From the Head and Neck Cancer Research Program, Guys,
King's, and St. Thomas' Dental Institute and the
** Division of Molecular and Medical Genetics, Pediatrics Research Unit,
Guy's Campus, King's College London, London SE1 9RT, United
Kingdom, the § Cold Spring Harbor Laboratory, Cold Spring
Harbor, New York 11724, and the
Imperial Cancer Research
Fund Skin Tumor Laboratory, the Royal London Hospital, London
E12AT, United Kingdom
Received for publication, September 29, 2000, and in revised form, November 20, 2000
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ABSTRACT |
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The proliferating cell nuclear antigen
(PCNA) is a highly conserved protein required for the assembly of the
DNA polymerase delta (pol DNA replication is a fundamental biological process that is vital
for cell proliferation. The mechanism of DNA synthesis and its
regulation is highly complex requiring the interplay of many proteins
that cooperate to duplicate the genetic information for the next
generation rapidly and accurately (1-4). Factors required for DNA
replication in eukaryotes have been identified using replication of
simian virus 40 (SV40) as a model system because all of the proteins
required for SV40, except the viral large T antigen, are host-provided
(5-7). Systematic reconstitution of SV40 replication in
vitro using highly purified proteins has provided important insights into their functions (8). Our current understanding of DNA
replication in eukaryotes suggests that polymerase A vital element of the DNA replication machinery is the ability of DNA
pol Besides being required for coordinated leading and lagging strand DNA
synthesis at a replication fork (25), PCNA has also been implicated in
a variety of cellular processes such as cell cycle control (26),
nucleotide excision repair (27, 28), postreplication mismatch repair
(29, 30), base excision repair (31), chromatin function (32-34), RNA
transcription, and cytosine-5 methylation (35) (for reviews see Refs.
2, 4, 20, 36). PCNA therefore not only acts as a clamp for DNA
polymerases, but it is also a multifunctional protein that may link
multiple protein-protein interactions in replication, repair,
recombination, and cell cycle regulation.
The primary structure of PCNA is highly conserved in the animal kingdom
(31, 37, 38), and related proteins have been found in plant (39), yeast
(40, 41), and virus (42). Recent mutation analyses of both human and
yeast PCNA have shown that PCNA is remarkably resistant to amino acid
substitution, but these studies have defined protein interaction sites
on the surface of PCNA (23, 43-51). However, deletions in any part of
the molecule including the N and C termini distort the tertiary
structure, rendering the protein of limited use in functional assays.
The Saccharomyces cerevisiae homolog of PCNA, pol30, shares
35% sequence identity with the human PCNA (hPCNA) and is able to
enhance the processivity of the mammalian pol We introduced mutations in the highly conserved region of PCNA and also
generated a set of novel human-S. cerevisiae PCNA hybrids.
These PCNAs were examined for their ability to stimulate pol Materials--
Restriction and DNA-modifying enzymes were
obtained from Roche Molecular Biochemicals (Germany) and from Promega.
Radioactive chemicals were bought from Amersham Pharmacia
Biotech and ICN (U. K.). Synthetic oligonucleotides were
supplied by Imperial Cancer Research Fund laboratories, U. K., and
purified by urea-PAGE before use. (dA)290-539 and
oligo(dT)12-18 were obtained from Amersham Pharmacia
Biotech. The 69-nucleotide hairpin DNA used in the RF-C stimulation
assay was constructed by ligating two oligonucleotides following the
method described previously (9). Singly primed M13 DNA, M13mp18, was
prepared according to the method described earlier (56). Anti-PCNA
antibodies, PC-9 and PC-10, were obtained by growing respective
hybridomas in Dulbecco's modified Eagle's medium + 10% fetal calf
serum. Supernatants from the confluent cultures were stored in 0.2%
(w/v) sodium azide until used. All other chemicals and reagents used in
this study were molecular biology grade and were obtained from BDH/Merck and Sigma.
Plasmid pSV011 was constructed by ligating a
HindIII-SphI fragment containing the SV40 origin
of replication into pUC18 as described earlier (25), and the construct
was purified by Triton X-100 lysis of recombinant bacteria followed by
CsCl gradient centrifugation. Construction of plasmids for the
expression of the human, pThPCNA, and S. cerevisiae,
pTyPCNA, PCNA has been described previously (41, 53).
Preparation of Replication Proteins--
DNA pol Site-directed Mutagenesis and Preparation of Human-S. cerevisiae
PCNA Hybrids--
Mutations were introduced into the respective
cDNA for the wild type (wt) h- and cPCNA using the plasmid pThPCNA
and pTyPCNA by polymerase chain reaction-mediated site-directed
mutagenesis using the Quick Change mutagenesis kit (Stratagene)
according to the manufacturer's instructions. The primers used for
generating mutants and the restriction sites created are shown in Table
I. Four human and five S. cerevisiae PCNA mutants were prepared using this method. The
mutations were confirmed by nucleotide sequencing.
The mutant cDNAs for h- and cPCNA were subjected to double
restriction enzyme digestion using the restriction sites created during
mutagenesis (see Table I) and the BamHI site present at the
3'-end in all constructs. The insert and vector DNAs were ligated in
different combinations such that the hPCNA inserts were swapped with
the corresponding segment of cPCNA. This resulted in 10 different
constructs containing part of the hPCNA fused with a part of cPCNA (see
Table II), which were confirmed by nucleotide sequencing. The hybrids
were named by two letters followed by a number in which the first
letter was derived from the species contributing the N terminus
followed by the species contributing the C terminus. For example,
hybrids with the human sequence at the N terminus were named HC whereas
those with S. cerevisiae sequence at their N terminus were
named as CH. The contribution of the two PCNAs in each hybrid is shown
in Table II.
