(Received for publication, December 12, 1996)
From the Program in Molecular Biology, Replication factor C (RFC) and proliferating cell
nuclear antigen (PCNA) are processivity factors for eukaryotic DNA
polymerases Replication factor C (RFC)1 is a
component of processive eukaryotic DNA polymerase holoenzymes. The
processive elongation of DNA chains by pol The five RFC subunits show high homology to each other, and a cluster
of seven conserved boxes, referred to as RFC boxes II-VIII, has been
defined (16, 19). The RFC boxes contain conserved motifs of 3-16 amino
acids in length, and the distances between these boxes are similar in
all subunits. The RFC boxes III and V contain conserved sequences
characteristic for nucleotide-binding proteins. The function of the
other boxes are unknown. In the four small subunits, these boxes are
located in the N-terminal half of the polypeptide while the C-terminal
sequences are unique to each subunit. The large subunit p140 contains
additional N-terminal sequences including another conserved domain, RFC
box I, that shows homology to prokaryotic DNA ligases and
poly(ADP-ribose) polymerase (14, 15, 19). (A summary of the relative
positions of the RFC boxes present in the p140 subunit is shown in Fig. 8.) Despite the sequence redundancy between the subunits, all five are
required to form a stable and active complex (17, 20).
The functions of each of the five subunits are presently poorly
understood due to the difficulties in isolating each subunit in a
soluble and native state. Although all of the subunits contain conserved ATP binding motifs, only the p40 subunit has been shown to
bind ATP (5, 12). The N-terminal half of the p140 subunit, containing
the ligase homology domain, binds DNA (5, 15, 21, 22), while the p40
and p140 subunits have been shown to interact with PCNA (7, 22).
However, the relevance of these observations to the function of the RFC
complex remains unclear.
We have reported the expression of the five cloned human RFC genes
using an in vitro transcription/translation system (17). A
complex was formed containing all five subunits that was active in
supporting RFC-dependent replication. Studies on the
interactions between the five subunits indicated a cooperative
mechanism in RFC assembly. A core complex consisting of the p36, p37,
and p40 subunits was detected, and it was shown that the p38 and p140 subunits were dependent upon each other for interaction with the core
complex.
We have exploited these findings to determine the role played by the
individual subunits in the function of the RFC complex. For this
purpose, we have deleted regions of each of the subunits in the
five-subunit complex. Here we report studies with the p140 large
subunit. We have defined a region in this subunit required for RFC
complex formation and have examined RFC complexes containing deleted
p140 variants for their ability to bind DNA primer ends, to bind PCNA,
load PCNA onto nicked circular duplex DNA, and to support
RFC-dependent elongation of singly primed M13 DNA by pol The DNA templates for in vitro
transcription/translation of the five subunits of hRFC were as
described previously (17).
pET16p140C555 was derived from pET16ap140 by excising a
BspMI-Bst BI fragment from the p140 coding
sequence. The ends were filled in and religated. Expression from this
vector results in a polypeptide spanning amino acids 1-555 of p140
plus three additional amino acids, TKK, at the C terminus.
pET16p140N555 was obtained from pET16ap140 by removing a
NcoI-BspMI fragment, filling in the ends, and
religation. This vector expressed amino acids 555-1148 of p140 with an
additional methionine for translational start at the N terminus.
pET19p140N604, -N687, -N776, -N822, and -N877 were cloned from
pET16ap140 by polymerase chain reaction (Expand High Fidelity,
Boehringer Mannheim) using the following primers: p140N604 N
terminus, 5 In vitro
transcription/translation was performed as described (17). Linearized
template DNA (0.4 µg) was added to a 10-µl transcription/translation reaction mixture. The translation products were quantitated by Western blotting of the p37 and p40 subunits and by
comparing the level of [35S]methionine incorporated into
these and other subunits.
Interactions
between subunits were studied by coexpressing the subunits in question
in the in vitro transcription/translation system. When a
linearized template was used, each subunit was expressed individually,
then mixed immediately after an 1 h incubation of the in
vitro translation reactions, followed by further incubation for 30 min at 30 °C. Polyclonal antiserum (1 µl) against one of the
subunits was prebound to a 5 µl suspension of protein A-Sepharose beads (Upstate Biotechnology Inc.) for 1 h on ice. Beads were washed with equilibration buffer (RIPA: 50 mM Tris/HCl, pH
8.0, 250 mM NaCl, 5 mM EDTA, 1 mM
DTT, 0.5% Nonidet P-40, and 1% BSA). Aliquots of the in
vitro translation reactions were added to the immunobeads and
complexes were adsorbed for 1 h on ice with frequent shaking. The
beads were then washed three times with 0.3 ml of RIPA and twice with
0.3 ml of RIPA without BSA and EDTA. Bound proteins were eluted with 25 µl of SDS-PAGE loading buffer and aliquots analyzed by SDS-10% PAGE.
