On the Specificity of Interaction between the Saccharomyces
cerevisiae Clamp Loader Replication Factor C and Primed DNA
Templates during DNA Replication*
Manju M.
Hingorani
and
Maria Magdalena
Coman
From the Wesleyan University, Molecular Biology and Biochemistry
Department, Middletown, Connecticut 06459
Received for publication, July 8, 2002, and in revised form, October 1, 2002
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ABSTRACT |
Replication factor C (RFC) catalyzes assembly of
circular proliferating cell nuclear antigen clamps around primed DNA,
enabling processive synthesis by DNA polymerase during DNA replication and repair. In order to perform this function efficiently, RFC must
rapidly recognize primed DNA as the substrate for clamp assembly, particularly during lagging strand synthesis. Earlier reports as well
as quantitative DNA binding experiments from this study indicate,
however, that RFC interacts with primer-template as well as single- and
double-stranded DNA (ssDNA and dsDNA, respectively) with similar high
affinity (apparent Kd 
10 nM). How then can RFC distinguish primed DNA sites from excess ssDNA and dsDNA
at the replication fork? Further analysis reveals that despite its high
affinity for various DNA structures, RFC selects primer-template DNA
even in the presence of a 50-fold excess of ssDNA and dsDNA. The
interaction between ssDNA or dsDNA and RFC is far less stable than
between primed DNA and RFC (koff > 0.2 s
1 versus 0.025 s1,
respectively). We propose that the ability to rapidly bind and release
single- and double-stranded DNA coupled with selective, stable binding
to primer-template DNA allows RFC to scan DNA efficiently for primed
sites where it can pause to initiate clamp assembly.
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INTRODUCTION |
Replicative DNA polymerases synthesize DNA with high processivity
in collaboration with two key accessory proteins: a circular clamp and
a clamp loader complex (see Fig. 1; reviewed in Refs. 1 and 2). These
accessory proteins are conserved in structure and function among a
variety of organisms including bacteriophage (e.g. T4 gp45
clamp and gp44/62 clamp loader), bacteria (e.g. Escherichia coli 
clamp and 
complex clamp loader),
archaebacteria (e.g. Pyrococcus furiosus
proliferating cell nuclear antigen
(PCNA)1 clamp and replication
factor C (RFC) clamp loader), and eukaryotes such as
Saccharomyces cerevisiae and humans (PCNA and RFC) as well
(see Ref. 3 for a review of recent crystal structures). Clamps are
ring-shaped multisubunit proteins with an inner diameter of ~35 Å that is large enough to encircle duplex DNA (e.g.
homotrimeric PCNA) (4). When bound to polymerase and DNA, the clamp
forms a sliding tether that stabilizes polymerase on the
primer-template and facilitates processive DNA replication (5). A
polymerase-clamp complex, such as S. cerevisiae Pol
·PCNA, can extend DNA with a processivity of hundreds of
nucleotides per template binding event, a substantial increase from the
6-12-nucleotide processivity of Pol alone (6, 7).
The circular clamp is opened and assembled around primer-template DNA
by the clamp loader, a multiprotein machine whose action is fueled by
ATP binding and hydrolysis. Clamps must be present at primed DNA sites
for polymerase to bind them and initiate processive DNA synthesis (7,
8). Efficient clamp assembly is particularly important during lagging
DNA synthesis when a clamp is required at the initiation of every
Okazaki fragment (Fig. 1). Given an estimated Okazaki fragment size of
100-200 nucleotides in eukaryotes and a Pol ·PCNA-catalyzed DNA
synthesis rate of ~100 nucleotides/s, clamp assembly is required once
every 1 or 2 s during lagging strand synthesis (7). The
replicative polymerase, Pol III holoenzyme, from the well examined
E. coli model system has a similar requirement for timely
clamp assembly as it completes a 1000-2000-nucleotide Okazaki fragment
every 1-2 s (reviewed in Ref. 2). Thus, clamp loaders have to rapidly
recognize primed sites on template DNA and assemble clamps on them for
efficient initiation and completion of DNA replication.
The S. cerevisiae clamp loader, RFC, is composed of five
proteins: RFC1 (95 kDa), RFC2 (40 kDa), RFC3 (38 kDa), RFC4 (36 kDa), and RFC5 (40 kDa). All five proteins are essential for viability according to deletion analysis (9-14) (reviewed in Ref. 15). The five
proteins share sequence homology among each other and with clamp loader
proteins from other organisms (e.g. and ' proteins of
E. coli complex and gp44 protein of bacteriophage T4
gp44/62; reviewed in Refs. 14 and 16). Human RFC is very similar to the
S. cerevisiae RFC complex, with one large subunit (p140) and
four small subunits (p37, p36, p40, and p38) that correspond to RFC1,
-2, -3, -4, and -5, respectively (15). The homology occurs chiefly in
seven regions, RFC boxes II-VIII, of which boxes III and V have the
most well defined function as ATP-binding Walker A and B motifs,
respectively. The large RFC1 subunit contains an additional box I at
the amino terminus, which shares homology with DNA ligases and is known
to bind DNA but appears unnecessary for clamp loading (17).
Recent crystal structures of the E. coli complex clamp
loader, 3' (18, 19), and the small RFC subunit
from archaebacterium P. furiosus (20) reveal striking
structural similarities as well among clamp loaders from different
organisms. Each subunit is composed of three domains, a helical
carboxyl-terminal domain III and amino-terminal domains I and II,
connected by flexible linker regions. The complete E. coli
3' clamp loader structure reveals five subunits
arranged in a circle that is closed by interactions among the
C-terminal domains but open at the N-terminal domains (the RFC sketch
in Fig. 1 is modeled after the 3' structure). The
subunit contains a clamp-binding element in N-terminal domain I
that contacts a hydrophobic pocket on the clamp and triggers clamp
opening. The crystal structure of a · clamp complex indicates that the clamp fits "flat" up against the clamp loader with its C-terminal face adjacent to the N-terminal face of the clamp loader (3,
21). Fig. 1 shows a sketch of RFC·PCNA
derived from the proposed 3'· complex
structure. Presumably, the primer-template DNA enters the circular
clamp through the open interface; however, it is not clear where or how
the clamp loader binds DNA to facilitate interaction between the clamp
and DNA. The P. furiosus small RFC subunit has the same
overall fold as the E. coli complex subunits (20), and
electron microscopic images of the human RFC complex (22) and archaeal
small RFC subunit (23) show the subunits in a circular arrangement,
indicating that the eukaryotic/archaeal clamp loaders adopt a similar
quaternary structure as the bacterial clamp loader and may therefore
employ similar mechanisms of action.
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Fig. 1.
A model eukaryotic DNA replication fork.
The diagram depicts the minimal protein activity required at
a DNA replication fork, including the DNA-unwinding helicase,
single-stranded DNA-binding protein (RPA), primase (Pol  ), leading
strand polymerase (Pol ), lagging strand polymerase (Pol and/or
Pol  ), circular sliding clamp (PCNA), and the clamp loader (RFC),
which must catalyze PCNA assembly at multiple primed sites during
lagging DNA strand synthesis.
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The mechanism of clamp assembly has been examined to varying degrees of
detail for E. coli, phage T4, and human/ S. cerevisiae clamp loaders. In the E. coli complex,
ATP binding to the subunits induces a change in conformation that
exposes , allowing it to bind and open the clamp (24, 25). A
recent study of the interactions between complex and its substrates
indicates that the clamp loader binds and then primer-template DNA
with high affinity, and DNA binding specifically triggers ATP
hydrolysis and release of linked topologically to DNA (26).