Expression and Purification of Recombinant PCNA--
Plasmids
constructs encoding wt, mutant, and hybrid PCNA were transformed into
BL21(DE3) cells for expression of protein. An overnight culture
(0.75 ml) was used to inoculate 200 ml of LB + ampicillin and grown at
37 °C until A600 reached to 0.6. The protein
expression was induced with 1 mM
isopropyl-1-thio-
The procedure for extraction and purification of PCNA from recombinant
bacteria was carried out at 4 °C. Bacteria from 200-ml cultures were
harvested at 3,000 rpm for 15 min, resuspended into 4 ml of lysis
buffer (25 mM Tris-HCl, pH 7.4, 25 mM NaCl, 1 mM EDTA, 0.01% Nonidet P-40, 2 mM benzamidine,
2 µM pepstatin A, 10 mM NaHSO3, 1 mM phenylmethylsulfonyl fluoride, and 1 mM
DTT), and sonicated to disrupt cells and reduce viscosity. The mixture was adjusted to 0.2 M NaCl, cleared by centrifugation, and
loaded onto a Q-Sepharose column (1.2 × 11 cm) equilibrated with
0.2 M NaCl and buffer A (50 mM Tris-HCl, pH
7.4, 20% glycerol, 2 mM EDTA, 0.02% Nonidet P-40, 2 mM benzamidine, 2 µM pepstatin A, 10 mM NaHSO3, 1 mM
phenylmethylsulfonyl fluoride, and 1 mM DTT). After washing
the column, bound PCNA was eluted with a 0.2-0.7 M linear
NaCl gradient in buffer A. The PCNA-containing fractions, identified by
SDS-PAGE, were pooled and dialyzed against 25 mM KPO4, pH 7.0, and buffer B (10 mM
KPO4, pH 7.0, 10% glycerol, 0.01% Nonidet P-40, 2 mM benzamidine, 2 µM pepstatin A, 10 mM NaHSO3, 1 mM
phenylmethylsulfonyl fluoride, and 1 mM DTT) and loaded
onto an S-Sepharose column (1.2 x 11 cm). The PCNA-containing flow-through fractions were pooled and loaded onto a hydroxyapatite column (2.5 × 11 cm) equilibrated with 25 mM
KPO4 and buffer B and eluted with a 0.025-0.5
M linear phosphate gradient in buffer B. The
PCNA-containing fractions from the hydroxyapatite column were pooled,
dialyzed against 1.2 M NaCl and buffer C (50 mM
Tris-HCl, pH 7.4, 2 mM EDTA, 0.02% Nonidet P-40, 2 mM benzamidine, 2 µM pepstatin A, 10 mM NaHSO3, 1 mM
phenylmethylsulfonyl fluoride, and 1 mM DTT), and 4 ml of
the sample was loaded onto a phenyl-agarose column (1.2 × 11 cm).
The protein was eluted with a decreasing linear salt gradient from 1.2 to 0 M NaCl in buffer C, and the PCNA-containing fractions
were pooled, dialyzed against 20% sucrose, 25 mM NaCl, and
buffer A and stored in aliquots at Electrophoresis, Immunoblotting, and Chemical
Cross-linking--
SDS-PAGE of PCNA proteins was carried out on 12%
polyacrylamide gels using the conditions described elsewhere (61).
Western blotting was performed using nitrocellulose membranes as
described earlier (62). Native gel electrophoresis was carried out on 8-25% gradient gels using the Phast System (Amersham Pharmacia Biotech) according to the manufacturer's recommendations.
Chemical cross-linking was carried out using ethylene
glycol-bis-succinimidyl succinate as described previously (63).
Gel Filtration Analysis--
Purified wt, mutant, and hybrid
PCNA preparations (100 µg) were applied onto a Superose 12 HR10/30
(Amersham Pharmacia Biotech) gel filtration column equilibrated with 25 mM Tris-HCl, pH 7.4, 5 mM EDTA, and 250 mM NaCl. The column was calibrated by eluting a sample of
blue dextran and five native molecular mass markers, lactate
dehydrogenase (145 kDa), bovine serum albumin (66 kDa), ovalbumin (43 kDa), carbonic anhydrase (31 kDa), and cytochrome c (12 kDa). The elution volume for each marker was the time taken to elute
the peak fraction of that sample. The peak fractions (300-500 µl)
for each PCNA were collected, concentrated using CentriconTM
microconcentrators (Amicon), and the presence of PCNA was confirmed by
SDS-PAGE.
Stimulation and Processivity Assay for DNA Pol
The processivity assay was essentially as described above except that
the reactions contained limiting amounts of PCNA (0.5-5 ng) and 0.0075 unit of pol RF-C ATPase Assays--
RF-C ATPase activity was measured
according to a protocol described previously (57). The reaction mixture
(20 µl) contained 30 mM HEPES pH 7.5 buffer, 7 mM MgCl2, 0.5 mM DTT, 0.1 mg/ml
bovine serum albumin, 100 µM ATP, 3 pmol of
[ DNA Replication Assays--
M13 DNA synthesis reactions were
carried out by incubating singly primed M13mp18 DNA with pol
The SV40 DNA replication assays in vitro were performed as
described previously (16) using S100, fraction IA, fraction II, large T
antigen, and SV40 origin of replication containing plasmid, pSV011. The
optimal amounts of extracts and proteins required in the assay were
determined empirically by titration. Reaction mixtures were assembled
using PCNA, pSV011, fractions IA and II, large T antigen, nucleotide
mixture, and creatine phosphate/creatine phosphokinase on ice. The
samples were incubated at 37 °C for 1 h; the controls were left
on ice. After terminating the reaction with 20 mM EDTA, the
32P incorporation was determined using DE81 paper. The
remaining portion of the reaction mixture from either M13 or SV40 assay was deproteinated with proteinase K and extracted with
phenol/chloroform. After precipitating with ethanol, the replication
products were analyzed on an agarose gel, fixed in a mixture of 10%
methanol + 10% acetic acid, dried, and autoradiographed.
Other Methods--
The techniques used for genetic manipulation
of bacteria were performed using standard protocols (64). Protein
concentrations were determined by the BCA method (Sigma) using BSA as
standard. Nuclease contamination in PCNA preparations was tested by
incubating each PCNA protein (20 ng-2 µg) with 1 µg of pGEX-2T for
1 h at 37 °C followed by separation on an agarose gel. All gel
pictures and blots were scanned and assembled using the Adobe PhotoShop version 5.
Expression and Purification of PCNA Mutants and Hybrids--
We
have produced 4 hPCNA (HU) and 5 cPCNA (CE) mutants, each bearing a
mutation that created a unique restriction site within the coding
region (Table I). The sites for mutation were selected, as far as
possible, to fall in the region connecting different
The pThPCNA and pTyPCNA plasmids expressing h- and cPCNA, respectively,
were used in site-directed mutagenesis as described under
"Experimental Procedures." The predicted structure of the hybrids
is shown in Fig. 1 and Table II. The
relative mobility of the wt h- and cPCNA on SDS-PAGE yielded an
apparent molecular mass of 36 kDa and 29 kDa, respectively. The wt
hPCNA, HU1, and HU3 had identical mobility on SDS-PAGE, whereas the
electrophoretic mobilities of the hybrids varied between that of wt h-
and cPCNA (Fig. 2). All constructs
produced soluble PCNA proteins when grown in bacteria on a smaller
scale; however, when a large scale cultures were employed, four of the
constructs, HC2, HC3, HC4, and CH1, lost their ability to produce
soluble protein efficiently, which could have been the result of
inclusion body formation. These constructs were therefore excluded from
this study.
