(Received for publication, November 27, 1996, and in revised form, January 30, 1997)
From the Program in Molecular Biology, Replication factor C (RFC) and proliferating cell
nuclear antigen (PCNA) are processivity factors for eukaryotic DNA
polymerases Processive DNA synthesis catalyzed by DNA polymerases
(pol)1 The RFC complex consists of five distinct subunits with apparent
molecular masses of 140, 40, 38, 37, and 36 kDa (4, 13). The amino acid
sequences of the five subunits exhibit a high degree of homology to
each other (14-17). In particular, the four small subunits p40, p38,
p37, and p36 possess striking similarities. The N-terminal halves of
these polypeptides are homologous, and seven conserved boxes (RFC boxes
II-VIII) have been defined within this region (18). The C-terminal
sequences of each subunit, however, are unique. The large subunit p140
also possesses RFC boxes II-VIII, located in the middle of p140
flanked on both sides by non-homologous sequences. The N-terminal
extension of p140 contains RFC box I, which shows homology to
prokaryotic DNA ligases (16, 19). RFC boxes III and V in all five
subunits contain consensus motifs for the binding of purine
nucleotides. The presence of extensive regions of sequence homology in
the subunits has raised the question as to whether all five subunits
are required to form an active RFC complex or whether the subunits in
the complex are functionally redundant. Studies on the properties of
the individual subunits are still incomplete, and it is unclear whether
observations made with each subunit alone reflect its function when
assembled in the RFC complex (5, 7, 19, 20). We have shown previously that after in vitro translation, all five human RFC subunits
are required to form a stable, active complex (21). We have now carried
out deletion analyses of the five RFC subunits to identify regions
required for complex formation and for RFC-dependent DNA replication. Here we report the results obtained with the four small
subunits. We show that C-terminal sequences, unique to each of the
subunits, are required for complex formation, explaining why the
subunits cannot substitute for each other in the formation of a
five-subunit complex. Progressive removal of the conserved RFC boxes
from the p36, p37, and p40 subunits reduces but does not abolish the
ability of the formed RFC complexes to support DNA replication. A
region around RFC box II was found to contribute to the function of
each subunit. Although the N termini, containing the RFC boxes, are
highly homologous in all subunits these regions cannot replace one
other. Thus, the role of the conserved region of each subunit in the
RFC complex seems to be unique for replication.
The DNA templates used for the in vitro
transcription and translation of the five subunits of human RFC were as
described previously (21).
N-terminal deletion mutants of the four small RFC
subunits were obtained by PCR amplification of fragments using cloned
cDNAs of each subunit as the template (Expand High Fidelity,
Boehringer Mannheim). The PCR-primers used for the construction of the
mutant subunits were: p40N56, N terminus
5 C-terminal deletion mutants of the four small subunits were generated
by cleavage of the coding DNA sequence in the previously described
expression vectors (pET5ap40, pET19bHisp38, pET3cp37, and pET19bHisp36;
Ref. 21) with the following restriction endonucleases: p40C350,
BglI; p40C337, StuI; p38C340, FokI;
p38C293, EcoO109I; p37C334, HincII; p36C338,
StuI; and p36C323, SacI. The linearized vector
fragments encoded C-terminally deleted subunits ending with the
indicated amino acid. The vector fragments formed after restriction
were used directly as templates for in vitro transcription and translation reactions.
Regions
of the p37 and p40 subunits, spanning from their respective N terminus
to a conserved heptapeptide motif in RFC box VIb of both subunits
(ALRRTME), were exchanged between the two subunits. The conserved DNA
sequence for this motif in both subunits contains a recognition site
for the restriction endonuclease NcoI. This site was chosen
for the construction of transitions between the two subunits. PCR
fragments, spanning the coding sequence of p40 from amino acids 1 to
155 and from 156 to 354, were generated, respectively, using the
following primers: p40(1-155), N terminus 5 In
vitro transcription/translation reactions were performed as
described (21). When linearized templates were used, 400 ng of the
digested vector fragments were added to 10 µl of
transcription/translation reaction mixture. The translation products
were quantitated by Western blotting of the p37 and p40 subunits and by
measuring the level of [35S]methionine incorporated into
these and other subunits.