Notably, the complex also binds single-stranded DNA with high
affinity in the presence or absence of the clamp (26), raising
questions about how the clamp loader recognizes primed DNA but not
ssDNA as the substrate for clamp assembly. In the case of bacteriophage T4 clamp loader, gp44/62, the proposed clamp loading mechanism includes
ATP binding to the four gp44 subunits in the complex, followed by
interaction between gp44/62 and gp45 clamp, hydrolysis of two ATP
molecules, interaction with DNA, hydrolysis of the remaining two ATP,
and finally release of the clamp·DNA complex (27). A more recent
study suggests that only one ATP molecule is hydrolyzed during gp45
assembly on DNA; thus, the precise mechanism of how gp44/62 couples ATP
binding and hydrolysis to clamp assembly on DNA is not yet clear (28).
Additionally, there is not enough information available on the
interaction between gp44/62 and different DNA structures to clarify how
the clamp loader selects primer-template DNA as the site for clamp assembly.
In the case of eukaryotic clamp loaders, questions about DNA selection
are even more pertinent, given the numerous reports of human/S.
cerevisiae RFC binding to double-stranded DNA (29-32), primer-template DNA with either a 3' primer-template junction (33, 34)
or a 5' phosphorylated primer-template junction (35), and
single-stranded DNA (33, 36, 37). These results suggest a relatively
low specificity of RFC binding to primer-template DNA, and since the
concentration of primed sites is expected to be much lower than that of
single- and double-stranded regions in replicating DNA, how does the
clamp loader rapidly recognize a primed DNA site for clamp assembly? An
early footprinting experiment with S. cerevisiae RFC and a
hairpin DNA substrate with a 3' recessed end demonstrated convincingly
that the clamp loader binds at the primer-template junction (33), but
the same study also found that RFC binds ssDNA and to some extent dsDNA
as well (gel mobility shift experiments). The addition of
single-stranded binding protein, RPA, to the reaction reduced the
interaction between an RFC·PCNA complex and ssDNA (33). A more recent
examination of RFC-DNA interaction by surface plasmon resonance
revealed similar inhibition of RFC binding to ssDNA by RPA (38).
However, the extent of RPA binding to single-stranded template DNA
during replication in vivo is not known, and it may not be
sufficient to facilitate highly specific interaction between RFC and
primer-template DNA. Other studies have shown that RFC binds dsDNA and
that its ATPase activity is stimulated by dsDNA (31, 39), although this
result is contradicted by reports that RFC binding to the 3'
primer-template junction is not competed by dsDNA and that the large
subunit of RFC in fact binds ssDNA (37). Single-stranded DNA binding
activity of human and Drosophila melanogaster RFC has also
been observed by electron microscopy, and in this study RFC appeared to
have no preference for 3' or 5' primer-template junctions (36). Given the variety of DNAs and techniques utilized by different research groups to examine RFC·DNA complexes, a study of the literature does
not yield adequate data for a quantitative comparison of clamp loader
interactions with different DNA structures. Thus, the question of how
RFC selects only primed DNA for clamp assembly although it apparently
binds single- and double-stranded DNA remains to be answered.
Efficient assembly and function of a DNA replicase depends to a large
extent on the ability of replication proteins to correctly and rapidly
recognize DNA structures relevant to their activity. Here we have
addressed the question of how the RFC clamp loader distinguishes
between binding to primed DNA and single- or double-stranded DNA during
PCNA clamp assembly. A quantitative analysis reveals that RFC binds all
three DNA structures with high affinity and yet selects primed DNA
almost exclusively in the presence of excess single- or double-stranded
DNA, possibly via a primed DNA-specific change in its
conformation/activity that is integral to the clamp assembly mechanism.
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EXPERIMENTAL PROCEDURES |
Proteins, DNA, and Buffers--
Overexpression and
purification of the complete five-subunit S. cerevisiae RFC
complex, as well as truncated RFC complex (RFC1:
2-283) and PCNA
(gifts from Dr. Mike O'Donnell, Rockefeller University, New York) are
described here briefly; the detailed procedure will be published
elsewhere.2 Genes for all
five RFC subunits, RFC1, -2, -3, -4, and -5, were cloned from S. cerevisiae genomic DNA into a single pET vector, each under the
control of a T7 promoter. The proteins were overexpressed in E. coli cells that also produced tRNAs for arginine, isoleucine, and
leucine to correct for codon bias and purified by ion exchange chromatography over Q Sepharose and SP Sepharose columns. PCNA was
cloned similarly into a pET vector, except with an amino-terminal His
tag and cAMP-dependent protein kinase tag, which allowed
single-step purification by nickel column chromatography and
32P labeling of PCNA, respectively. E. coli SSB
was a gift from Dr. Mike O'Donnell (40). Protein concentrations were
measured by the Bradford assay and by absorbance at 280 nm in 6 M guanidinium hydrochloride, using calculated extinction
coefficients: RFC, 163520; trRFC, 162120; PCNA, 5600. Trypsin was
purchased from Sigma, and T4 polynucleotide kinase and
cAMP-dependent protein kinase were purchased from New
England Biolabs. Bacteriophage T7 DNA polymerase (5'-3' exonuclease
mutant) was a gift from Dr. Smita Patel (Robert Wood Johnson Medical
School, New Jersey).
Oligodeoxyribonucleotides were synthesized by Integrated DNA
Technologies and purified by denaturing polyacrylamide gel
electrophoresis. The sequences are as follows: 30-mer, 5'-CTG GTA ATA
TCC AGA ACA ATA TTA CCG CCA-3'; 30A-mer, 30-mer plus 3' A; 30cmpT, 5'-T
TGG CGG TAA TAT TGT TCT GGA TAT TAC CAG-3'; 81-mer, 5'-GAG CGT TTT TTC
CTG TTG CAA ACG ATT GGC GGT AAT ATT GTT CTG GAT ATT ACC AGC AAG GCC GAT
AGT TTG AGT TCT TCT-3'; 56-mer, 5'-GAG CGT TTT TTC CTG TTG CAA ACG ATT
GGC GGT AAT ATT GTT CTG GAT ATT ACC AG-3'; 56-mer-5p, 5'-TTG GCG GTA
ATA TTG TTC TGG ATA TTA CCA GCA AGG CCG ATA GTT TGA GTT CTT CT-3'.
Primer-template and duplex DNAs were prepared by mixing 30-mer or
30A-mer with the requisite complementary strand at a ratio of 1.1:1,
respectively, in 20 mM Tris-HCl, pH 7.5, 150 mM
NaCl, followed by heating to 95 °C (2 min) and slow cooling for >6
h. Single-stranded M13 mp18 ssDNA was purified and annealed with a
30-nucleotide primer as described (41). ATP and ATPS were purchased
from Sigma; [-32P]ATP, [-32P]ATP, and
[-32P]dATP were purchased from PerkinElmer Life
Sciences. Nitrocellulose membranes and PEI-cellulose TLC plates were
purchased from Schleicher and Schuell and EM Science, respectively. The
buffers are as follows: buffer H, 30 mM HEPES, pH 7.5, 4 mM MgCl2, 5% glycerol; gel filtration buffer,
buffer H plus 0.1 mg/ml bovine serum albumin, 1 mM DTT, 0.1 mM EDTA, 100 mM NaCl; protease buffer, buffer H
plus 0.1 mM EDTA, 2 mM DTT; T7 Pol buffer, 40 mM Tris-HCl, pH 7.5, 14 mM MgCl2, 50 mM NaCl, 1 mM EDTA, 1 mM DTT,
0.1 mg/ml bovine serum albumin; SDS gel-loading buffer, 50 mM Tris-HCl, pH 7.5, 100 mM DTT, 2% SDS, 0.1%
bromphenol blue, 10% glycerol.