Soluble PCNA extracted from large scale cultures were purified to near
homogeneity as judged by silver staining of SDS-PAGE gels (Fig. 2).
Starting from a 200-ml culture, the yield of purified PCNA was 1-2 mg
from different constructs except for HU3 and CH4, which gave only 400 and 360 µg of purified protein, respectively. The lower yield of CH4
could be caused by the aggregation tendency of this hybrid as reported
for other mutants (23, 45); however, the same did not apply to HU3 that
was predominantly trimeric in solution (see Fig.
3). These preparations were free of
nucleases because no degradation of plasmid DNA was observed by up to
200 µg/ml PCNA (not shown), which was about 5-fold higher than the amount used in DNA replication assays.
Immunoblotting of the hybrids with anti-PCNA antibodies (65) produced
reactivity of PC-9 with CH2, CH3, and CH4, but not with HC1, HC5, and
CH5, whereas PC-10 reacted with CH2 but did not react with HC1, HC5,
CH3, CH4, and CH5 (data not shown). The mutants HU1 and HU3 reacted
with both of the antibodies, as did the wt hPCNA. The reactivity of
these antibodies with the hybrids is consistent to their epitope
locations reported earlier (66).
PCNA Mutants and Hybrids in Solution Can Assemble into
Oligomers--
To test whether the PCNA mutants and hybrids can form
trimers in solution we determined their elution profile on a gel
filtration column calibrated with five native proteins of known
molecular sizes. Wt h- and cPCNA preparations separated into a major
peak corresponding to an apparent molecular mass of 98 kDa (trimer) and
a minor peak corresponding to 29 kDa (monomer). The mutants HU1 and HU3
and hybrids HC5 and CH3 also gave a major trimer peak and a minor
monomer peak. The hybrids HC1 and CH5 produced more monomers than
trimers, and in CH2 most of protein eluted at the position of a dimer.
The hybrid CH4 had a small proportion of trimers, but most of the
protein eluted in the void volume. The proportion of oligomeric species
in different PCNA preparations is listed in Table
III. The data suggest that in solution
our PCNA preparations, with the exception of CH4, exist in equilibrium among monomers, dimers, and trimers. Thus our initial expectation that
hybrids of PCNA from the two species would assemble into trimers was
vindicated. However, the tertiary structure of some of the hybrids was
distorted as indicated by a lower proportion of trimers.
The pattern of PCNA oligomerization was investigated further by native
gel electrophoresis using an Amersham Pharmacia Biotech Phast System.
In this gel system the trimer band could be clearly distinguished from
the fast moving dimer and monomer bands (45). Consistent with our gel
filtration data we observed trimeric species in the wt h- and cPCNA,
HU1, HU3, HC5, and CH3. The HC1 hybrid remained predominantly a
monomer, and CH5 was a mixture of monomers and trimers. However, minor
PCNA species were not detected in this system, which could be the
result of the low sensitivity of Coomassie staining. The hybrid CH2
produced a smear with no defined band for monomers or trimers. This
suggested that trimers in CH2 were unstable and that the PCNA molecule
might spontaneously unfold and refold. CH4 was the only hybrid to have
large aggregates with no monomer and only traces of trimer (Fig. 3, in
the actual gel a trace of trimer was seen which is not very clear in
the photograph). The aggregates in CH4 were much more homogeneous in
size than for the Y114A mutation reported earlier (45). The presence of
dimers in CH2 and large aggregates in CH4 was established further by
ethylene glycol bis-succinimidyl succinate-induced cross-linking (data
not shown).
Function of Mutants and Hybrids Using PCNA-induced DNA Polymerase
Most of the PCNA Hybrids Were Active in RF-C-mediated DNA
Clamping--
The interaction between RF-C and PCNA in the presence of
DNA and ATP results in ATP hydrolysis and the formation of a sliding clamp. The ATPase activity of RF-C is stimulated by PCNA in the presence of primed DNA. The RF-C ATPase assay thus measures the ability
of PCNA to interact with RF-C and DNA. We examined the loading of PCNA
mutants and hybrids onto primed DNA by RF-C. The assay was conducted at
four different PCNA concentrations (100, 200, 400, and 800 ng) and to
compare the data from different sets, the PCNA-dependent
stimulation of RF-C ATPase activity for different preparations was
expressed as percentage of the wt hPCNA (Fig. 6). The RF-C stimulation with cPCNA was
similar to that in the absence of PCNA, suggesting that cPCNA was
inactive in this assay. All PCNA hybrids tested showed
PCNA-dependent stimulation of RF-C ATPase, but to different
extents. The least stimulation was seen with the hybrids HC1 and HC5,
both showing about 10% of PCNA-dependent RF-C stimulation
compared with the wt hPCNA. The two mutants HU1 and HU3 and the other
hybrids, CH2, CH3, and CH5, with the exception of CH4, achieved
40-70% of RF-C stimulation. As with the wt PCNA, the percentage of
RF-C stimulation did not increase with PCNA concentration for any of
the hybrid PCNA preparations, except for the hybrid CH4. In CH4 the
PCNA-dependent stimulation of RF-C ATPase was proportional
to the PCNA concentration and was much higher than for the wt
hPCNA.
To determine the ATP turnover for the interaction of RF-C with PCNA, we
performed RF-C ATPase assays at 800 ng of PCNA for 15 and 30 min.
Previous studies (9, 23, 67) have shown that an incubation of 30 min
was sufficient for ATP hydrolysis during RF-C·PCNA·DNA complex
formation. Although an incubation of 30 min was sufficient for ADP
production to reach steady state, the level of ATPase activity with
mutants and hybrids, with the exception of CH4, was less compared with
hPCNA (data not shown). This is consistent with the
dose-dependent experiments described above and shows that
the PCNA preparations, with the exception of HC1 and HC5, were active
in RF-C dependent clamping of DNA.