After separation, gels were fixed in 25% 2-propanol, 10% acetic acid,
soaked in luminographic enhancer (Amplify, Amersham), dried, and
exposed for autoradiography. The amount of RFC bound to the beads was
quantitated after SDS-PAGE analysis by phosphorimager (Fuji), using
known amounts of the p37 and p40 subunits as standards.
The following proteins
were prepared from HeLa cell extracts as described: HSSB (23), PCNA
(24), pol RFC
complexes formed from 25 µl of in vitro translation
reactions were isolated with immunobeads as described for
immunoprecipitation. In each case, the amount of RFC adsorbed to the
beads was quantitated as described above. After the beads were washed,
14.5 µl of the following reaction mixture was added directly to the
washed beads: 40 mM Tris/HCl, pH 7.5, 7 mM
magnesium acetate, 0.5 mM DTT, 0.01% BSA, 2 mM
ATP, 100 µM each of dATP, dGTP, and dTTP, 20 µM [ Reactions in which RFC isolated from HeLa cells was used were carried
out under the above conditions either in the absence of, or after
binding to immunobeads as described above. Products formed were
analyzed as described above.
The substrate used for DNA binding was a
hair-pinned DNA structure, which was described previously and used to
measure the binding of RFC to a DNA primer end (5, 26). The hairpin was formed from a synthetic 96-mer oligonucleotide after annealing in 20 mM Tris/HCl, pH 7.4, 50 mM NaCl, 2 mM MgCl2. The DNA contained a biotin moiety at
the 5 PCNA was
overexpressed in Escherichia coli (24). Purified PCNA was
covalently bound to activated Affi-Gel-15 (Bio-Rad) at a concentration
of 10 mg/ml resin according to the manufacturer's protocol. For
control experiments, BSA was bound to the resin in the same way.
Aliquots of translation reactions expressing RFC complexes were added
to 5 µl of the affinity beads in 10 µl equilibration buffer (50 mM Tris/HCl, pH 8.0, 200 mM NaCl, 5 mM MgCl2, 2 mM ATP, 2 mM DTT, 0.25% Nonidet P-40, 10% glycerol, and 2.5% BSA).
Binding was carried out on ice for 4 h. The beads were then washed
twice with 0.3 ml of equilibration buffer containing 1% BSA and once
with equilibration buffer without BSA. Bound protein was eluted with 25 µl of SDS-PAGE loading buffer, and aliquots were separated on
SDS-PAGE. The gels were processed as described under
immunoprecipitation.
RFC complexes
formed from in vitro translation reactions were isolated
with immunobeads as described for immunoprecipitation. After the beads
were washed, the following reaction mixture was added to the washed
beads: 0.5 pmol of singly nicked pBluescript DNA and 2.6 pmol of
32P-labeled PCNA trimers (500-800 cpm/fmol) in 50 µl of
incubation buffer (20 mM Tris/HCl, pH 7.5, 100 mM NaCl, 8 mM MgCl2, 0.5 mM ATP, 4% glycerol, 5 mM DTT, and 40 µg/ml
BSA) (27, 28). The reaction was incubated for 15 min at 37 °C,
stopped on ice and applied to a 5-ml gel filtration column (Bio-Gel
A15m, Bio-Rad) equilibrated with incubation buffer. Fractions of 160 µl were collected at 4 °C, and 32P was quantitated by
Cerenkov counting.
To identify regions in the subunit p140 that are
required for complex formation, p140 deletion mutants were constructed,
expressed in vitro, and assayed for their ability to
assemble an RFC complex with the four small subunits. Fig.
1 shows the products obtained after in vitro
transcription and translation of cDNAs encoding full-length p140
(lane 1) and deleted variants of this subunit (lanes
2-5). Each reaction mixture yielded a labeled protein of the
expected size, although a substantial number of smaller products were
formed. These products are most likely due to aberrant translational starts at internal methionine codons present in the transcribed RNA.
An immunoprecipitation assay employing anti-p37 antibodies was used to
analyze the formation of the five-subunit complex with the p140
deletion mutants described in Fig. 1. Deletion analysis of the p140
subunit revealed a region close to the C terminus that was required for
complex formation with the small subunits of RFC. A C-terminal deletion
of the p140 subunit that ended at amino acid 1142 (p140C1142; Fig. 1,
lane 2) supported complex formation (lanes 9 and
10), as did full-length p140 (amino acids 1-1148,
lanes 1, 6, and 7). However, p140C976,
with another 166 C-terminal amino acids deleted, did not support RFC
complex formation (Fig. 1, lanes 3, 11, and
12). Consistent with this, the N-terminal half of the p140
subunit (p140C555), spanning amino acids 1-555 that included the DNA
ligase homology domain, was also unable to form the five-subunit RFC
complex (Fig. 1, lanes 4, 13, and 14).