PCNA Loading Assays--
PCNA (2 µM) was labeled
at an N-terminal kinase recognition site in a 100-µl reaction with 20 µCi of [-32P]ATP and 50 units of
cAMP-dependent protein kinase in NEB kinase buffer for
1 h at 37 °C. Excess [-32P]ATP was removed by
filtration through Centricon-10 (Millipore Corp.). In the loading
reaction, 0.03 µM 32P-PCNA was mixed with
0.03 µM DNA (primed or unprimed M13mp18 ssDNA coated with
8.5 µM SSB or duplex pBluescript plasmid) and 0.5 mM ATP in 60 µl of gel filtration buffer. The reaction
was initiated with 0.02 µM RFC or trRFC (RFC1:2-283),
incubated at 30 °C for 5 min, and then filtered over a 5-ml Bio-Gel
A-15m column equilibrated in gel filtration buffer. 200-µl fractions
were collected, and aliquots were quantitated by scintillation counting.
Quantitative DNA Binding Assays--
The DNA used in
nitrocellulose membrane binding assays was 32P-labeled as
follows: 0.05 µM annealed 30-mer/30cmpT was incubated in
a 100-µl reaction with 10 µCi of [-32P]dATP and
0.2 µM T7 DNA polymerase (premixed with a 5-fold molar excess of thioredoxin and 30 mM freshly prepared DTT) in T7
Pol buffer for 5 min at 37 °C. Next, 100 µM dATP was
added to the reaction, and after 5 min the reaction was quenched with
30 mM EDTA and 5 min at 90 °C. The DNA was separated
from free nucleotide by spinning through a Bio-Gel P-30 column
(Bio-Rad). The radiolabeled 3032P-A-mer/30cmpT DNA
(0.03 µM) was mixed with appropriate unlabeled DNAs to
prepare 30 µM stocks of single-stranded 30A-mer,
primer-templates 30A-mer/81-mer, 30A-mer/56-mer (3' primer-junction),
and 30A-mer/56-mer-5p (5' primer-junction) as well as duplex
30A-mer/30cmpT DNA, as described above.
The stoichiometry of interaction between RFC and primer-template DNA
was measured by nitrocellulose membrane binding assays in which a
constant amount of 32P-labeled DNA was titrated with
increasing concentrations of RFC. Nitrocellulose membranes were
pretreated with 0.5 N NaOH for 2 min and then washed
thoroughly with H2O and equilibrated in buffer H. The
reactions (15-µl total volume) containing 0.5 µM
32P-DNA and 0-3 µM RFC in buffer H were
incubated for 10 min at 25 °C, and 10-µl aliquots were filtered
through the membrane in a dot-blot assembly (Schleicher and Schuell).
The membrane was washed before and after filtration with 120 µl of
buffer H. 1-µl aliquots were spotted onto a separate membrane to
measure the total DNA in the reaction. Radioactivity on the membrane
was quantitated on a PhosphorImager (Amersham Biosciences), and
the molar amount of DNA bound to RFC was determined and plotted
versus RFC concentration. Experiments measuring equilibrium
dissociation constants for the RFC-DNA interaction were performed
similarly except with a constant amount of RFC (0.016 µM)
or trRFC (0.016 µM) and increasing 32P-DNA
(0-0.6 µM) in buffer H in the absence or presence of 0.5 mM ATPS or ADP; the effect of PCNA on RFC-DNA
interaction was measured in similar experiments with 1 mM
ATPS and 1 µM PCNA. The molar amount of DNA bound to
RFC was plotted versus DNA concentration. The binding
isotherms were fit to a quadratic equation to determine the apparent
dissociation constant for the interaction,
![[D·R]=0.5{K<SUB>d</SUB>+[D<SUB>t</SUB>]+[R<SUB>t</SUB>]−[(K<SUB>d</SUB>+[D<SUB>t</SUB>]](/content/vol277/issue49/fulltext/47213/img001.gif)
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(Eq. 1)
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where D·R represents the amount of DNA
bound to RFC, Dt and Rt are total
DNA and RFC concentrations, respectively, and Kd is
the dissociation constant.
Competitive DNA binding assays were performed by mixing 0.02 µM 32P-labeled substrate DNA with increasing
concentrations of unlabeled competitor DNA (0-1 µM) in
buffer H (15-µl total volume), followed by the addition of 0.016 µM RFC and filtration through a nitrocellulose membrane
after 10 min at 25 °C, as described above; complementary assays were
performed with 0.02 µM unlabeled substrate and 0-1 µM labeled competitor. The amount of DNA substrate bound
to RFC was plotted versus competitor DNA concentration, and
the data were fit to a hyperbola to determine K1/2, the competitor concentration at which half of the substrate remains bound to RFC.
The rate of dissociation of DNA from RFC was measured by incubating
0.016 µM RFC with 0.02 µM
32P-DNA substrate in buffer H for 10 min at 25 °C,
followed by the addition of 1 µM unlabeled competitor
DNA. At various times, 10-µl aliquots of the reaction were filtered
through a nitrocellulose membrane as described above
(32P-DNA bound to RFC at time 0 was measured in the absence
of competitor). The molar amount of DNA bound to RFC was plotted
versus time, and the data were fit to a single exponential
function to yield the dissociation rate,
koff.
![[D·R]=R<SUB>t</SUB>[<UP>exp</UP>(−k<SUB><UP>off</UP></SUB> t)]](/content/vol277/issue49/fulltext/47213/img003.gif)
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(Eq. 2)
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Trypsin Digest--
Tryptic digestion of RFC was performed at
both high and low NaCl concentrations, in the absence and in the
presence of various DNA substrates. The 65-µl reaction contained 2 µM RFC and either no DNA or 3 µM DNA in
protease buffer at 100 or 30 mM final NaCl concentration.
The digest was initiated by the addition of trypsin to a final
concentration of 0.4 ng/µl in the reaction and incubation at
37 °C. At varying times (0-15 min), 10-µl aliquots of the
reaction were quenched with 10 µl of SDS gel-loading buffer and
heated at 90 °C for 5 min prior to analysis on a 12% polyacrylamide
gel. The proteolytic products were visualized by staining with
Coomassie Blue.
ATPase Assays--
Steady-state ATPase activity of RFC was
assayed by monitoring hydrolysis of [-32P]ATP to
[-32P]ADP plus Pi. 0.2 µM
RFC was mixed with 1 mM ATP plus
[-32P]ATP, 1 µM PCNA, and 1 µM DNA (when in the reaction) in buffer H plus 0-400
mM NaCl at 25 °C. At varying times, 5-µl aliquots were
withdrawn, quenched with 5 µl of 0.5 M EDTA, and analyzed by polyethyleneimine-cellulose thin layer chromatography in 0.6 M potassium phosphate buffer, pH 3.4. The molar amount of
[-32P]ADP formed was quantitated on a
PhosphorImager and plotted versus reaction time to yield
the ATPase rate constants (kcat), which were
then plotted versus NaCl concentration.