PCNA Mutants HU1 and HU3 Showed Diminished Ability to Complement
M13 DNA Replication in Vitro--
Having shown the effects of the
mutants and hybrids on both pol Human-S. cerevisiae PCNA Hybrids Were Inactive in SV40 DNA
Replication in Vitro--
A more functional characterization of the
PCNA mutants and hybrids was carried out by reconstituting SV40 DNA
replication in vitro with purified PCNA and partially
fractionated cell extracts. In the reconstitution experiments,
replication of DNA was dependent on the presence of large T antigen and
the SV40 origin of replication, indicating that replicative rather than
repair synthesis was occurring (data not shown). The level of DNA
synthesis achieved by the mutants and hybrids was determined from the
amount of dAMP (in pmol) incorporated during a 1-h incubation. None of
the mutants, hybrids, or the cPCNA attained a level of replication
achieved with the wt hPCNA (Fig. 8).
Analysis of the replication products on an agarose gel revealed three
different categories, A, B, and C (Fig. 8). The replication products
with hPCNA and HU1 shown in category C were almost identical. HU3 and
cPCNA gave rise to replication products similar to those reported due
to uncoupled leading strand synthesis (Fig. 8; group B). Hybrids HC1,
HC5, CH2, CH3, CH4, and CH5 did not support DNA replication, and the
products seen were due to pol
We also investigated the possibility that complete DNA replication in
these reactions might be PCNA dose-dependent, hence the
replication reactions were carried out at three different concentrations of five of the PCNA hybrids. No obvious difference was
observed in any of the reactions with increasing amounts of PCNA (data
not shown). This suggested that the lack of complete DNA replication
described above was not caused by insufficient amounts of PCNA in these assays.
Previous mutagenesis studies have shown that deletion of any part
of PCNA can distort its tertiary structure, rendering it incapable of
association into trimers. Taking a lead from previous work, where cPCNA
was unable to complement hPCNA in SV40 DNA replication in
vitro (53), when both proteins existed as trimers in solution (17,
55), we hypothesized that hybrids of the two proteins would be
equivalent to large deletions within hPCNA without disrupting the
trimeric ring. This would allow identification of functional regions in
hPCNA involved in the assembly of the pol From the crystal structure of PCNA, the intermonomer interactions
involving The two mutants HU1 and HU3 stimulated pol All hybrids were able to assemble into trimers, albeit to different
extents, but only CH3 and CH5 stimulated pol The data on RF-C stimulation with CH2, CH3, and CH5 suggest that
increasing the proportion of cPCNA sequence does not change the ATPase
activity, implying that the major RF-C binding was located at the C
terminus (Fig. 6 and Table II). This is consistent with previous
mutagenesis studies (23, 70). However, the C terminus of neither pPCNA
(68) nor cPCNA (48) contains a RF-C binding site, which is consistent
with our finding that the hybrids HC1 and HC5 with the C terminus of
cPCNA showed the least RF-C binding (see Fig. 6). For RF-C stimulation,
the conformation of residues rather than a trimeric structure appears
to be more important. For instance, the hybrid CH2 that was
predominantly a dimer in solution expressed RF-C stimulation as much as
by HU1, HU3, and CH5, which were mainly trimeric (Fig. 6). This
suggests that a trimeric PCNA may not be a prerequisite for
RF-C-mediated clamping of DNA. However, it is possible that binding of
RF-C to PCNA could induce trimeric assembly and subsequent clamping.
Jonsson et al. (45) have shown that Y114A PCNA
aggregates did not compete with the trimeric PCNA for pol Assembly of the pol The three sites that we have identified on hPCNA located on N- and
C-terminal regions and the IDC loop appear to play an important role in
the stimulation of pol In summary, we have shown that the trimeric structure of PCNA is
maintained by associations among several ) holoenzyme. Because PCNAs from
Saccharomyces cerevisiae and human do not complement each
other using in vitro or in vivo assays, hybrids
of the two proteins would help identify region(s) involved in the
assembly of the pol
holoenzyme. Two mutants of human PCNA, HU1
(D21E) and HU3 (D120E), and six hybrids of human and S. cerevisiae PCNA, HC1, HC5, CH2, CH3, CH4, and CH5, were prepared
by swapping corresponding regions between the two proteins. In
solution, all PCNA assembled into trimers, albeit to different extents.
These PCNA variants were tested for stimulation of pol
and in
vitro replication of M13 and SV40 DNA as well as to stimulate the
ATPase activity of replication factor C (RF-C). Our data suggest that
in addition to the interdomain connecting loop and C terminus, an
additional site in the N terminus is required for pol
interaction.
PCNA mutants and hybrids that stimulated pol
and RF-C were
deficient in M13 and SV40 DNA replication assays, indicating that
PCNA-induced pol
stimulation and RF-C-mediated loading are not
sufficient for coordinated DNA synthesis at a replication fork.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(pol1
)
primase
synthesizes the first RNA/DNA primer on the leading strand which is
then followed by the binding of RF-C and loading of PCNA and a
polymerase switch from pol
to pol
. Thus pol
holoenzyme
performs processive leading strand synthesis, whereas pol
participates in RNA priming and lagging strand synthesis. However,
complete synthesis of Okazaki fragments also requires participation of
pol
holoenzyme (9-15).
to execute processive DNA synthesis allowing the polymerase to
move quickly along the template thousands of nucleotides without
dissociation. This is accomplished in Escherichia coli by
the
subunit of DNA pol III and in eukaryotes by PCNA, which is a
processivity factor for pol
(16). The crystal structure of the
subunit and PCNA showed a toroidal structure that can encircle the DNA
(17, 18). PCNA is a homotrimer in which each subunit consists of two
structurally related domains giving the molecule a 6-fold symmetry. The
center of the ring is positively charged, and it is large enough to
allow free passage of double-stranded DNA. PCNA is loaded onto DNA by
the action of RF-C (11, 19), a complex of five different subunits (20,
21), in an ATP-dependent manner. The RF-C·PCNA
complex then tethers pol
onto the template to assemble a highly
processive pol
·PCNA·RF-C complex also known as the pol
holoenzyme. However, in the absence of RF-C, PCNA can still load onto
linear DNA at a double-stranded end, albeit at much lower efficiency
(22). The orientation of the PCNA ring provides two surfaces, one
facing the primer-template junction required for pol
and RF-C
binding and the other surface facing the double-stranded DNA for
binding of other proteins (23, 24). The molecular interactions between
different subunits of various proteins in the holoenzyme have only been
partially studied.