These experiments were carried out with antibodies against the p37
subunit, so that immunoprecipitation of p140 variants incapable of
supporting reconstitution of the five-subunit complex resulted in
coprecipitation of the p36·p37·p40 core complex but not the p38
subunit or the p140 variant (Fig. 1, lanes 12 and 14; and control, omitting p140, lanes 17 and
18). p140C555 migrates through SDS-polyacrylamide gels as a
band of 75 kDa although the calculated molecular mass of this fragment
is 62 kDa. This aberrant migration is also observed with the
full-length p140, a polypeptide of calculated molecular mass of 128 kDa
that migrates through gels as a band of 140 kDa.
The C-terminal half of the p140 subunit (p140N555), spanning amino
acids 555-1147, migrates at its calculated mass of 66 kDa. RFCp140N555
includes the RFC homology boxes II-VIII and was sufficient for complex
formation with the small subunits (Fig. 1, lanes 5, 15, and 16).
To further delineate the region within the C terminus critical for
complex formation, N-terminal deletions of the p140 subunit were
examined starting at amino acids 604 after RFC box II, 687 after box
IV, 776 after box VI, 822 after box VIII and 877 in the C terminus
(p140N604, -N687, -N776, -N822, and -N877, respectively; Fig.
2, lanes 1-6). As shown, the large subunit
derivative p140N822 supported formation of the five-subunit RFC
complex, while p140N877 did not (Fig. 2, lanes 16-19). This
indicated that a region between amino acids 822 and 1142, within the
p140 C-terminal region that is not conserved among the RFC subunits, is
necessary and sufficient for RFC complex formation.
The replication activity of RFC complexes formed with
the N-terminally deleted variants of p140 was examined. Complexes,
assembled from in vitro translation reactions, were isolated
on immunobeads, and their ability to support RFC-dependent
DNA elongation was measured. The efficiency of the replication reaction
with RFC bound to immunobeads was markedly reduced (approximately
6-fold) compared with reactions with soluble RFC (Fig.
3A, compare lanes 1 and
2), which was partly due to the limited efficiency of
immunoprecipitation (25-35%). The elongation of singly primed
single-stranded M13 DNA with immobilized RFC, catalyzed by pol Replication activity of RFC complexes formed
with N-terminally deleted variants of p140. A, assay for
activity of RFC immobilized on immunobeads. Elongation of a single
primer on single-stranded circular M13 DNA was performed as described
under "Materials and Methods"; the replication products were
separated by denaturing gel electrophoresis. A reaction in which 30 fmol of RFC, isolated from HeLa cells (hRFC), was added in
lieu of immunobeads is shown (C, lane 1). hRFC
bound to immunobeads (100 fmol input) with antibodies against p37 or
the immunobeads alone was used in place of RFC in the reaction
(lanes 2 and 3). The products of an in
vitro translation reaction (25 µl), coexpressing the five RFC
subunits (ivtRFC), were bound to a 5-µl suspension of
protein A-Sepharose beads containing antibodies against the p37
subunit. After washing, the immunobeads were used in a complete
reaction (lane 4), or in reactions without PCNA, pol
The measurement of RFC activity of complexes linked to
immunobeads showed poor linearity as a function of RFC
concentration in the M13 elongation assay (Fig. 3B). For
this reason, these experiments were repeated using
poly(dA)4000·oligo(dT)12-18 as the
template in an RFC-dependent replication reaction.
RFCp140N555 supported poly(dT) synthesis 5 times more effectively than
RFCp140 (data not shown). In this assay, the synthesis of poly(dT)
showed a more linear response to RFC linked to immunobeads (10-40
fmol). Similar observations were made with these complexes isolated
from in vitro translation mixtures by phosphocellulose
chromatography (data not shown). Thus assays using different DNA
substrates and different isolation procedures indicated that the RFC
complex containing the N-terminally deleted p140 was more active than the full-length p140 RFC complex.
To further identify regions within the p140
subunit required for replication, the N-terminally deleted variants of
p140, p140N604 to p140N822, which lack some of the conserved RFC boxes
but still form the five-subunit RFC complex, were examined for their
ability to support DNA synthesis in the elongation reaction. As shown in Fig. 4, deletion of RFC box II from the p140 subunit
(RFCp140N604) resulted in loss of replication activity, delineating a
domain between amino acids 555 and 604 that is essential for
replication activity.
Deletion of the last six amino acids of p140 (p140C1142) did not affect
the activity of the RFC complex formed with this subunit (data not
shown).
For its function in replication, RFC must possess at least
three activities: the ability to bind to DNA primer ends, to interact with PCNA, and to load PCNA onto DNA in an ATP-dependent
reaction. To determine why the RFCp140N604 complex is inactive in DNA
synthesis, we examined which of these functions is disrupted.