When
interactions between subunits were examined, each subunit was expressed
individually. After incubation of the in vitro translation
reactions for 1 h, reaction mixtures were combined and incubated
for an additional 30 min at 30 °C. Polyclonal antiserum (1 µl)
against one of the subunits was bound to a 5-µl suspension of protein
A-Sepharose beads (Upstate Biotechnology Inc.) for 1 h on ice.
Beads were then 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 adsorbed to the beads were eluted with 25 µl of
SDS-PAGE loading buffer, and aliquots were separated by electrophoresis
on SDS-polyacrylamide gels (10%) or gradient gels (7.5%-20%). After
separation, gels were fixed in 25% 2-propanol, 10% acetic acid,
soaked in luminographic enhancer (Amplify, Amersham), dried,
and exposed for autoradiography. Quantitation was performed using
a phosphorimager (Fuji).
The following proteins
were prepared from HeLa cell extracts as described: HSSB (22), PCNA
(23), pol RFC
complexes formed from in vitro translation reactions (25 µl) were isolated with immunobeads as described under
immunoprecipitation of translation products. After the beads were
washed, 19.5 µl of the following reaction mixture was added directly
to the washed beads: 40 mM Tris/HCl, pH 7.5, 7 mM MgCl2, 2 mM ATP, 0.5 mM DTT, 0.015% BSA, 40 mM creatine phosphate,
50 mM [methyl-3H]dTTP (350 cpm/pmol), 100 ng of poly(dA)4000·oligo(dT)12-18 annealed at a nucleotide ratio of 20:1, 320 ng of HSSB, 50 ng of PCNA,
and 50 fmol of pol Reactions containing RFC isolated from HeLa cells were carried out by
adding the reaction mixture (19.5 µl) to a purified fraction of RFC
(0.5 µl, 30 fmol). Reactions were incubated and analyzed as described
above.
RFC complexes
formed from in vitro translation reactions (25 µl) 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) (11, 25). 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 present in the four small subunits
of RFC that are required for complex formation, deletion mutants in each of the subunits were constructed, expressed in vitro,
and the products assayed for their ability to assemble an RFC complex. Fig. 1A shows the results obtained with the
p40 subunit. Lanes 1-5 present the products obtained after
in vitro transcription and translation of cDNAs encoding
full-length and deleted variants of the p40 subunit. Each reaction
mixture yielded a labeled protein of the expected size, although
smaller products were formed, which 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 p40 deletion
mutants. A C-terminal deletion of the p40 subunit that ended at amino
acid 350 (p40C350; Fig. 1A, lane 2) supported complex formation (lanes 9 and 10), as did
full-length p40 (amino acids 1-354, Fig. 1A, lanes
1, 6, and 7). However, p40C337, a mutant
containing a deletion of 17 C-terminal amino acids, did not support RFC
complex formation (lanes 3, 11, and
12). In this case, where the p40 subunit did not enter the
RFC complex, a complex containing the remaining four RFC subunits was
immunoprecipitated with an efficiency one-tenth of that observed in the
presence of full-length p40. This less stable complex was also obtained in the absence of the p40 subunit (Fig. 1A, lanes
17 and 18) as described previously (21). In contrast to
deletions at the carboxyl end, deletions from the N terminus of p40 to
amino acid 56 or 241 (p40N56 and p40N241, lanes 4 and
5) did not affect formation of the five-subunit RFC complex
(lanes 13 and 14 and lanes 15 and
16). This indicates that a C-terminal region of p40, between amino acids 241 and 350, mediates its interaction with the other RFC
subunits to form the five-subunit complex.
Deletion analysis of the p38 subunit is presented in Fig.