(52), but it is
unable to complement hPCNA in SV40 DNA replication in vitro
(53). This suggests that S. cerevisiae PCNA (cPCNA) has very
low affinity for mammalian pol
and RF-C, even though both yeast and
mammalian PCNAs exist as trimers (54, 55). We therefore argued that by
swapping different regions between h- and cPCNA, it should be possible
to generate hybrids that were equivalent to large deletions in hPCNA
while retaining a trimeric structure. Functional analyses of these
hybrids would help to define region(s) of hPCNA involved in DNA replication.
and
RF-C or to function in DNA replication assays. The stimulation of RF-C
and pol
involves associations on at least three sites located at
the N and C termini, and at the interdomain connector (IDC) loop of
PCNA. We have observed induction of RF-C-catalyzed ATPase activity by a
PCNA hybrid that was predominantly dimeric, suggesting that PCNA
trimers may not be necessary for stimulation of RF-C ATPase. Our data
also suggest that assembly of the pol
holoenzyme may require
participation of other proteins at a replication fork.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
from calf
thymus, RPA from E. coli, and SV40 large T antigen from
Sf9 insect cells infected with recombinant baculovirus were
purified as described previously (15). Human RF-C was expressed and
purified from baculovirus infected Sf9 cells as described earlier (57). The cytosolic replication extract (S100) from human 293 cells was prepared as described (7). Fractionation of S100 into
fractions II and IA has been described previously (16). Fraction II
contained multiple components including DNA polymerases
and
,
RF-C, topoisomerases I and II, DNA ligases, and other essential DNA
replication proteins; RPA was the main component of fraction IA (9, 16,
58-60).
Sequence of oligonucleotides used in site-directed mutagenesis of h-
and cPCNA
Contribution of the h- and cPCNA in different hybrids used in this
study along with the origin of IDC loop
-D-galactopyranoside and grown
for 3-6 h at 37 °C.
70 °C until used.
--
The DNA
pol
stimulation assay used in this study was a slightly modified
version of that described previously (12, 13). The reaction mixtures
(12.5 µl) contained 30 mM HEPES/NaOH pH 6.6 buffer, 7 mM MgCl2, 0.5 mM DTT, 0.1 mg/ml
bovine serum albumin, (dA)540, oligo(dT)16, in
a ratio of 19:1 in nucleotides, 40 µM dTTP, 0.17 pmol of
[
-32P]TTP, 0.15 unit of calf thymus pol
, and
different concentrations of PCNA. The reactions were incubated at
37 °C for 15 min, and 2 µl was spotted on DE81 paper (Whatman),
washed in 0.5 M Na2HPO4, and the
incorporated radioactivity was measured in a scintillation counter.
in the presence of 1.7 pmol of
[
-32P]TTP. The incorporated radioactivity was
determined as described above, and the rest of the reaction mixture was
analyzed on a 2% alkaline agarose gel and visualized by autoradiography.
-32P]ATP (800 Ci/mmol), 0.25 pmol of RF-C, 0.4 µmol
of hairpin DNA with 5'-single-strand extension, and different
concentrations of PCNA. The reaction mixtures were incubated for 1 h at 37 °C and stopped by 10 mM EDTA. One-tenth (2 µl)
of the reaction mixture was spotted onto a polyethyleneimine-cellulose
plate that had been pretreated with 1 M formic acid. The
plates were then developed in a mixture of 1 M formic acid
and 0.5 M LiCl for 60 min. After autoradiography the amount
of ADP produced was determined by scanning the plates on a Storm 860 PhosphorImager (Molecular Dynamics).
, RF-C,
PCNA, and RPA as described previously (56).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
sheets or
helices. Two of the hPCNA mutants, HU2 and HU4, had silent mutations
and were used only for making hybrids; the other two, HU1, containing
the mutation D21E in the A1B1 loop on the inner surface of the trimer,
and HU3, containing the mutation D120E at the beginning of the IDC
loop, were conservative changes. These mutants and hybrids were
employed in functional analyses, whereas the cPCNA mutants were used
only for preparing hybrids.
View larger version (21K):
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Fig. 1.
Schematic representation of the polypeptide
folding in a native PCNA molecule. Panel A, the
elements within the two topologically identical domains N1 and C1 are
labeled using the nomenclature described by Krishna et al.
(17). The location of the mutation D21E in HU1 is shown by an
asterisk, which, in the folded structure, lies on the inner
surface of the trimeric ring. The mutation D120E in HU3, shown by a
diamond, is located in the IDC loop. Panel B,
structure of monomeric PCNA for the wt human, S. cerevisiae,
and the hybrids. The hPCNA polypeptide is shown by a combination of
red and pink; cPCNA is shown by a combination of
cynol and blue. The N termini of the h- and cPCNA
are shown, respectively, by pink and cynol; the C
termini are shown by red and blue.
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Fig. 2.
Purification of recombinant wt, mutant, and
hybrid PCNA from E. coli. Plasmid constructs
containing wt, mutant, and hybrid PCNA were transformed in BL21(DE3)
E. coli cells, and protein expression was induced with 1 mM isopropyl-1-thio- -D-galactopyranoside.
The lysate from a 200-ml culture was adjusted to 0.2 M
NaCl, filtered through 0.22 µM before subjecting to
Q-Sepharose, S-Sepharose, hydroxyapatite, and phenyl-Sepharose columns
as described under "Experimental Procedures." The purification of
PCNA at various stages of the protocol was followed by subjecting a
small fraction through SDS-PAGE and staining the gel with Coomassie
Blue. Panel A, a representative gel of the PCNA hybrid CH3
during different stages of purification. Panel B,
silver-stained SDS gel of the purified PCNA proteins used in this
study. A 10-µl sample containing 3-5 µg of purified PCNA was
analyzed in each case.
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Fig. 3.
Analysis of PCNA proteins on a native
gel. Wt PCNA (h- and cPCNA), mutants (HU1 and HU3), and the hybrid
proteins were dialyzed in buffer A containing 25 mM NaCl
and 20% glycerol. 1 µg of each PCNA protein and molecular markers
were taken in 8 µl of loading buffer, and 4 µl of this mixture was
run on an 8-25% polyacrylamide gel for 45 min and stained with
Coomassie Blue. Lanes M1 and M2 indicate native
protein markers: thyroglobulin (669 kDa), ferritin (440 kDa), catalase
(232 kDa), lactate dehydrogenase (140 kDa), BSA (67 kDa), and ovalbumin
(45 kDa).