First, DNA binding properties of the RFC subunits and complexes were
studied. p140, formed in the in vitro translation reaction, bound to a DNA primer end formed by a hair-pinned DNA containing a
recessed 3
DNA binding by RFC complexes is shown in Fig. 5B. The
complex containing full-length p140 bound specifically to the primer end (Fig. 5B, lanes 1-5). RFCp140N555 bound with
reduced efficiency (lanes 6 and 7), as expected
from the observed behavior of free p140N555 (see Fig. 5A).
RFCp140N604, the complex inactive in replication, bound with equal
efficiency as RFCp140N555 (lanes 8 and 9). DNA binding of the RFC complex was lost only after the p140 deletion extended to amino acid 776 (lanes 10-13). Likewise,
p140N687 alone, rather than in the RFC complex, bound to primer ends,
while p140N776 did not, suggesting that stable DNA binding of the RFC
complex is mediated mainly by the large subunit. C-terminal deletion of p140N555 to amino acid 976 resulted in the loss of DNA binding by the
subunit (data not shown). This localizes the region critical for DNA
binding activity in the C-terminal half of p140 between amino acids 687 and the C terminus.
These results indicated that the inability of RFCp140N604 to support
DNA synthesis was not due to its deficiency in binding DNA primer
ends.
We compared the abilities of
RFC complexes containing full-length or N-terminally deleted p140 to
bind PCNA. The RFCp140N604 complex interacted with PCNA but not with
BSA covalently linked to a solid phase, as did the RFC complexes
containing the full-length large subunit and p140N555 (Fig.
6). This suggests that the replication defect in
RFCp140N604 is not solely due to its inability to interact with PCNA.
However, it cannot be excluded that multiple contact sites between PCNA
and RFC are required and that one of these sites may be absent in the
RFCp140N604 complex.
p140 and the deleted variants p140N555 and p140N604 alone interacted
with PCNA as did the single subunits p36 and p38 (data not shown),
suggesting that the interactions between PCNA and RFC are likely to be
complex, involving more than one subunit of RFC.
We determined whether
the RFC complex formed with the p140N604 subunit that was inactive in
replication was able to load PCNA onto DNA. The loading of
32P-labeled PCNA onto nicked circular DNA was assayed by
separating the [32P]PCNA-DNA complex from free
[32P]PCNA after the loading reaction by filtration
through a sizing column. As shown in Fig. 7, RFCp140N604
was inactive in loading PCNA, whereas the RFCp140N555 complex
catalytically loaded PCNA onto DNA. RFCp140N555 immobilized on beads
(40 fmol) loaded 1.1 pmol of PCNA onto DNA, and the reaction was
dependent on the addition of ATP (data not shown). A similar amount of
RFCp140 loaded 0.2 pmol of PCNA. This indicated that the RFCp140N555
complex was 5 times more active in the PCNA loading reaction than RFC
containing the full-length p140 subunit, consistent with the
differences observed in the elongation reaction of primed DNA
substrates (Fig. 3). Similarly, the unloading reaction, in which
[32P]PCNA is released from the
[32P]PCNA-DNA complex, was catalyzed more
efficiently by RFC containing p140N555 than full-length p140 (data not
shown).
These results suggest that the loss of replication activity observed
with RFCp140N604, which possessed DNA and PCNA binding activities, was
most likely due to its inability to load PCNA onto DNA.
Deletion analysis of the large subunit p140 of human RFC presented
in this report provides a step toward the functional characterization of this polypeptide. A summary of the p140 variants constructed, as
well as the activity of RFC complexes containing these variants, is
presented in Fig. 8. A region was defined within amino
acids 822-1142 that is required for the formation of the RFC complex. All of the small subunits also require sequences close to their C
termini for complex formation (29), suggesting that the unique sequences in these regions govern the interactions between the five
subunits. This might explain why all five subunits are required to form
the RFC complex despite the redundancy in their conserved regions. The
only other stable complex formed by the RFC subunits consists of the
three subunits p36, p37, and p40. This core complex is inactive in
replication, although it contains some of the enzymatic properties of
RFC.2
In the yeast Saccharomyces cerevisiae, the genes coding for
the homologous RFC complex have been identified (3, 19, 20, 31-36).
The genes for the corresponding subunits of yeast and human include
highly homologous RFC boxes as well as several conserved C-terminal
regions. These homologous C-terminal sequences may include sites
critical for subunit interactions that have been conserved through
evolution.
The p140 subunit was shown to contain two independent DNA binding
domains. Apart from a DNA binding activity that has been mapped to the
ligase homology domain (15, 21, 22), a separate region located in the
C-terminal half of the subunit was found to recognize primer ends. This
confirms previous observations that different nonoverlapping parts of
this protein can bind DNA (37). Both the p37 subunit and the
p36·p37·p40 core complex have been shown to bind specifically to
DNA primer ends under conditions less stringent than those used to bind
p140 (7).2 However, further studies will be necessary to
establish the significance of these DNA binding activities.