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RESULTS |
Rapid assembly of clamps at primed DNA sites is important to
expedite genomic DNA replication, since clamps are essential for
processive DNA polymerase activity. In eukaryotes, the RFC clamp loader
loads PCNA clamps onto DNA in a reaction fueled by ATP binding and
hydrolysis. In an earlier study, RFC was observed to footprint a 3'
primer-template junction, which suggested that the clamp loader
specifically recognizes this DNA structure as the site for clamp
assembly (33). However, the same report as well as other studies of
clamp loaders from S. cerevisiae, D. melanogaster, mice, and humans indicate that RFC binds
single-stranded DNA and double-stranded DNA as well (reviewed in Ref.
15). How can RFC distinguish a primed DNA site as the target for clamp assembly from the background of single- and double-stranded DNA at the
replication fork? The experiments described below address this question
by quantitatively analyzing the interactions between RFC and various
DNA substrates.
S. cerevisiae RFC Purified from E. coli Assembles PCNA Clamps
Preferentially on Primed DNA Templates--
Previous reports on
overexpression and purification of S. cerevisiae RFC from
E. coli indicated that the RFC subunits are insoluble when
expressed individually. However, when genes for the small RFC subunits
were co-expressed on a single plasmid in S. cerevisiae, a
soluble complex was detectable (42). More recently, a truncated version
of the RFC complex with 273 amino acids deleted (of 861 total) from the
amino terminus of the RFC1 subunit has been expressed and purified from
E. coli (43). This truncated version of RFC is active in
catalyzing PCNA clamp assembly on DNA; however, it is not yet
known whether all of its properties are identical to those of
the wild-type RFC clamp loader. We have purified the full-length,
wild-type RFC complex from E. coli with a yield of close to
7 mg of pure protein/liter of cells. Fig. 2A shows an SDS-PAGE analysis
of the RFC complex, which has all five subunits present in a 1:1
stoichiometric ratio. (The cloning and purification strategies will be
described in detail elsewhere.)2
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Fig. 2.
E. coli-produced RFC loads PCNA
clamps on primed DNA. A, an SDS-PAGE analysis of
S. cerevisiae RFC complex comprising five subunits in 1:1
stoichiometry. B, PCNA clamp assembly on DNA, assayed by
incubating 0.03 µM 32P-PCNA with 0.02 µM RFC and 0.03 µM SSB-coated circular
primed M13 ssDNA in the presence ( ) or absence ( ) of 0.5 mM ATP, unprimed M13 ssDNA with ATP ( ), and circular
duplex pBluescript DNA with ATP ( ) for 5 min at 30 °C, followed
by gel filtration as described under "Experimental Procedures."
PCNA loaded on DNA elutes in fractions 7-12 and free PCNA elutes in
fractions 14-25.
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The clamp loading activity of recombinant RFC was examined
by measuring the assembly of 32P-labeled PCNA onto circular
DNA (Fig. 2B). When incubated with singly primed, circular
M13mp18 ssDNA coated with SSB, RFC loads the clamp onto DNA in the
presence of ATP. The large PCNA·DNA complex elutes early from a gel
filtration column (fractions 7-12), whereas free PCNA elutes later
(fractions 14-25). The reaction is absolutely dependent on the
presence of ATP (Fig. 2B) and RFC (data not shown), since no
PCNA loading is evident in the absence of either of these reactants.
Furthermore, RFC does not load PCNA on ssDNA in the absence of a
primer, whether the DNA is coated with SSB (Fig. 2B) or not
(data not shown); earlier studies have shown that E. coli
SSB and eukaryotic RPA are indistinguishable in this PCNA loading assay
(43). Thus, RFC catalyzes PCNA assembly specifically at a primed DNA
site, as expected for its function in vivo. Fig.
2B also shows that PCNA is loaded to some extent on
supercoiled circular DNA substrates (the same occurs with nicked DNA;
data not shown). Clamp assembly on circular duplex DNA may occur at
secondary structures with single-stranded/double-stranded DNA junctions
that mimic a primer-template, or perhaps S. cerevisiae RFC
can load PCNA onto duplex DNA, albeit with much less efficiency than on
primed DNA (as suggested for human RFC) (29). The clamp loading assay
shows that S. cerevisiae RFC purified from E. coli is active and exhibits a clear preference for loading PCNA
onto a primed DNA template.
RFC Binds Single-stranded, Double-stranded, and Primer-Template
DNAs with High Affinity--
The interaction between RFC and DNA was
measured quantitatively using 32P-labeled DNA in
nitrocellulose membrane filtration assays. First, we measured the
stoichiometry of RFC binding to primer-template DNA, in order to
determine the active-site concentration of this recombinant, E. coli-expressed eukaryotic clamp loader. The primer-template is a
31-nucleotide (nt) primer annealed to an 81-nt template to form a 31-nt
duplex with 25-nt ssDNA overhangs on either side. The single-stranded
DNA substrate is the 31-nt primer, and double-stranded DNA is the
primer annealed to its complement. The DNA lengths were chosen to be in
excess of the reported RFC footprint of 12 bases on the 5'
single-stranded overhang and 8-15 bases on the duplex region of a
primer-template DNA (33). During assay development, we noted that RFC
has high affinity for a 5'-phosphate residue on any DNA structure
(single-stranded, double-stranded, or primer-template); a similar
property of human and D. melanogaster RFC1 amino-terminal domains for recognizing 5'-phosphate on duplex DNA has been reported recently (35). In order to eliminate possible misinterpretation of
5'-phosphate recognition as interaction between RFC and DNAs other than
the primer-template, the DNA substrates used in this study were
32P-labeled by incorporation of [-32P]dATP
at the 3'-end by phage T7 DNA polymerase. Fig.
3A shows a titration of 0.5 µM 32P-primer-template DNA with increasing
concentrations of RFC. All DNA in the reaction is bound with 0.53 µM RFC in the reaction, indicating that 95% of the
protein is active for interaction with DNA.
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Fig. 3.
RFC binds a variety of DNA substrates with
high affinity. A, 32P-labeled
primer-template DNA (0.5 µM) binding to RFC (0-3
µM), assayed by nitrocellulose membrane filtration assays
as described under "Experimental Procedures." The molar amount of
bound DNA plotted versus RFC concentration shows a 1:1
stoichiometry for the RFC-DNA interaction. B, a titration of
0.016 µM RFC with 0-0.1 µM different
32P-labeled DNAs yields an apparent Kd
of 12 ± 2, 7 ± 1, and 7 ± 1 nM,
respectively, for RFC binding to 31/81 primer-template DNA (),
31-mer ssDNA (), and 31-mer duplex DNA (), respectively.
C, a similar experiment performed with 0.2 µM
RFC. D, the experiment performed with 0.016 µM
RFC and primer-template DNA in the presence of 0.5 mM
ATPS () and ADP ( ); Kd = 7-9
nM. E, 0.016 µM truncated RFC
(missing the N-terminal 283 amino acids of RFC1 that are not essential
for clamp assembly) binding primer-template DNA (), 31-mer ssDNA
(), and 31-mer duplex DNA () with high affinity similar to full
RFC (apparent Kd = 13, 6, and 5 nM,
respectively).