1B. The products obtained after in vitro
transcription and translation of full-length and deleted variants of
p38 are shown in lanes 1-4. p38C340, with 16 C-terminal
amino acids deleted, migrated slightly slower than full-length p38
during SDS-PAGE (lanes 1 and 2). The same effect
was observed after deletion of C-terminal amino acids of the p36
subunit (see below). This deletion in p38, which yielded p38C340, did
not affect its ability to form the RFC complex, as revealed by
immunoprecipitation of the five-subunit complex containing either
full-length p38 or this p38 variant (lanes 5 and
6 and lanes 8 and 9). Deletion of an
additional 47 amino acids from the C terminus led to a subunit variant,
p38C293, that did not support RFC complex formation (lanes
10 and 11). Since immunoprecipitations were carried out
with antibodies against the p37 subunit, the RFC core complex,
containing the three subunits p36, p37, and p40, was coprecipitated,
but not the large subunit p140 or the p38 variant (control, omitting
p38; lanes 14 and 15) (21). This indicates that a
region close to the C terminus of the p38 subunit is required for
complex formation, as observed for the p40 subunit. However, deletion
of 19 N-terminal amino acids of p38 (p38N20) resulted in a subunit
variant incapable of supporting RFC complex formation (lanes
12 and 13). These findings suggest that, in contrast to
the p40 subunit, a region close to the N terminus of p38 is required
for complex formation as well as a region at the C terminus. Whether
these regions are involved directly in complex formation or essential
to maintain a proper conformation for subunit interactions is presently
unknown.
Similar experiments were performed with deletion mutants of the p37
subunit (Fig. 1C). In vitro
transcription/translation products of full-length p37 and deletion
mutants of the subunit are presented in lanes 1-5.
Immunoprecipitation of a mixture of the five RFC subunits, including
full-length p37, with antibodies against the p37 subunit yielded a
complex containing all five subunits (lanes 6 and
7). When the full-length p37 subunit was replaced by
p37C334, a deletion mutant lacking 29 C-terminal amino acids
(lane 2), no complex was formed and the p37 variant alone was precipitated (lanes 9 and 10). This indicates
that a region close to the C terminus of this subunit is required for
complex formation with the other RFC subunits, as found with the p40
and p38 subunits. N-terminal deletions of p37 that removed the first 53 or the first 179 amino acids of this subunit (p37N54 and p37N180, lanes 3 and 4) did not affect complex formation
with the other RFC subunits (lanes 11 and 12 and
lanes 13 and 14). When 247 N-terminal amino acids
from the p37 subunit were deleted (p37N248, lane 5), its
ability to support complex formation was lost (lanes 15 and 16). These results defined a region within the p37 subunit
between amino acids 180 and the C terminus, which is required for
interaction with the other RFC subunits. The two cross-reacting labeled
protein bands of 31 and 25 kDa, present in some of the lanes, were
nonspecific background bands produced during the in vitro
translation reaction (see also Fig. 1, A and
D).
Deletion analysis of the p36 subunit is presented in Fig.
1D. The products obtained after in vitro
transcription and translation of cDNAs encoding full-length and
deleted derivatives of the p36 subunit are shown in lanes
1-5. An immunoprecipitation assay, as described for the other
small subunits, was employed to measure RFC complex formation with
these variants. Full-length p36 (lane 1), as well as p36C338
that lacked two C-terminal amino acids (lane 2), supported
formation of the five-subunit complex (lanes 6 and
7 and lanes 9 and 10), but p36C323,
deleted of an additional 15 C-terminal amino acids, did not
(lanes 11 and 12). This indicates that, similar
to the other small subunits, a region close to the p36 C terminus was
indispensable for RFC complex formation. Deletion from the N terminus
of p36 to amino acids 40 or 225 (p36N40 and p36N225) did not affect its
ability to support RFC complex formation (lanes 13 and
14 and lanes 15 and 16).
In summary, these results demonstrate that sequences near to the C
termini of all of the four small subunits are required for formation of
the five subunit RFC complex. In the case of the p36, p37, and p40
subunits, a region within the C terminus of each subunit was sufficient
for complex formation, whereas sequences close to the N terminus of the
p38 subunit were also essential.
We next examined the effects of deletions in the
RFC small subunits on the ability of the RFC complex to support the
PCNA-dependent elongation reaction catalyzed by pol
Fig. 2 summarizes the activities of RFC complexes that contain the
various deleted small subunits. An RFC complex assembled from the
C-terminally deleted subunits p36C338, p38C340, and p40C350, together
with full-length p37 and the p140 C-terminal fragment, supported DNA
synthesis as effectively as the RFC complex formed with full-length
small subunits. Thus the C-terminal regions of the p36, p38, and p40
subunits that are dispensable for complex formation are also not
required for the replicative activity of RFC.