Ratio of monomeric, dimeric, and trimeric PCNA recovered from gel
filtration column
Activity--
The ability of the different PCNA proteins to
stimulate calf thymus pol
activity on a synthetic DNA template was
studied. Incubation of pol
with the wt hPCNA stimulated the enzyme
in a dose-dependent fashion as reported previously (23).
Compared with the hPCNA, the wt cPCNA did not express significant pol
stimulatory activity (Fig. 4). The
two mutants, HU1 and HU3, also stimulated pol
in a
dose-dependent fashion, but they had about 40% stimulatory
activity compared with the wt hPCNA. This is interesting because the
two mutations were located in different regions of the molecule. The
D21E mutation in HU1 is located toward the N terminus, and the D120E
mutation in HU3 is located in the IDC loop. These two sites appear to
play a role in PCNA-induced stimulation of pol
activity. As shown
in Fig. 4, the hybrids HC1, HC5, CH2, and CH4, did not stimulate pol
, but CH3 and CH5 stimulated the enzyme in a
dose-dependent fashion. The degree of pol
stimulation by CH3 was similar to that achieved with hPCNA, whereas CH5 had much
reduced stimulatory activity. The ability of the wt, mutant, and hybrid
PCNA to enhance the processivity of pol
was determined by analyzing
the size distribution of the replication products on alkaline agarose
gel electrophoresis. As shown in Fig. 5,
the hybrids HC1, HC5, CH2, and CH4 did not induce processive DNA
synthesis, which was consistent with them being unable to interact
efficiently with pol
. The PCNA proteins that stimulated pol
,
HU1, HU3, CH3, and CH5, appeared processive, but their activity, except for CH3, was reduced compared with the wt hPCNA. This suggested that
HU1, HU3, and CH5 were impaired in their initial interaction with pol
but not in the processive DNA synthesis. The data indicate involvement of IDC loop and both the N- and C-terminal domains in the
interaction of PCNA with pol
. The processivity of pol
with CH3
was not very different from that with hPCNA, but it was certainly more
active than the wt hPCNA (see Fig. 5). Taken together, the data suggest
that formation of PCNA·pol
complex involves multiple site
interactions.
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Fig. 4.
Function of PCNA variants with calf thymus
DNA pol as determined by PCNA-induced
stimulation of DNA polymerase activity. Reactions were carried out
as described under "Experimental Procedures" with increasing
concentrations of PCNA, and incorporation of radioactivity after
incubation at 37 °C for 15 min was determined by spotting 2 µl of
the reaction mixture on DE81 paper. The amount of dTMP (in pmol)
polymerized was plotted against the amount of PCNA used in the
reaction. Each graph in panels A, B,
and C represents results of a minimum of at least three
independent experiments. The standard error bars represent
95% confidence intervals on the means. The PCNA proteins used in
different reactions are shown by individual symbols in each
graph.
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Fig. 5.
Effect of PCNA mutants and hybrids on the
processivity of DNA pol . In this set of
experiments the reaction components were adjusted such that the dTMP
incorporation corresponded to a DNA synthesis of less than 0.3 nucleotide from each primer end. A reaction mixture containing 0.5 ng
of the indicated PCNA protein, 0.0075 unit of pol
, and 1.7 pmol of
[
-32P]TTP in a total volume of 50 µl was incubated
at 37 °C for 15 min. The products were analyzed on a 2% agarose gel
under alkaline conditions and revealed by autoradiography. The length
of the products represents the processivity of DNA pol
in the
presence of indicated PCNA proteins. Lane M contained DNA
fragments obtained with
DNA digested with HindIII, and
NP was a control reaction without PCNA.
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Fig. 6.
Characterization of RF-C ATPase activity
induced by PCNA mutants and hybrids. The RF-C-catalyzed ATPase
assays were performed in a 20-µl reaction containing 0.4 µmol of
hairpin DNA and 0.25 pmol of RF-C as described under "Experimental
Procedures." The amount of ADP produced by RF-C in the absence of
PCNA (NP) and in the presence of increasing amounts (100, 200, 400, and 800 ng) of each of the PCNA protein after incubation at
37 °C for 1 h was determined using a Storm 860 PhosphorImager.
The degree of stimulation of RF-C ATPase activity by different PCNA
preparations was compared with the wt hPCNA. The activity of hPCNA
under identical conditions was taken as 100%.
stimulation and RF-C loading, we
tested their ability to support replication of a singly primed circular
DNA, M13mp18. In this reaction assembly of the holoenzyme is achieved,
and the assay measures the overall interaction of PCNA with RF-C and
DNA as well as with pol
. From the incorporation of
[32P]dAMP, formation of a quaternary complex of wt hPCNA
with RF-C, pol
, and the DNA was evident. The cPCNA was inactive in
this assay (Fig. 7A). The
mutants HU1 and HU3 supported DNA replication, but the hybrids were
inactive. The overall activity of both mutants was similar to each
other but only about 30% compared with the wt hPCNA. However, an
analysis on an alkaline agarose gel revealed that the replication
products with HU1 were shorter than those obtained with hPCNA (Fig.
7B). HU3 replication products were even shorter than with
HU1, and they resembled those described previously when PCNA or RF-C
was omitted from the replication reaction (9, 25). Clearly, the effect
of mutation in HU3 was more dramatic than in HU1 and highlighted the
role of the IDC loop in the assembly of the pol
holoenzyme.
Furthermore, the inability of CH3 and CH5 hybrids to induce DNA
synthesis in this assay suggests that the intermolecular interactions
at a replication fork are perhaps not the same as those required for
the stimulation of pol
and RF-C using synthetic substrates.
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Fig. 7.
Influence of PCNA mutants and hybrids on M13
DNA replication in vitro. DNA synthesis on singly
primed M13 template was carried in 30 mM HEPES/KOH pH 7.5 buffer, 7 mM MgCl2, 50 mM NaCl, and
1 mM DTT using 4 µg/ml M13mp18 DNA, 10 ng of pol , 10 µg/ml RPA, and 1 unit of RF-C in a 20-µl reaction mixture with a 20 µg/ml concentration of the indicated PCNA. Panel A, amount
of dAMP (in pmol) incorporated in the presence of different PCNA
proteins. Panel B, replication products were analyzed on a
2% agarose gel run under alkaline conditions and visualized by
autoradiography. Lanes 1 and 2 contained DNA
fragments of known sizes by digesting
DNA with Bst II and
the plasmid pBR322 with MspI, respectively. Lane
NP shows the reaction products in the absence of PCNA. Lanes
hPCNA and cPCNA contained, respectively, the wt human
and S. cerevisiae PCNAs. The single-stranded full-length
linear DNA (ssL) and a possible pause site (I)
are shown by arrows.