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Next, we assayed RFC binding to single-stranded, double-stranded, and
primer-template DNAs order to compare quantitatively its affinity for
these different DNA structures. A constant amount of RFC (0.016 µM) was titrated with increasing concentrations of
32P-DNA, and the binding isotherm yielded the maximum
RFC·DNA complex formed and an apparent Kd for the
interaction (Fig. 3B). To our surprise, RFC appears to bind
primer-template, ssDNA, and dsDNA to the same extent and with similar
high affinity (apparent Kd = 12, 7, and 7 nM, respectively), implying that the clamp loader does not
distinguish between these different DNA structures (nonspecific DNA
binding to the membrane is less than 0.5% of the total DNA bound to
RFC).
One possible caveat in the above experiment is that at low nanomolar
concentrations, the RFC subunits may not form a stable pentamer. This
issue is of particular concern given reports that the large RFC subunit
alone can bind single-stranded and double-stranded DNA. Therefore, we
performed the same experiment with RFC concentrations up to 0.5 µM (at low micromolar concentrations, RFC migrates as a
stable complex in a gel filtration column; data not shown). At all
concentrations tested, we observed similar high affinity binding of DNA
substrates to RFC (shown in Fig. 3C for 0.2 µM RFC). The DNA binding assays were performed initially in the absence of
nucleotide cofactors, and a recent surface plasmon resonance study of
the interaction between a truncated version of RFC and primed DNA
suggests that the protein·DNA complex is more stable in the presence
of ATPS (38). Thus, DNA binding was measured also in the presence of
0.5 mM ATPS and ADP (ATPS is not hydrolyzed by RFC
under the assay conditions; data not shown). No significant difference
in RFC binding to the three DNAs was detectable in the absence or
presence of these nucleotides (shown in Fig. 3D for the
primer-template; Kd = 7-9 nM; ssDNA and
dsDNA data not shown). The slightly higher amount of RFC·DNA detected in the presence of ATPS may reflect a subtle effect of the
nucleotide on the stability of the complex. Previous reports indicate
that the amino-terminal domain of RFC1, which is considered unnecessary for clamp assembly based on deletion analysis, binds double-stranded DNA and possibly single-stranded DNA as well (reviewed in Ref. 15). In
order to determine whether the high affinity RFC binding to ssDNA and
dsDNA we observe is peculiar to the RFC1 amino-terminal domain or a
property of the PCNA loading-active domains, we assayed trRFC
(containing a truncated version of RFC1:2-283) for DNA binding as
above; the trRFC complex catalyzes PCNA assembly on primed DNA similar
to full-length RFC (data not shown). As observed for full-length RFC,
trRFC binds the three DNAs with high affinity (Fig. 3E;
Kd = 13, 6, and 5 nM for
primer-template, ssDNA, and dsDNA, respectively). The maximum amount of
dsDNA bound to trRFC is lower (~50% of total) and may indicate a
role for the N-terminal domain of RFC1 in stabilizing the interaction
between RFC and dsDNA. Thus, an initial quantitative analysis of the
DNA binding activity of RFC does not reveal significant selectivity for
primer-template DNA over other DNA structures.
Despite Their High Affinity for RFC, ssDNA and dsDNA Cannot Compete
with Primed DNA--
Given the contrast between the need for RFC to
distinguish primer-template DNA from single- and double-stranded DNA
and initial evidence that RFC binds all three DNAs with high affinity
(Fig. 3B), we continued the investigation further to
determine whether the interaction between RFC and primed DNA is somehow
different from that between RFC and ssDNA and dsDNA. To measure
directly whether RFC exhibits preference for one DNA structure over the others, we assayed RFC binding to primer-template DNA in the presence of increasing concentrations of competitor single- or double-stranded DNA in the reaction.
As shown in Fig. 4A, when RFC
(0.016 µM) is incubated with 32P-labeled
primer-template (0.02 µM) premixed with unlabeled ssDNA (0-1 µM), nearly all of it forms RFC·primed DNA
complex, even in the presence of 1 µM ssDNA (at 3 µM ssDNA competitor, primed DNA binding is reduced to
about 40%; data not shown). A complementary experiment with 0.02 µM unlabeled primer-template and 0-1 µM
32P-labeled ssDNA shows very weak binding of ssDNA to RFC
under these conditions (K1/2 = 160 nM), relative to ssDNA binding in the absence of primer-template DNA (Kd = 7 nM; Fig. 3B). Similar
results are obtained with double-stranded DNA as the competitor.
Experiments with 32P-labeled primer-template DNA and
32P-labeled dsDNA, respectively, show RFC binding to primed
DNA even in the presence of 1 µM dsDNA and very low
affinity of dsDNA for RFC in the presence of the primer-template (Fig.
4B). trRFC exhibits the same profile as full-length RFC in
these assays, indicating that resistance of the clamp
loader-primed DNA complex to competition with ssDNA and dsDNA is
not dependent on the amino-terminal DNA-binding domain of RFC1 (data
not shown).
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Fig. 4.
Single- and double-stranded DNA cannot
compete effectively with primed DNA for binding RFC. A
and B, competitive DNA binding assays that measure RFC
(0.016 µM) binding to a mixture of 0.02 µM
32P-labeled primer-template plus 0-1 µM
unlabeled 31-mer single-stranded DNA (A, ) or 31-mer
double-stranded DNA (B, ), 0.02 µM
unlabeled primer-template plus 0-1 µM
32P-labeled ssDNA (A,  ) or dsDNA
(B, ) or 0-1 µM 32P-labeled
ssDNA (A, ) or dsDNA (B, ) alone.
C, RFC (0.016 µM) binding to 0.02 µM 32P-primer-template DNA plus 0-1
µM primer-template competitor (), 0.02 µM 32P-ssDNA plus 0-1 µM ssDNA
competitor (), and 0.02 µM 32P-dsDNA plus
0-1 µM dsDNA competitor (). The data fit to a
hyperbola yield a K1/2 value of 0.051, 0.037, and
0.042 µM, respectively. D,
32P-ssDNA (0-1 µM) binding to RFC (0.016 µM) alone () or in the presence of 0.02 µM unlabeled primer-template () or dsDNA ();
K1/2 = 0.16 and 0.055 µM for ssDNA
binding in the presence of primer-template and dsDNA, respectively.
E, 32P-ssDNA (0.02 µM) binding to
RFC (0.016 µM) in the presence of 0-0.4 µM
unlabeled primer-template DNA () or ssDNA ();
K1/2 = 0.003 and 0.037 µM with
primer-template and ssDNA competitor, respectively. F, a
titration of 0.005 µM RFC with 0-0.1 µM
32P-labeled primer-template alone () and in the presence
of 0.03 µM unlabeled primer-template () or ssDNA
(); K1/2 = 0.045 and 0.009 µM with
primer-template and ssDNA competitor, respectively
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A series of experiments detailed below test further this surprising
lack of competition between DNA substrates with similar affinity for
RFC. First, experiments were performed with 0.016 µM RFC,
0.02 µM 32P-labeled ssDNA, dsDNA, or
primer-template DNA as substrate and increasing amounts of unlabeled
self as competitor, to test whether the membrane filtration assay and
experimental design support detection of simple competitive DNA binding
to RFC (Fig. 4C). For homologous competition between labeled
and unlabeled ligand, K1/2 = [labeled ligand] + Kd, where K1/2 is the competitor
concentration at which substrate binding is lowered by 50%. This
predicts K1/2 0.03 µM under these
experimental conditions (Kd = 7-12 nM
for the three DNAs) (Fig. 3B). The competitive binding
curves in Fig. 4C yield K1/2 = 0.051, 0.037, and 0.042 µM for primer-template, ssDNA, and
dsDNA, respectively. Thus, each DNA competes effectively with itself,
implicating reasons other than the assay technique for the observed
lack of competition between primer-template and ssDNA or dsDNA for
binding RFC.