The RFC complex formed with p40 N-terminally deleted to amino acid 56 (p40N56) supported replication about half as efficiently as the complex
formed with full-length p40. Further deletion of the p40 subunit to
amino acid 241 (p40N241) did not significantly alter the activity of
the complex compared with the complex formed with p40N56. N-terminal
deletion of the p37 subunit to amino acid 54 (p37N54) resulted in a
10-fold decrease of replication activity compared with RFC formed with
full-length p37. Extension of the p37 deletion to amino acid 180 (p37N180) did not alter this residual activity. When the p36 subunit
was N-terminally deleted, the activity of the resulting RFC complex was
reduced 2-fold compared with the activity observed with full-length
p36. No significant differences were observed with complexes containing
p36 deleted either to amino acid 40 or to amino acid 225 (p36N40 and
p36N225, respectively).
In summary, RFC complexes formed with N-terminally deleted variants of
the small subunits p36, p37, and p40 supported replication less
efficiently than RFC formed with wild-type subunits. Deletion of a
relatively short N-terminal part of the subunits, which in each case
included the RFC homology box II, resulted in the maximal loss of
replication activity. Deletions of the other RFC boxes (III-VI of the
p37 subunit and all of the remaining boxes of the p36 and p40
subunits), including the purine nucleotide binding consensus motifs,
did not lead to further loss of replication activity.
The five-subunit RFC complex formed with both N-terminally deleted p40
and p36 subunits (p40N241 and p36N225) supported replication 5-fold
less efficiently than the RFC complex formed with wild-type p40 and p36
(Fig. 2). RFC complexes formed with either one of the p40 or p36
deletions were 2-fold less active than the wild-type RFC complex. Thus,
the loss of activity observed with the RFC complex containing the two
deleted subunits appeared to be additive.
We
determined whether the RFC complexes formed with N-terminally deleted
small subunit variants that showed reduced replicative activity were
impaired in their ability to load PCNA onto DNA. The loading of
32P-labeled PCNA onto nicked circular duplex DNA was
assayed by separating the [32P]PCNA-DNA complex formed
from free [32P]PCNA after the loading reaction by
filtration through a sizing column. RFC complexes, formed from in
vitro translation reactions, were assembled with the p140
C-terminal fragment and isolated by adsorption onto immunobeads. As
shown in Fig. 3, RFC complexes containing the p40 or p36
subunit deleted of the RFC homology box II (p40N56 or p36N40) were only
about 10% active in PCNA loading compared with the wild-type complex.
RFC containing p40N56 (40 fmol) loaded 0.18 pmol of PCNA (as the
trimer) onto DNA while the complex with p36N40 loaded 0.16 pmol PCNA.
The RFC complex (40 fmol) containing the full-length small subunits
loaded 1.5 pmol of PCNA onto DNA. When the p37 subunit in the RFC
complex was deleted of its N-terminal region, including homology box II (p37N54), the PCNA loading activity was barely detected. Thus, the
reduction in PCNA loading activity of the RFC complexes after deletion
of the N termini of the small subunits paralleled the observed
reduction in replicative activity but the effects were more pronounced.
In the replication assay, a single PCNA trimer on DNA can lead to
processive pol
The N-terminal regions of the small subunits are
highly homologous, particularly within the RFC boxes. To examine
whether these N-terminal regions of the small subunits are
interchangeable, we constructed two chimeric subunits in which the
conserved N termini between p37 and p40 were exchanged. The resulting
chimeric subunits, named p37N/40C and p40N/37C, contained the p37 N
terminus (amino acids 1-164) with the p40 C terminus (amino acids
156-354) and the p40 N terminus (amino acids 1-155) with the p37 C
terminus (amino acids 165-363), respectively. These chimeric subunits
were expressed in vitro, and the products formed are shown
in Fig. 4A (lanes 2 and
3). We tested first whether the chimeric subunits could
replace the wild-type p37 and p40 subunits in supporting formation of
the five subunit RFC complex. An immunoprecipitation assay was carried
out with antibodies against the p36 subunit. The p37N/40C chimera
replaced the p40 subunit (lanes 8 and 9) and the
p40N/37C supported complex formation in lieu of the p37 subunit
(lanes 11 and 12). However, the p37N/40C and
p40N/37C chimeras did not replace the p37 and p40 subunits,
respectively, in keeping with the essential role of the C-terminal
sequences of the respective subunit for RFC complex formation (data not shown). A five subunit complex in which both the p37 and p40 subunits were replaced by the chimeric variants was also formed (lanes 14 and 15).