When RFC complexes were formed with N-terminally deleted variants of
the p140 subunit, it was found that the N-terminal half (amino acids
1-555) of p140 was not required for RFC to load PCNA onto DNA and to
support elongation of primed DNA templates. The resulting complex
(RFCp140N555) was 2-5 times more active than wild-type RFC in
catalyzing PCNA loading and the elongation reaction. Two hypothetical
explanations for this effect should be mentioned. The N-terminal half
of p140 contains a strong DNA binding activity, whereas the C-terminal
half binds DNA less effectively, but sufficiently to support RFC
activity. The catalytic turnover of RFC may be enhanced by the absence
of the strong but dispensable N-terminal DNA binding activity. The p140
N-terminal half also contains two potential CDC2 kinase sites (15)
whose phosphorylation status may regulate RFC activity. RFCp140N555
lacks these sites and thus might escape this regulation. A similar
mechanism has been described for mammalian DNA ligase I (38), where the
enzymatic activity, located in the C terminus, is blocked by
interaction with the N terminus. This blockage was released after
phophorylation or alternatively by deletion of the N terminus. Further
studies with RFCp140N555 purified from baculovirus-infected insect
cells will be necessary to examine this effect in more detail.
The in vivo function of the p140 N-terminal half remains
speculative. Although the DNA binding activity of this region may not
be essential for RFC function in replication, it might direct RFC to
sites involved in other DNA transactions, such as DNA repair or
recombination. Furthermore, while the studies presented here show that
the p140 N-terminal half is dispensable for the elongation reaction,
more elaborate interactions between the replication proteins probably
take place at a replication fork, which might involve the N terminus of
the p140 subunit.
In S. cerevisiae several mutations in the gene encoding the
RFC large subunit (cdc44) have been identified that cause
cold sensitivity, elevated levels of spontaneous mutations and
increased sensitivity to DNA damaging agents. One such mutation was
located in the DNA ligase homology domain (30, 39). Further studies in
yeast will help to elucidate the in vivo function of the
p140 N-terminal half.
Further deletion of the p140 N terminus into the RFC box II resulted in
an RFC complex (RFCp140N604) devoid of replication activity. This loss
in replication activity was shown to be due to the inability of
RFCp140N604 to load PCNA onto DNA, although it cannot be excluded that
interactions of RFC with other replication proteins (pol PCNA loading onto DNA requires opening of the trimeric ring structure
of PCNA and disruption of the contacts on at least one of the
interfaces between the PCNA monomers. It is likely that more than one
contact site between RFC and PCNA is necessary to accomplish the
structural changes essential for this reaction. The small subunits p36,
p38, and p40 have also been found to interact with PCNA
(7),3 and it is possible that a site
between amino acids 555 and 604 within p140 is critical for PCNA
loading but was not revealed due to the presence of other interacting
sites.
ATP binding and hydrolysis by RFC are essential for its function in
PCNA loading and replication. These properties of the inactive
RFCp140N604 complex could not be measured using in vitro translated material due to high background values, but are unlikely to
be altered since the conserved putative ATP binding motifs of p140 were
not affected in this variant.
Another possible explanation for the loss of replication function in
RFCp140N604 is that the absence of amino acids 555-604 of p140 leads
to a structural distortion in the RFC complex that renders it inactive.
However, deletion of a similar fragment, containing RFC box II, from
any of the small subunits (p36, p37, or p40) did not lead to a
comparable inactivation of the RFC complex (29).
In summary, we have defined a region around RFC box II in the large
subunit, which when deleted inactivated RFC in loading PCNA. However,
the reasons for this loss of function and a general understanding of
how the RFC complex works as a molecular machine assembling the PCNA
ring around DNA await elucidation of its three-dimensional structure.
We thank Charles E. Stebbins for a
preparation of PCNA used in this study and Dr. Z.-Q. Pan for continuous
helpful discussions.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
and
. RFC contains multiple activities, including
its ability to recognize and bind to a DNA primer end and load the
ring-shaped PCNA onto DNA in an ATP-dependent reaction.