In the next experiment, RFC was titrated with increasing concentrations
of 32P-labeled single-stranded DNA, alone and in the
presence of 0.02 µM unlabeled double-stranded or
primer-template DNA. Given that ssDNA and competitor DNA
(double-stranded or primer-template DNA) exhibit the same affinity for
RFC, if the two DNAs bind the same site on RFC, simple homologous
competition predicts a K1/2 of ~0.03
µM for ssDNA binding with 0.02 µM
competitor DNA in the reaction. The binding isotherm for ssDNA in the
presence of dsDNA is slightly right-shifted relative to the one with
ssDNA alone and yields a K1/2 of 0.055 µM. In contrast, the K1/2 for ssDNA
binding in the presence of 0.02 µM primer-template is
0.16 µM. Thus, ssDNA and dsDNA compete effectively with
each other for binding RFC, but even a small amount of primer-template
in the reaction appears to substantially reduce RFC binding to ssDNA
(and dsDNA), indicating a distinctive interaction between RFC and
primed DNA.
In complementary experiments, we measured binding of
32P-labeled ssDNA (0.02 µM) to RFC (0.016 µM), in the presence of increasing amounts of unlabeled
ssDNA or primer-template DNA as competitor (0-1 µM).
Fig. 4E shows that when ssDNA is the competitor,
K1/2 for the interaction is 0.037 µM
(i.e. at a competitor concentration of Kd
plus labeled DNA, the amount of labeled ssDNA bound to RFC is reduced
by half (shown also in Fig. 4C)). In contrast, when
increasing amounts of primer-template competitor are added to the
reaction, the K1/2 is 0.0035 µM
(i.e. only 0.0035 µM primed DNA is required to
reduce labeled ssDNA (0.02 µM) binding to RFC (0.016 µM) by half). These results confirm that there is no
simple competition between ssDNA and primed DNA for RFC and suggest
that primed DNA may bind RFC with high affinity whether RFC is free or
in the presence of single-stranded (or double-stranded) DNA. In the
next experiment, this prediction is tested directly by measuring
binding of 32P-labeled primer-template DNA (0-0.1
µM) to RFC (0.005 µM), alone or in the
presence of 0.03 µM unlabeled primer-template DNA or ssDNA (Fig. 4F). As expected from the binding isotherm in
Fig. 3B, the 32P-labeled primer-template alone
binds RFC with high affinity; Kd = 8 nM.
With 0.03 µM unlabeled primer-template in the reaction,
the binding isotherm shifts to the right and yields a
K1/2 of 0.045 µM (expected
K1/2 0.04 µM). In striking
contrast, however, the primer-template binding isotherm in the presence
of 0.03 µM unlabeled ssDNA is virtually identical to that
with no other DNA in the reaction and yields a K1/2
of 9 nM (expected K1/2 0.04 µM). It appears that the 0.03 mM ssDNA in the
reaction is invisible with respect to the interaction between primed
DNA and RFC.
The results described above are intriguing because they indicate that
the RFC·primed DNA complex is resistant to competition with ssDNA or
dsDNA despite similar high affinity of these DNAs for RFC. Possible
explanations for these results include the following: (a)
the DNAs bind RFC with significantly differing affinity (this explanation is not consistent with the measured apparent equilibrium constants); (b) ssDNA and dsDNA may bind a different site on
RFC than primed DNA; (c) the DNAs may bind the
same/overlapping site on RFC and the primer-template binds RFC even if
the site is occupied with ssDNA or dsDNA, but not vice
versa; (d) primed DNA forms a highly stable complex
with RFC that is unavailable for interaction with ssDNA or dsDNA;
(e) primed DNA induces a conformational change in RFC that
lowers its affinity for other DNA structures. Experiments described
below examine these possible explanations for the high specificity of
primed DNA-RFC interaction.
RFC·Primed DNA Complex Exhibits Higher Stability Relative to
Other RFC·DNA Complexes--
We utilized a partial tryptic digest
assay to detect possible differences between RFC·ssDNA, RFC·dsDNA,
and RFC·primed DNA complexes (e.g. the presence of
distinct DNA binding sites or conformational changes specific to each
DNA substrate). As shown in Fig.
5A, panel 1, the
RFC1 subunit is susceptible to tryptic digest over time and is largely
degraded within 15 min in the absence of DNA (the smaller RFC subunits
appear curiously resistant to proteolysis). When RFC is bound to
primer-template DNA, however, the large RFC subunit becomes
significantly more resistant to proteolysis, and about half of the
original protein remains undigested after 15 min (Fig. 5A,
panel 2). Interestingly, the same experiment performed with
ssDNA (Fig. 5A, panel 3, 81-mer template; data not shown for 31-mer primer) or dsDNA (Fig. 5A, panel
4) showed no protection of the RFC1 subunit from proteolysis.
Initially, we took these data to mean that on binding primer-template
DNA, RFC undergoes a conformational change that makes it more resistant to tryptic digest or that the primer-template directly blocks tryptic
digestion at its site of interaction. In either case, the effect
appeared specific to primer-template DNA.
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Fig. 5.
Partial tryptic digest and ATPase assays
reveal primed DNA-specific changes in RFC. A, RFC (2 µM) was subjected to proteolysis by trypsin over time, in
the absence of DNA (1) or in the presence of 31/81
primer-template (2), 81-mer ssDNA (3), and 31-mer
dsDNA (4) in a reaction containing 100 mM NaCl
and analyzed by SDS-PAGE as described under "Experimental
Procedures." Similar assays were performed at a lower NaCl
concentration of 30 mM, with no DNA (5),
primer-template (6), ssDNA (7), and dsDNA
(8), in the reaction. B, the ATPase rate of RFC
(0.2 µM) at varying NaCl concentrations in the absence of
DNA () or in the presence of primer-template (), ssDNA (), and
dsDNA (). The maximum steady-state ATPase rate is 0.45 s1 with primed DNA at 150 mM NaCl
concentration.
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We noted, however, that the high concentration of RFC (2 µM) utilized in the proteolytic digest introduced more
NaCl into the reaction than was present in the DNA binding experiments
(100 versus ~30 mM, respectively). Therefore,
the digest was repeated at 30 mM NaCl (after lowering salt
concentration in the RFC preparation by dialysis). As shown in Fig.
5A, under the less stringent conditions, RFC1 is still
susceptible to proteolysis in the absence of DNA (panel 5)
and is protected in the presence of primer-template DNA (panel
6), with similar kinetics as in panels 1 and
2 at high NaCl, respectively. However, under these
conditions, ssDNA (panel 7) and dsDNA (panel 8)
protect RFC1 from trypsin as well. Thus, it appears that all three DNAs
confer similar protease resistance on RFC1, presumably by binding at
the same site on RFC and possibly even inducing similar changes in
conformation. It is clear, however, that the RFC·primed DNA complex
is more stable to NaCl concentration and therefore different from the
other two complexes. Relatively high resistance of RFC·primed DNA to
NaCl was observed also in nitrocellulose binding experiments, although
Kd values could not be determined accurately at high
NaCl, since the binding did not reach saturation.