We then examined the activity of RFC complexes formed with these
chimeric subunits in the RFC-dependent elongation reaction. Fig. 4B compares complexes that are deleted in the N termini
of p37 or p40 with complexes that contain the heterologous N termini of
the chimeric subunits. As described above, deletion of the N terminus
of p40 (p40N241) led to an RFC complex that was half as active as the
complex formed with the full-length p40 subunit. A RFC complex formed
with p37N/40C in place of p40N241 only slightly increased the
replication activity. Deletion of the p37 N terminus in the RFC complex
(p37N180) resulted in 10-fold reduction in activity. The RFC complex
containing the p40N/37C chimera in place of the N-terminally deleted
p37 increased the activity of the complex only 2-fold. This suggests
that the functions of the N termini of the p37 and p40 subunits are
unique and are not interchangeable between the subunits. Finally, an
RFC complex was assembled that contained both p37N/40C and p40N/37C. In
this complex, the N termini of both p37 and p40 subunits were present
but their locations were exchanged. The activity of this complex was
not significantly different from the activity of a complex in which the
N termini of both of these subunits were deleted (p40N241, p37N180)
(Fig. 4B), indicating that the presence and distinct
location of the N termini of the subunits are essential.
Deletion analysis of the four small subunits of hRFC, presented in
this report, extends the understanding of the structural and functional
organization of the RFC complex. The ability of each of these variants
to support the assembly of the five-subunit RFC complex and
RFC-dependent DNA synthesis are summarized in Fig.
5. Sequences close to the C terminus of all subunits
were essential for complex formation. In the case of the p36, p37, and
p40 subunits, a region within their C-terminal half was defined that
was sufficient for formation of the RFC complex. In contrast, the p38
subunit also required sequences close to its N terminus for complex
formation. The p36, p37, and p40 subunits form a stable complex and the
interaction of the p140 subunit with this core complex requires the
presence of an intact p38 subunit (21). A C-terminal fragment of p140
is also sufficient for complex formation with the small subunits (31),
leading to a model in which p38 acts as a bridge between the core
complex and the large subunit.
When the activities of RFC complexes containing N-terminally deleted
small subunits were studied, it was found that a region close to the N
terminus, containing RFC box II, was critical for the function of each
of the subunits. Deletion of this region reduced the ability of the
resulting RFC complex to support DNA synthesis. Deletion of more or all
of the remaining RFC boxes in any of the subunits had no further
effect. Deletion of a region including RFC box II of the large subunit
p140 yielded a complex devoid of replicative activity (31), an
observation that further substantiates the importance of RFC box
II.
For its function in replication, RFC possesses 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. Detailed studies on the loss of function after deletion of
RFC box II in the large subunit p140 have been carried out (31). DNA
primer end binding and PCNA binding were not altered in the inactive
complex, but PCNA loading was abolished. Similar experiments were
performed with RFC complexes containing the small subunit deletion
mutants described here. DNA binding and PCNA binding were not altered
(data not presented), but the loading of PCNA onto nicked circular DNA
was reduced in a manner that paralleled the reduction in replication
activity. This suggests that the N termini (including RFC box II) of
the p36, p37, and p40 subunits may be involved in the PCNA loading
reaction.
ATP binding and hydrolysis are essential functions of the RFC complex.
Interestingly, the subunits of the core complex (p36, p37, and p40)
possess these activities (5, 14, 26).2
Deletion of the putative nucleotide binding motifs located in RFC boxes
III and V of these subunits individually, however, did not completely
inactivate the RFC complex. Furthermore, deletion of these potential
ATP binding sites in both p36 and p40 subunits yielded an RFC complex
that was still partially active (p40N241, p36N225). This suggests that
the binding and hydrolysis of ATP may be carried out by more than one
subunit. Thus the three subunits p36, p37, and p40 may have in part
common functions within the RFC complex. However, deletion of the
N-terminal region of the p37 subunit reduced the replication activity
of RFC to a greater extent than similar deletions in the p36 or p40
subunits. Furthermore, the N-terminal regions of the p37 and p40
subunits did not substitute for each other in restoring RFC activity.