-primase activity (Fig. 8; group A).
Thus the wt hPCNA and HU1 were able to accomplish complete DNA
synthesis, and HU3 and the hybrids were inactive.
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Fig. 8.
Influence of PCNA mutants and hybrids on the
SV40 DNA replication in vitro. The SV40 DNA
replication in vitro was performed using S100 cell extract,
fractions IA, II, purified large T antigen, and a SV40 origin
containing plasmid pSV011 as described under "Experimental
Procedures" with different PCNA proteins indicated in the figure.
PCNA was mixed with 6 µg/ml pSV011, 600 µg/ml fraction II, 79 µg/ml large T antigen, 900 µg/ml fraction IA, 40 mM
HEPES, pH 8, 8 mM MgCl2. 0.5 mM
DTT, 25 µM dATP, 0.1 mM each dCTP, dGTP,
dTTP, 3 mM ATP, 0.2 mM CTP, GTP, UTP, 40 mM creatine phosphate, 0.12 unit creatine
phosphokinase in a total volume of 12.5 µl. The samples were
incubated at 37 °C for 1 h, and the controls were left on ice.
After stopping the reaction with 20 mM EDTA, the mixture
was freed of proteins, and the replication products were analyzed on an
agarose gel. The gel was fixed, dried, and autoradiographed. Lane
M refers to HindIII DNA size markers. NP
is a control reaction in the absence of PCNA. The replication products
were classified into groups A, B, and C based on the pattern seen on
the gel.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
holoenzyme at a
replication fork. We therefore produced two human PCNA mutants, HU1
(D21E) and HU3 (D121E), and six hybrids of human and S. cerevisiae PCNA (see Fig. 1). We have characterized these proteins
for their ability to oligomerize and interact with DNA, pol
, and
RF-C using M13 and SV40 DNA replication in vitro.
I1 and
D2 sheets appear important in trimer formation
(17, 55). Consequently, mutations located in
I1, such as Y114A in
hPCNA (45) and S115P in cPCNA (46), are reported to disrupt trimer
formation. However, trimer formation by CH3 suggests that the presence
of the
D2 sequence of human and the
I1 of S. cerevisiae at the intermonomer interface does not interfere with
trimeric structure. In CH3, almost the complete domain 1 of cPCNA was
fused to domain 2 of hPCNA. However, replacing
A2,
B2, and
C2
sheets of hPCNA in CH3 by the corresponding sheets of cPCNA, as in CH4,
distorted the tertiary structure (Figs. 1 and 3). Similarly, replacing
the N-terminal sequences as in CH2 and HC1 led to a lower proportion of
trimers. These data indicate that intramolecular interactions between
certain
sheets of h- and cPCNA in domain 1 and 2 may not be
compatible with trimer formation. The C terminus of hPCNA in CH5 gave a
higher proportion of monomers than the converse hybrid HC5, indicating
a role for the C terminus in monomer-trimer equilibrium. The N and C
termini in PCNA are buried into the structure, and deletion of these
residues (23, 44) or replacing them by another PCNA sequence, as
described in this study, is likely to disrupt inter-
sheet
interactions. Our data are therefore consistent with a recent study
where mutations L68S and G69D in
F1 of Schizosaccharomyces
pombe PCNA (pPCNA) produced only dimers, suggesting that other
parts of the molecule, besides intermonomer interface, play a role in
the stabilization of trimers (51).
and RF-C by about 40%
compared with the wt hPCNA (see Figs. 4 and 6), suggesting a role for
Asp-21 and Asp-120 in these interactions. The mutation D21E in HU1 is
located just outside
A1, and this region has never been implicated
in pol
or RF-C stimulation. In the crystal structure, however,
Asp-21 is close to the central loop
(41DSSHV45), which is shared by pol
and
RF-C (23, 24). Furthermore, the proximity of Asp-21 to helix
A1,
which formed part of the central ring for DNA clamping, might also
affect pol
and RF-C stimulation. The mutation D120E in HU3 was part
of IDC loop (118MDLDVEQLGIPEQEYSC134), which
has been implicated in pol
stimulation in several studies (23, 49).
However, the IDC loop by itself was not sufficient for pol
stimulation because the hybrid HC5 containing the IDC loop of human
origin was inactive. More than 50% reduction in RF-C stimulation with
HU3 may indicate a role for the IDC loop in RF-C-induced clamping.
Involvement of the IDC loop in RF-C stimulation has not been suggested
in the literature. However, a mutation close to the IDC loop, A112T,
and another within the loop, S135F, have been shown to suppress the
cold-sensitive mutants in Cdc44p, the largest subunit of S. cerevisiae RF-C (67). Although the mechanism for the suppression
of cdc44 cold sensitivity is not clear, the data indicate a
role for the IDC loop in the process.
. The hybrid CH5, with
the last 50 amino acids replaced by the corresponding region of the
hPCNA (see Fig. 1), stimulated pol
, but the converse hybrid HC5 was
inactive. This indicates that the C-terminal region of hPCNA was a high
affinity site for pol
. This is consistent with previous work where
the pol
binding site in hPCNA (23) and in pPCNA (68, 69) has been
located at the C terminus. However, the inability of CH2 and CH4 to
stimulate pol
when both had a C-terminal sequence of hPCNA could be
due to a lower proportion of trimers in these preparations (see Fig.