Is the ability of RFC to load PCNA clamps specifically on primed DNA
related to the observed resistance of RFC·primed DNA complex to NaCl?
We searched for a possible connection by assaying the effect of
different DNA substrates on RFC ATPase activity, which is coupled to
its clamp loading activity. Steady-state ATPase assays were performed
with RFC (0.2 µM), PCNA (1 µM), and
[-32P]ATP (1 mM) in the presence of ssDNA,
dsDNA, or primer-template DNA (1 µM) at varying NaCl
concentrations. As shown in Fig. 5B, the RFC ATPase rate is
stimulated by the presence of any DNA substrate by at least 2-fold over
no DNA (at low NaCl, kcat = 0.1 s1
without DNA and 0.25 s1 with DNA). As NaCl concentration
increases, RFC ATPase activity in the presence of primed DNA increases
to kcat = 0.45 s1 (peak at 150 mM NaCl), but the activity in the presence of ssDNA or
dsDNA remains constant or declines (kcat = 0.2 s1 at 150 mM NaCl). Thus, primed DNA binding
has a distinctive effect on RFC activity, which corresponds to the
NaCl-stable nature of the RFC·primed DNA complex.
Release of Primed DNA from RFC Is Slow Compared with Other DNA
Structures--
Results from competitive DNA binding and partial
proteolysis assays indicate that RFC·primed DNA complex is more
stable than other RFC·DNA complexes. The next experiment tests this
stability directly, by measuring the rate of dissociation of the three
DNAs from RFC (Fig. 6). RFC (0.016 µM) was preincubated with 32P-labeled DNA
(0.02 µM) and then chased with 1 µM
unlabeled self over time (1 µM DNA is a sufficient chase
according to the data in Fig. 4C). At time 0, ~80% of RFC
in the reaction is bound by labeled DNA, which dissociates over time.
The experiment performed with 32P-labeled ssDNA substrate
and unlabeled ssDNA chase shows that the DNA dissociated completely
from RFC at the first measurable time point, which yields an off rate
equal to or faster than 0.2 s1. Double-stranded DNA
exhibits a similar, rapid rate of dissociation from RFC (Fig.
6A). In contrast, primer-template DNA dissociates from RFC
at a rate of 0.025 s1, at least 10-fold slower than
single- or double-stranded DNA. The RFC·primed DNA complex is highly
stable compared with the other RFC·DNA complexes, and this property
probably contributes to specificity of the interaction. It should be
noted, however, that the half-life of the RFC·primed DNA complex is
~28 s; therefore, the 10-min incubation in the competition
experiments of Fig. 4 should be more than adequate time for single- or
double-stranded DNA in the reaction to gain access to free RFC.
However, as shown in Fig. 6B, when 1 µM ssDNA
or dsDNA is used as chase for 32P-labeled primer-template,
there is almost no loss of the primer-template from RFC over time
(tested up to 30 min). Thus, even with a high excess of ssDNA or dsDNA
in the reaction, RFC preferentially binds and maintains its interaction
with primed DNA.
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Fig. 6.
Interaction of RFC with primed DNA is more
stable than with ssDNA or dsDNA. Release of
32P-labeled DNA (0.02 µM) from RFC (0.016 µM) was measured over time in the presence of 1 µM unlabeled DNA chase by nitrocellulose membrane
filtration assays, as described under "Experimental Procedures."
A, 32P-labeled primer-template (),
32P-labeled ssDNA (), and 32P-labeled dsDNA
() chased with excess unlabeled self as competitor. The dissociation
rates are 0.025 ± 0.003 s1,  0.2
s1, and 0.2 s1, respectively.
B shows that if 32P-labeled primer-template is
chased with excess unlabeled ssDNA () or dsDNA (), the
RFC·primed DNA complex persists over time, in contrast to the chase
with unlabeled primer-template DNA ().
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Primer-Template DNAs with Either 5' or 3' Single-stranded Overhang
Can Mimic Full Primed DNA to Varying Extents--
The assays described
thus far have been performed with primer-template containing a 31-nt
duplex region flanked by 25-nt 5' and 3' single-stranded DNA overhangs.
What features of this DNA are important for specific recognition by
RFC: the 3' primer-template junction, the 5' junction, or perhaps
arrangement of double- and single-stranded regions adjacent to each
other? We addressed this question by measuring binding of
primer-template DNAs with either a 25-nt 5' ssDNA overhang (3' primer
junction) or 25-nt 3' ssDNA overhang (5' primer junction) to RFC. Both
DNAs bind RFC with high affinity (apparent Kd = 10 nM; data not shown), and ssDNA cannot compete effectively
against either primer-template for RFC in competition experiments with
0.016 µM RFC, 0.02 µM 32P-labeled primer-template, and 0-1 µM
unlabeled ssDNA (Fig. 7A). In
complementary experiments (Fig. 7B), when
32P-labeled ssDNA (0.02 µM) is competed with
unlabeled 5' primer junction or 3' primer junction DNA (0-1
µM), only 0.004 and 0.003 µM of the
competitor, respectively, is required to reduce ssDNA binding to RFC by
half (as observed with the full primer-template DNA competitor; Fig.
4E). In contrast, the same experiment performed with only
the 56-nt template strands of the 5' or 3' primer junction DNAs yields
a K1/2 of 0.035 µM as expected from
simple competition between the 31- and 56-mer single-stranded DNAs.
Thus, it appears that a single-stranded plus double-stranded DNA
structure, with a 5' or 3' ssDNA overhang, is sufficient for specific
interaction with RFC.
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Fig. 7.
A 5' or 3' primer-template junction supports
specific interaction between RFC and primed DNA. A,
binding of 0.02 µM 32P-ssDNA (),
32P-31/56 5' primer junction DNA (), or
32P-31/56 3' primer junction DNA () to RFC (0.016 µM) in the presence of 0-1 µM unlabeled
ssDNA competitor. B, a complementary experiment in which
32P-ssDNA 0.02 µM binding to RFC (0.016 µM) is competed with 0-0.4 µM unlabeled
31/56 5' primer junction DNA (), 56-mer template ssDNA (), 31/56
3' primer junction DNA (), and the corresponding 56-mer template
ssDNA (). For both 56-mer single-stranded DNAs, the
K1/2 is 0.035 µM, indicating simple
competition between the 31- and 56-mer ssDNAs. In contrast, both 3' and
5' primer junction DNA competitors yield a K1/2 of
0.004 µM. C, the ATPase activity of RFC (0.2 µM) measured in the presence of 5' primer junction DNA
() or 3' primer junction DNA (), as described under
"Experimental Procedures." The peak steady-state ATPase rate is
0.45 s1 for the 5' primer junction (similar to full
primer-template DNA) and 0.25 s1 for 3' primer junction
(similar to ssDNA).
