 40 by direct pull-down assays (see below). Fig. 6,
C and D, also shows the effect of His-RF-C 140 fragment B on DNA replication, confirming the results already obtained
with the GST-RF-C 140 fragment B (10). His-RF-C 140 fragment B
inhibited RF-C-dependent DNA synthesis by about 90% and,
therefore, appears to interfere strongly with the RF-C·PCNA interaction. This strong interaction with PCNA was even reflected in
RF-C-independent DNA replication where RF-C 140 fragment B, unlike the
RF-C 36 and RF-C 40, caused inhibition of nucleotide incorporation and
thus likely trapping PCNA from pol
.
A Pull-down Assay Supports the Interaction Data Obtained with KPA and DNA Replication
To support the data obtained with KPA and
with the in vitro DNA replication experiments, we took
advantage of the fact that RF-C 140, RF-C 140 fragment B, RF-C 36, and
RF-C 40 are all His tagged so that they could be bound to
Ni-NTA-coupled resin, allowing detection of PCNA binding with pull-down
assays (see "Experimental Procedures"). We found that RF-C 140, RF-C 140 fragment B, and RF-C 36 were able to bind to PCNA in the
absence of ATP, whereas RF-C 40, under the conditions used, was not
(Fig. 7). This was consistent with the results obtained
in the KPA and in the DNA replication experiments. Inclusion of 1 mM ATP did not significantly alter the amount of PCNA
pulled down by RF-C 140, RF-C 140 fragment B or RF-C 36, but a weak
PCNA binding activity (less than 1% compared to RF-C 140, RF-C 140 fragment B, and RF-C 36) appeared with RF-C 40 (data not shown),
thereby indicating that RF-C 40 was properly folded.
Since its first isolation from human cells (2), RF-C has been the
subject of several studies aimed at revealing its structure, function
and properties. Several functions of this replication factor have been
identified, but the precise mechanisms hidden behind its function
remain a subject of speculation. It appears that many features of RF-C
action in DNA replication are comparable with the complex of
E. coli, a prokaryotic counterpart. Indeed, in spite of
great evolutionary distance, sequences and functions are similar
between these two protein complexes. There are, however, details
specific to the eukaryotic replication machinery. A main goal of this
work was to begin characterizing the molecular interactions between
RF-C and PCNA by first determining the region within PCNA that is bound
by RF-C.
The subunits comprising the RF-C complex carry out a variety of
activities. Human RF-C 40 has been shown to bind ATP (9, 35). RF-C 37 was proposed to take part in the binding of RF-C to the primer termini
of both leading and lagging strands (14). RF-C is a DNA- and
PCNA-activated ATPase, an activity that has been linked to the RF-C 3 subunit in yeast (36), which corresponds to human RF-C 36. Recently, it
was shown that RF-C 140 specifically binds to PCNA, and a PCNA binding
region was located between aa 478-712 (10). This PCNA binding region
contains the boxes II-IV that are conserved in all RF-C subunits. This
region also shows significant similarities to the E. coli
pol III subunits /
(37, 38) and
(39) and to the
bacteriophage T4 gp44 protein (40).
In the crystal structure of PCNA, the C termini protrude from one face of the ring (20), appearing as candidates for interaction with other proteins, including RF-C. A modified form of PCNA, the C termini of which could be artificially phosphorylated with 32P, and different subunits and fragments of RF-C have been used to screen for possible interactions. We have shown that PCNA and calf thymus RF-C can interact in a way that makes the C termini of PCNA less accessible to cAMP-dependent kinase. A 2-fold molar excess of RF-C over PCNA caused a 50% inhibition of phosphorylation. This suggests that RF-C binds to PCNA close to the C termini of the latter. In this interaction, the large subunit is certainly involved. In fact, incubation of cphPCNA with the PCNA binding region of human RF-C 140 resulted in a consistent inhibition of PCNA phosphorylation, while the DNA binding region, in agreement with its inability to bind PCNA, did not have the same effect. A high degree of protection of the C termini from phosphorylation was also obtained with RF-C 36, suggesting that this subunit interacts with the same region of PCNA. The lower phosphorylation inhibition (50%) reached with the RF-C 36 compared with the 80% inhibition obtained with the RF-C 140 fragment B could be explained with a lower affinity of the small subunit to the same site on PCNA. It is unclear why RF-C 140 fragment B and fragment A + B do not behave in the same way, but a similar effect has been observed in other studies (10). It may be that the DNA binding region exerts some negative steric effect on the PCNA binding region. The fact that the native pentameric RF-C complex, as well as D. melanogaster RF-C 140 and the PCNA binding region of human RF-C 140, could not significantly protect the N termini of PCNA from phosphorylation are evidence for an interaction of RF-C at the C-side of the PCNA ring. The binding of D. melanogaster RF-C 140 to human PCNA gives rise to an important observation, namely the strong conservation in the mode of interaction of PCNA and RF-C throughout eukaryotic evolution. In fact, the RF-C complex from Saccharomyces cerevisiae, mouse, human, and D. melanogaster are all able to interact with human PCNA, as expected from the high degree of sequence similarity observed in the PCNA binding region.
In RF-C 140, the DNA binding region and the PCNA binding region are adjacent (10) so that formation of a DNA/RF-C complex could lead to a higher affinity of RF-C to PCNA and to a subsequent formation of the DNA/PCNA/RF-C complex. RF-C 140 could bind first to DNA and PCNA and facilitate, by a concerted action, the binding of the small subunits that alone would have a lower affinity for the ring. Small subunits other than RF-C 36 might also be involved in the PCNA interaction since, as mentioned before, they show significant sequence similarity to each other and, more importantly, with the PCNA binding region of RF-C 140 (Fig. 5).
In summary, our results show that RF-C interacts with a distinct face
of the PCNA ring, namely with the C-side. This interaction certainly
involves the large subunit; among the small subunits, RF-C 36 also
interacts with PCNA at the C-side. Our attempts to determine a site of
interaction between RF-C 40 were not successful. Although we could
detect ATP-dependent binding of His-RF-C 40 to PCNA, the
interaction seen was too weak to have an effect in KPA or in
vitro DNA replication assays. Indeed, preliminary experiments with
the yeast two-hybrid system failed to show an interaction between RF-C
40 and PCNA,3 further suggesting that RF-C
40 may not interact strongly with PCNA when removed from the RF-C
complex. If RF-C covers the C-side of PCNA (as implied by the
protection from phosphorylation of cphPCNA but not of nphPCNA), it
seems plausible that the other ring side would be free for interactions
with other proteins, e.g. with pol . Another possibility
would be that pol
competes for PCNA interaction with RF-C and,
thus, also binds to the C-side of the ring. That this is the case for
the prokaryotic replication complex was shown by Naktinis et
al. (15). However, addition of pol
to the KPA did not show any
difference in protection between cph- and nphPCNA (data not shown). It
is, therefore, not yet possi-ble to speculate about the structure of
the possible PCNA/RF-C/pol
complex.