3). This suggests that a certain proportion of PCNA trimers in solution was necessary in addition to a pol
binding site for effective polymerase stimulation. It is interesting to note that hybrid CH3,
which formed perfect trimers, also showed high pol
stimulation and
appeared to be more active than the wt hPCNA (see Figs. 1, 4, and 5). A
PCNA mutant with such characteristics has not been described in the
literature. The pol
stimulation assay measures the interaction of
PCNA with pol
and the DNA in the absence of RF-C. The interaction
of PCNA with DNA depends on the charge distribution on the central
helices in PCNA which is conserved across species. All trimeric PCNA
hybrids would have DNA clamping similar to hPCNA. The much higher pol
stimulatory activity associated with CH3 could therefore indicate a
stretch of cPCNA in CH3 acquiring a conformation with much higher
affinity for pol
.
and RF-C,
and based on this they concluded that RF-C did not have a role in
aiding trimer formation. However, it may be that for RF-C to aid trimer
assembly the RF-C binding must be preserved, and the aggregated PCNA
might have lost these sites. Although monomeric PCNA mutants have been
shown inactive in RF-C mediated clamping in vitro (23, 46),
one of them, S115P, assembled the pol
holoenzyme in the presence of
6% polyethylene glycose and also supported S. cerevisiae
cell growth, suggesting that nontrimeric PCNAs that have retained their
ability to associate can be loaded by RF-C (46). RF-C-induced loading
of CH2 as shown in this study indicates that RF-C can sequester
nontrimeric PCNA in presence of DNA. RF-C-mediated loading of the two
dimeric mutants L68S and G69D described recently is not known as they
were not used in the loading assay (51). The basis for the very high PCNA-dependent RF-C ATPase activity observed in the PCNA
hybrid CH4 is not clear. It could be caused by structural alterations introduced by swapping over the polypeptide segments.
holoenzyme complex at a replication fork is a
reflection of the ability of PCNA to load on to DNA by RF-C and
subsequent association of pol
into a highly processive enzyme. Thus
interaction among different components of the ternary complex including
RF-C, PCNA, pol
, and DNA can be measured in one holoenzyme assay.
Using the M13 replication assay we found that only HU1 supported some
replicative activity, whereas HU3 along with the hybrids was inactive.
This indicated that stimulation of pol
and RF-C-induced DNA
clamping by the mutants and hybrids CH3 and CH5 could not activate DNA
synthesis at a replication fork. It may be argued that hybrids CH3 and
CH5 cannot interact simultaneously with pol
and RF-C in this assay
because identical sites are involved. However, pol
and RF-C do
interact simultaneously with the wt hPCNA perhaps because they can
share redundant structure, which may be lost in the hybrids. Another
possibility could be that assembly of the pol
holoenzyme involves
binding of other proteins and that loss of this binding in the hybrids
could block DNA synthesis. Involvement of other proteins in holoenzyme
assembly has been suspected previously from the IDC loop mutants unable to interact with pol
but had no influence on DNA replication (48).
Besides PCNA, pol
, and RF-C, the only other protein in M13 assay is
RPA. Although a direct interaction between RPA and PCNA has not been
reported, a recent study has shown that RPA forms a common touch-point
with pol
, RF-C, and PCNA at a replication fork. Among these, an
association of RPA with the RF-C·PCNA complex has been proposed to
play an important role for the holoenzyme function (71). It is possible
that the complex between RF-C and PCNA mutants or hybrids acquires a
conformation that is not compatible with RPA binding. Alternatively,
RPA may have cryptic sites for PCNA which are accessible only at the
replication fork. Incomplete accessibility of these sites in PCNA
variants could have prevented RPA binding. Our results therefore
suggest a role of RF-C and RPA far beyond loading of PCNA and coupling the unwinding of the double helix with the initiation of DNA synthesis (72, 73). The results from SV40 DNA synthesis were similar to the M13
assay except for HU1, which was more active in SV40 than in the M13
assay. The discrepancy between the two assays could be caused by a
factor(s) in the SV40 reaction mixture able to compensate for the loss
of binding in the M13 assay. This is likely given the fact that SV40
DNA replication required a far more complex mixture of proteins. Taken
together, the data from SV40 and M13 DNA replication assays suggest
that the PCNA hybrids and the mutant HU3 were unable to assemble a
functional DNA pol
holoenzyme.
, RF-C, and also in the assembly of the
holoenzyme. Besides PCNA, the proteins involved in the holoenzyme
assembly pol
, RF-C, and RPA are oligomers of heterosubunits. Mammalian DNA pol
is an oligomer of three different subunits, 125 kDa, 48 kDa, and a recently identified 66-kDa subunit (74). RF-C has
five subunits, 140, 36, 37, 38, and 40 kDa, but only the largest
subunit binds PCNA (57). RPA is also a heterotrimer of p70, p34, and
p11 (75). PCNA provides a platform for dynamic assembly and disassembly
of these proteins at a replication fork. To enable an ordered flow of
events during DNA replication it is perhaps important that there is
more than one binding site for each protein on PCNA.
sheets located in domains
1 and 2 of the molecule. We have identified three regions on hPCNA,
located at the N- and C-terminal domains and the IDC loop, which are
required to stimulate pol
and RF-C activity, but the site at the
C-terminal domain appears to be the high affinity site for the two
proteins. Interestingly, PCNA dimers, which were inactive in pol
stimulation but showed significant RF-C-induced loading, suggested that
trimeric PCNA may not be a prerequisite for RF-C-mediated clamping of
DNA. Our data indicate participation of other proteins, one of them
could be RPA, for successful DNA synthesis at a replication fork. The
identification and roles of these proteins at a replication fork will
enable us to have a complete understanding of the biochemical steps
necessary for the assembly of pol
holoenzyme.
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to GKT Dental Institute for providing the space and facilities for this project. Ian Goldsmith of Clare Hall Laboratories, Imperial Cancer Research Fund, kindly supplied synthetic oligonucleotides used in this study. We also thank Dr. R. Evans and Dr. C. Joannou, Division of Biomolecular Sciences, King's College London, for helping us with the gel filtration and Phast electrophoresis experiments and Patricia Purkis for technical help. We also acknowledge Drs. R. Evans and A. Yeudall for helpful comments on this manuscript.
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FOOTNOTES |
---|
* This work was supported by the Special Trustees of Guy's Hospital, Guy's Dental Funds Committee, and the Wellcome Trust.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Present address: Dept. of Biochemistry and Molecular Biology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565 0871, Japan.
To whom correspondence should be addressed: Dept. of
Craniofacial Development, GKT Dental Institute, Floor 28, Guy's
Hospital, King's College London, London SE1 9RT, United
Kingdom. Tel.: 44-207-955-4992; E-mail:
ahmed.waseem@kcl.ac.uk.
Published, JBC Papers in Press, November 27, 2000, DOI 10.1074/jbc.M008929200
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ABBREVIATIONS |
---|
The abbreviations used are: pol, polymerase; RF-C, replication factor C; PCNA, proliferating cell nuclear antigen; hPCNA, human PCNA; cPCNA, S. cerevisiae PCNA; PAGE, polyacrylamide gel electrophoresis; RPA, replication protein A; wt, wild type; DTT, dithiothreitol; interdomain connector.
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