PCNA then tethers the polymerase to the template allowing processive
DNA chain elongation. Human RFC consists of five distinct subunits
(p140, p40, p38, p37, and p36), and RFC activity can be reconstituted
from the five cloned gene products. To characterize the role of the
large subunit p140 in the function of the RFC complex, deletion mutants were created that defined a region within the p140 C terminus required
for complex formation with the four small subunits. Deletion of the
p140 N-terminal half, including the DNA ligase homology domain,
resulted in the formation of an RFC complex with enhanced activity in
replication and PCNA loading. Deletion of additional N-terminal amino
acids, including those constituting the RFC homology box II that is
conserved among all five RFC subunits, disrupted RFC replication
function. DNA primer end recognition and PCNA binding activities,
located in the p140 C-terminal half, were unaffected in this mutant,
but PCNA loading was abolished.
and
requires the two
accessory factors PCNA and RFC (1-7). PCNA is a ring-shaped
homotrimeric protein that encircles DNA and interacts with DNA
polymerases, ensuring that they maintain a high affinity interaction
with the template until completion of DNA synthesis (8-10). RFC binds
DNA at a primer end and loads PCNA onto the DNA in an
ATP-dependent reaction. Subsequent RFC-catalyzed ATP
hydrolysis is required for pol
to join this complex prior to
initiation of chain elongation (2-5). Human RFC, first isolated as a
protein complex required for SV40 replication in vitro,
consists of five subunits that migrate during SDS-PAGE as protein bands
of 140, 40, 38, 37, and 36 kDa (2, 11). The genes encoding each of
these five subunits have been cloned, and the five polypeptides encoded
by these genes have been shown to reconstitute RFC activity
(12-18).
Fig. 8.
Summary of the p140 subunit deletions and
their effects on complex formation and RFC activities. The RFC
homology boxes are indicated and numbered in the
uppermost bar, representing full-length p140. The names
given for the deleted p140 variants indicate the first or last amino
acid present at the N or C terminus, respectively. n.d., not
determined; N/A, not applicable, since these fragments do
not form RFC complexes; *, the DNA binding activity of p140C555 alone
is presented.
[View Larger Version of this Image (13K GIF file)]
.
Templates for in Vitro Transcription/Translation of RFC
Subunits
-CATGCCATGGACCAGAGCTGTGCCAACAAACTCC-3
; p140N687 N
terminus, 5
-CATGCCATGGCGATTGTTGCTGAGTCACTGAAC-3
; p140N776 N terminus,
5
-CATGCCATGGTTGAACAGATTAAGGGTGCTATG-3
; p140N822 N terminus,
5
-CATGCCATGGCACGAAGTAAAGCATTAACCTATGAC-3
; p140N877 N terminus,
CATGCCATGGATTATTCAATAGCACCCCCTCTTC-3
. The primer for the C terminus of
all constructs was 5
-CGGGGTACCTCATTTCTTCGAACTTTTTCCTTTTCC-3
. Polymerase chain reaction products were cleaved with the
restriction endonucleases NcoI and KpnI and
ligated into the NcoI and KpnI sites of a
modified pET19b vector (Novagen). This generated an artificial
methionine in front of the p140 sequence starting at the respective
amino acid indicated. Expression from these vectors ends at the C
terminus of p140 (amino acid 1148). The validity of these constructs
was confirmed by DNA sequencing. p140C1142 and p140C976 were generated
by cutting pET16ap140 within the p140 coding sequence with the
restriction endonucleases XmnI and AlwNI, respectively. The linearized vector fragments were used directly in
in vitro transcription/translation reactions.
, and RFC (25).
-32P]dCTP (10,000 cpm/pmol), 10 fmol
of singly primed circular M13 DNA, 320 ng of HSSB, 50 ng of PCNA, and
50 fmol of pol
. Reactions were incubated for 30 min at 37 °C
with frequent shaking and stopped with 10 mM EDTA. Aliquots
(2 µl) were withdrawn and added to 0.1 ml of solution containing 0.67 mg/ml salmon sperm DNA and 67 mM sodium pyrophosphate, and
the mixture was precipitated with 1 ml of 5% trichloroacetic acid.
After 15 min on ice, the precipitate was filtered through glass fiber
filters (Whatman) and acid-insoluble replication products were
quantitated by liquid scintillation counting. To 10 µl of the
remaining replication products, loading dye was added, and the mixture
was subjected to alkaline agarose gel electrophoresis. Gels were dried
and exposed for autoradiography. The amount of full-length replication
products formed was quantitated by phosphorimager analysis.
-end. Oligo(dA-dC)26, also biotinylated at its 5
-end,
was used as a single-stranded DNA substrate. Supercoiled pBlueBac II
plasmid DNA was used as the double-stranded DNA competitor, where
indicated. To a reaction mixture containing about 25 fmol of in
vitro translated RFC subunits or RFC complexes, 10 pmol of the DNA
substrate was added and the mixture was incubated at 30 °C for 10 min. Avidin agarose beads (5 µl, Vector) in 10 µl of equilibration
buffer (20 mM Tris/HCl, pH 7.5, 100 mM NaCl, 2 mM MgCl2, 1 mM ATP, 2 mM DTT, 0.1% Nonidet P-40, and 1% BSA) was added to the
reaction. Biotinylated DNA was bound to the avidin beads by incubating
the mixture for 20 min on ice, with shaking. The beads were then washed
three times with 0.3 ml of wash buffer (50 mM Tris/HCl, pH
7.5, 200 mM NaCl, 2 mM MgCl2, 1 mM ATP, 2 mM DTT, 0.5% Nonidet P-40, and 1%
BSA) and once with wash buffer without BSA. Bound protein was eluted
with 25 µl of SDS-PAGE loading buffer. Elution could also be
performed with 0.2 M NaOH or less efficiently by treatment
with nuclease S1. Aliquots of the eluate were separated on SDS-PAGE and
the gels processed as described under immunoprecipitation.