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Next, we tested the effect of the two different primer-template
junctions on the ATPase activity of RFC. Fig. 7C shows that the 5' primer junction DNA (3' ssDNA overhang) stimulates RFC ATPase
activity similar to full primer-template DNA (peak ATPase rate is 0.45 s1 at 150 mM NaCl). In contrast, in the
presence of 3' primer junction DNA (5' ssDNA overhang), the ATPase rate
remains at levels observed with single-stranded DNA substrate. Thus,
RFC can distinguish the two half-primer-template DNA structures from
single- or double-stranded DNA and also appears capable of
distinguishing between the two. Since it is not clear yet exactly how
DNA binding is linked to individual steps in the ATPase pathway, the
implication of this difference between the 3' and 5' primer-template
junction DNAs awaits investigation.
PCNA Suppresses RFC Binding to ssDNA and dsDNA--
It is not yet
completely clear whether RFC binds PCNA or DNA first in the
clamp-loading pathway. If PCNA is bound first to RFC, it may also
influence the ability of RFC to recognize primed DNA as a target for
clamp assembly. We examined this possibility by performing DNA binding
experiments with PCNA (1 µM) and ATPS (1 mM) in the reaction with 0.2 µM RFC and 0-1
µM 32P-labeled DNA (30 mM NaCl);
the experiments were performed at high RFC concentration to ensure
stable RFC·PCNA complex formation. Fig.
8A shows that primer-template
DNA binding to RFC is unaffected by the presence of PCNA, but binding
of ssDNA (Fig. 8B) and dsDNA (Fig. 8C) is
significantly lower (note that inclusion of PCNA does not increase NaCl
concentration in the reaction). Thus, the RFC clamp loader can
discriminate against single- and double-stranded DNA when free or in
complex with the PCNA clamp.
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Fig. 8.
PCNA increases selectivity of RFC for primed
DNA. A-C, respectively, show the effect of PCNA on
interaction between RFC and DNA in assays measuring
32P-primer-template, 32P-ssDNA, and
32P-dsDNA binding to RFC (0.2 µM) in the
absence () and in the presence () of PCNA (1 µM)
plus ATPS (1 mM).
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DISCUSSION |
The RFC clamp loader is a critical component of the protein
machinery responsible for DNA replication and repair/recombination. It
catalyzes assembly of circular PCNA clamps onto primed DNA sites, where
they are bound by replicative DNA polymerases and used as sliding
tethers for rapid, processive DNA synthesis (reviewed in Ref. 1).
Initiation of processive DNA synthesis is influenced by the efficiency
of clamp assembly on DNA, and rapid, specific recognition of
primer-template DNA is important for efficient RFC function. Rapid
clamp loader action is especially important during synthesis of lagging
strand DNA, since a clamp must be loaded about every second for the
initiation of each new Okazaki fragment. RFC is known to bind
primer-template DNA with high affinity, but it binds other DNA
structures, such as single- and double-stranded DNA, as well (reviewed
in Ref. 15). This raises the question of whether (and how) RFC can
distinguish a primed DNA site from the considerable background of
single- and double-stranded DNA during replication. In this study, we
examine RFC binding to different DNA substrates and demonstrate that
RFC selectively binds and forms a stable complex with primer-template
DNA even in the presence of a large excess of single- and
double-stranded DNA.
The affinity of RFC for primer-template DNA as well as ssDNA and dsDNA
was measured by nitrocellulose membrane filtration assays that provide
a quantitative measure of protein-DNA interactions. The apparent
Kd values are close to 10 nM for all
three DNA structures, indicating that RFC binds ssDNA and dsDNA with as
high affinity as primer-template DNA. The RFC1 subunit of RFC contains
an amino-terminal domain (~275 amino acids) that reportedly binds
both dsDNA and ssDNA but appears to be dispensable for its PCNA-loading
activity (43). In fact, many in vitro studies have been
performed with RFC deleted for the RFC1 amino-terminal domain, since it
appears unnecessary and its removal seems to improve protein stability
and activity (38, 44-47). Thus, it was possible that the high affinity
DNA binding we observed with the full-length, wild-type RFC complex is
unrelated to its clamp loading function. It should be noted, however,
that even if it were unrelated to clamp loading, tight binding of RFC
to DNA structures other than primer-template could titrate out the
clamp loader and negatively impact DNA replication efficiency in
vivo. We did, however, assay a truncated version of RFC
(trRFC = RFC1:2-283 and RFC2, -3, -4, and -5) for DNA binding
and observed that trRFC also binds ssDNA and dsDNA with similar
affinity as primer-template DNA. Additionally, nucleotide cofactors of
RFC (ATPS (for ATP) and ADP) do not appear to significantly affect
the interaction (as measured by nitrocellulose membrane filtration assays).
Competitive DNA binding experiments were performed next, in order to
assay directly if the presence of ssDNA or dsDNA in the reaction
reduces formation of the RFC·primer-template DNA complex. Surprisingly, ssDNA and dsDNA were unable to compete effectively with
the primer-template even at 50-fold higher concentrations (100-fold
higher than the apparent Kd). RFC appears to interact selectively with primed DNA among excess ssDNA and dsDNA, although it binds these DNAs with high affinity in the absence of
primed DNA. This selectivity could result from a highly stable interaction between RFC and primed DNA that renders RFC unavailable for
interaction with other DNA or because primer-template binding to RFC
lowers its affinity for other DNA or because primer-template binds RFC
with high affinity whether RFC is free or in complex with single- or
double-stranded DNA (but not vice versa).
The first hypothesis predicts a slow rate of dissociation of primed DNA
from RFC, the second hypothesis implies a primed DNA-specific change in
RFC conformation, and both the second and third hypotheses predict
tight binding of primer-template to RFC in the absence and presence of
ssDNA or dsDNA. These predictions were tested by measuring the rate of
dissociation of 32P-labeled primer-template from RFC in
chase experiments, by partial proteolysis of RFC to detect
DNA-dependent conformational changes, and by several
competitive DNA binding experiments. The chase experiments revealed
that dissociation of primer-template DNA from RFC is at least 10-fold
slower than that of single- or double-stranded DNA. Thus, the
RFC·primed DNA complex is much more stable than RFC·ssDNA or
RFC·dsDNA complexes, which probably contributes to the selectivity of
interaction between RFC and primed DNA. It should be noted that
differences in the dissociation rates (koff) imply that the nearly identical Kd values determined for the three DNAs from membrane filtration assays may not be absolutely correct; these assays may not necessarily measure binding under true equilibrium conditions, because the reactants could undergo
significant changes in concentration as the solution passes through the
membrane. It is also quite possible that the DNAs bind RFC with
different association rates (kon); there is
evidence that DNA-binding proteins (RPA, for example) can bind
different DNA structures with different bimolecular association rates
(48). It will be interesting to measure the "on" and
"off" rates of RFC-DNA interaction in solution to determine
whether RFC does bind primed DNA with a different "on" rate and
whether this plays a role in its selection as the site for clamp assembly.
The second possibility, that primed DNA binding to RFC induces loss of
affinity for single- and double-stranded DNA, is still likely, because
the half-life of RFC·primed DNA complex is only about 30 s
(koff = 0.025 s1), and this does
not explain complete resistance of the complex to competition by excess
ssDNA and dsDNA even on prolonged exposure (10 min; Fig. 6). A partial
tryptic digest indicates a change in RFC conformation on binding DNA;
the RFC1 subunit is predominantly affected, consistent with earlier
reports that RFC1 is the primary DNA-binding subunit in RFC. There are
no obvious differences in t
TOP
ABSTRACT
INTRODUCTION
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