Identification of a Region within p140 Required for Complex
formation
Fig. 1.
Influence of p140 deletions on formation of
the RFC complex. Full-length p140 and deletion mutants, labeled
with [35S]methionine after in vitro
transcription/translation, were separated on SDS-PAGE, and are shown in
lanes 1-5. Complex formation of p140 with the small
subunits was detected by immunoprecipitation of a mixture of all five
subunits (lane 6, 10% of the input material; lane
7, immunoprecipitate with antibodies against p37; lane
8, immunoprecipitate using preimmune serum). Requirements for
complex formation were studied by replacing p140 with the respective
deletion mutant as indicated (lanes 9, 11,
13, and 15, 10% of the input material;
lanes 10, 12, 14, and 16,
immunoprecipitate). An experiment omitting p140 is shown as a control
(lanes 17 and 18). Under the conditions used, the
p36 and p37 subunits were not resolved and are jointly labeled
p36, p37.
[View Larger Version of this Image (55K GIF file)]
Fig. 2.
Influence of sequential deletions of p140
from the N terminus on RFC complex formation. Products formed in
in vitro transcription/translation reactions are shown using
templates for p140 deletion mutants starting from amino acids 555, 604, 687, 776, 822, and 877, respectively (lanes 1-6). These
deletion mutants were coexpressed with the four small subunits
(lanes 7, 10, 12, 14,
16, and 18, respectively) and complexes
immunoprecipitated with antibodies against p37 (lanes 8,
11, 13, 15, 17, and
19). A control immunoprecipitation reaction using preimmune
serum is shown in lane 9.
[View Larger Version of this Image (51K GIF file)]
, is
dependent upon RFC, PCNA, and HSSB (Fig. 3A, lanes
1-8). The RFC complex formed with p140N555 (RFCp140N555) appeared
to support replication more efficiently than the complex formed with
full-length p140 (Fig. 3A, compare lanes 4 and
9). To quantitate this apparent difference, the RFC
complexes formed with full-length p140 (RFCp140) or p140N555 were
titrated by varying the amount of complex bound to immunobeads in the
assay reaction (Fig. 3B). The efficiency of
immunoprecipitation of RFCp140N555 was reproducibly twice the
efficiency observed with RFCp140 (compare Fig. 1, lanes 7 and 16) and the input material for the reactions was
adjusted accordingly. The activity of RFCp140N555 was approximately
2-fold greater than the activity of RFCp140 as measured by nucleotide
incorporation, and 5-fold when formation of full-length replication
products (7.2 kilobase pairs) in the singly primed M13 elongation
reaction was measured (Fig. 3B, lanes 4-6 and
7-9). These differences were reproducibly observed in three
additional experiments.
Fig. 3.
, or
HSSB (lanes 5-7). A mock in vitro translation reaction (25 µl), using template DNAs without the T7 RNA polymerase promotor (lane 8), and a reaction coexpressing the five
subunits of RFCp140N555 (25 µl, lane 9) were bound to
immunobeads. B, comparison of the activities of the RFC
complexes containing full-length p140 or p140N555. Lanes
1-3 are controls as in A. Products contained in 50, 10, and 2 µl of the in vitro translation reaction
(IVT) coexpressing RFCp140 (lanes 4-6), and 25, 5, and 1 µl of coexpressing RFCp140N555 (lanes 7-9) were
bound to immunobeads and used in the replication reaction. The amount
of RFC complex isolated on the beads in each case is indicated. An
autoradiograph of the alkaline agarose gel in which the replication
products were resolved is shown, and the quantitation of
[
-32P]dCTP incorporated into replication products is
given, as well as relative numbers for the amount of full-length (7.2 kilobase pairs) replication products formed (normalized against
full-length products present in lane 6).
[View Larger Version of this Image (27K GIF file)]
Fig. 4.
Replication activity of RFC complexes formed
with progressively increasing N-terminally deleted p140 variants.
Replication reactions with RFC complexes (40 fmol), isolated from
in vitro translation reactions, were performed as described
in Fig. 3A. dCTP incorporation was quantitated and is shown
for reactions with RFCp140N555, RFCp140N604, RFCp140N687,
RFCp140N776, RFCp140N822, and for a reaction without RFC.
[View Larger Version of this Image (14K GIF file)]
-end (Fig. 5A, lanes 1 and 2). p140 specifically interacted with the primer end of
this DNA substrate, since the binding was not competed by
double-stranded DNA (lane 3), and p140 weakly bound to
single-stranded DNA (lane 4). p140C555, containing the DNA
ligase homology domain, bound to the primer end with the same
efficiency as full-length p140 (lanes 6 and 7).
p140N555, lacking this domain, still contained DNA binding activity,
although the efficiency of binding was reduced (lanes 8 and
9). The four small subunits of RFC did not bind to DNA under
the conditions used (lanes 10-17).
Fig. 5.
DNA binding activity of RFC subunits and
complexes. A, DNA binding properties of the single subunits.
5 µl of in vitro translation reactions, expressing about
25 fmol of the respective subunits or different regions of p140, were
incubated with the indicated DNA substrates. The abbreviation
hp refers to the addition of 10 pmol of biotinylated
hair-pinned DNA containing a primer end, ss refers to
addition of 10 pmol of biotinylated single-stranded oligo(dA-dC)26, and ds refers to the addition of a
6-fold molar excess (in nucleotides) of double-stranded plasmid DNA (2 µg). Biotinylated substrates were bound to avidin beads, and bound protein was eluted and analyzed on SDS-PAGE. 10% of the material added
to the binding reaction is shown (lanes 1, 6,
8, 10, 12, 14, and
16), and the percentage of input material bound to avidin beads was quantitated and is indicated as % bound. B, DNA
binding activity of RFC complexes formed with full-length and
N-terminally deleted p140. Translation reaction mixtures in which the
four small RFC subunits were coexpressed with p140 (lanes
1-5) or deleted p140 variants (lanes 6-13) were used
in the binding reactions. Reactions were carried out as described in
A. In lanes 1, 6, 8, 10, and 12, 10% of the material added to the
binding reaction is shown. The elution of proteins bound to avidin
beads and autoradiography were described under "Materials and
Methods" under DNA binding assay.
[View Larger Version of this Image (38K GIF file)]
Fig. 6.
Interaction of RFC complexes containing
full-length or N-terminally deleted p140 with PCNA. In vitro
translation reactions expressing the small RFC subunits and either
p140, p140N555, or p140N604 (RFC, N555, and
N604 in the figure) were incubated with PCNA- or
BSA-affinity beads as described under "Materials and Methods." 10%
of the translation reaction mixture (lanes 1, 4, and 7), the material that eluted from the PCNA beads
(lanes 2, 5, and 8), and the material
that eluted from the BSA beads (lanes 3, 6, and
9) were analyzed on SDS-PAGE.
[View Larger Version of this Image (69K GIF file)]
Fig. 7.
Loading of PCNA onto nicked circular DNA by
RFC containing full-length or deleted p140 variants. Loading
reactions with RFCp140, RFCp140N555, and RFCp140N604 (designated
RFC, N555, and N604 in the figure),
isolated from in vitro translation reactions, were carried
out as described under "Materials and Methods." The elution profile
of [32P]PCNA from the sizing column is shown and
represents the amount of PCNA present in each fraction. Fractions
7-16 correspond to the excluded volume containing PCNA bound to
DNA. Fractions from the included region, containing free
[32P]PCNA and [-32P]ATP, are not
shown.
[View Larger Version of this Image (19K GIF file)]
or HSSB)
are also affected. RFCp140N604 bound to DNA primer ends and to PCNA
with the same efficiency as RFCp140N555.
*
These studies were supported by National Institutes of
Health Grants GM 38559 (to J. H.) and GM 38839 (to M. O'D.).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.
Enrolled in the graduate program at the Physiologisch-chemisches
Institut, Universität Tübingen, and supported by the German Academic Exchange Service (DAAD) through funds of the Zweites Hochschulsonderprogramm.
¶
Professor of the American Cancer Society. To whom
correspondence should be addressed: Program in Molecular Biology,
Memorial Sloan-Kettering Cancer Center, 1275 York Ave., Box 97, New
York, NY 10021.
1
The abbreviations used are: RFC, replication
factor C, also called activator 1; PCNA, proliferating cell nuclear
antigen; pol, DNA polymerase; HSSB, human single-stranded DNA-binding
protein, also called RPA; BSA, bovine serum albumin; PAGE,
polyacrylamide gel electrophoresis; DTT, dithiothreitol; RIPA,
radioimmune precipitation buffer.
2
J. Cai, E. Gibbs, F. Uhlmann, B. Phillips, N. Yao, M. O'Donnell, and J. Hurwitz, submitted for publication.
3
F. Uhlmann and J. Hurwitz, unpublished
results.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.