This establishes unique functions for each of the subunits within the
RFC complex, in addition to possible redundant features.
The DNA replication machinery of many different species relies on the
participation of a sliding clamp (PCNA in human and yeast, We thank Dr. Z.-Q. Pan for helpful discussions
and comments on the manuscript.
and
. RFC binds to a DNA primer end and loads PCNA
onto DNA in an ATP-dependent reaction. The five RFC
subunits p140, p40, p38, p37, and p36, all of which are required to
form the active RFC complex, share regions of high homology including
the defined RFC boxes II-VIII. RFC boxes III and V constitute a
putative ATP binding site, whereas the function of the other conserved
boxes is unknown. To study the individual subunits in the RFC complex and the role of the RFC boxes, deletion mutations were created in all
subunits. Sequences close to the C terminus of each of the small
subunits are required for formation of the five subunit complex. A
N-terminal region of the small subunits, containing the RFC homology
box II, plays a critical role in the function of these subunits,
deletion of which reduces but does not abolish RFC activity in loading
PCNA onto DNA and in supporting an RFC-dependent replication reaction. The N termini of p37 and p40, although highly homologous, are not interchangeable, suggesting unique functions for
the individual subunits.
or
in eukaryotes requires
two accessory protein factors, RFC and PCNA. PCNA, a homotrimeric
protein that forms a ring-shaped structure around DNA, is capable of
sliding along duplex DNA, a property consistent with its role as a
clamp that tethers the polymerase to the template (1-10). RFC is a
protein complex that binds PCNA and catalyzes its loading onto DNA as
well as its unloading from DNA (11, 12). In the loading reaction, RFC
first binds specifically to a DNA primer end and then recruits PCNA and
loads it onto DNA in a reaction that requires binding of ATP (4, 5).
Subsequent ATP hydrolysis is necessary for the polymerase to enter the
complex and for initiation of chain elongation (2, 3).
Templates for in Vitro Transcription/Translation of RFC
Subunits
-CATGTCATGACCATGAGCAGGCTAGAGGTCTTTG-3
and C terminus
5
-CGGGATCCTAAGGCGCATTTTCCCCCATCAG-3
; for p40N241, N terminus
5
-CATGCCATGGGATTTGGCTTCATTAACAGTGAG-3
and C terminus 5
-CGGGGTACCTAAGGCGCATTTTCCCCCATCAG-3
; for p38N20, N terminus 5
-CATGTCATGAAGGAGCAGGCGGCCCAGCTGC-3
and C terminus
5
-CGGGGTACCTCAGAACATCATGCCTTCCAATCC-3
; for p37N54, N terminus
5
-CATGACATGTTCCAGGAAGAAGTGGTTGCAGTG-3
and C terminus
5
-CGGGGTACCTTAACAATTCTGAGATAACTGCTGC-3
; for p37N180, N terminus
5
-CATGACATGTGTAACTATGTCAGTCGAATAATTG-3
(the C terminus primer used
was the same as that used for p37N54); for p37N241, N terminus
5
-CATGCCATGGCTACTCGATTAACAGGTGGAAAG-3
(C terminus primer as used for
p37N54); for p36N40, N terminus
5
-CATGTCATGATTCTGAGTACCATTCAGAAGTTTATC-3
and C terminus
5
-CGGGGTACCTAGGCCTCTGCAACAATCAGGTC-3
; for p36N225, N terminus
5
-CATGCCATGGGCCTTTGGGAAGGTGACAGAG-3
(C terminus primer as
described for p36N40). The PCR products were cleaved with the following
restriction endonucleases to yield cohesive ends: p40N56, Bsp HI and BamHI; p40N241, p37N248, and p36N225,
NcoI and KpnI; p38N20 and p36N40, Bsp
HI and KpnI; p37N54 and p37N180, AflIII and
KpnI. The processed fragments were ligated to
NcoI and KpnI, or NcoI and
BamHI sites of a modified pET19 vector (Novagen), respectively. Expression from these constructs yielded polypeptides starting at the indicated amino acid of the respective subunit with an
additional methionine for translational start at the N terminus. In all
cases, constructs were verified by DNA sequencing (U. S. Biochemical
Corp.).
-GCATCGATCATATGGAGGTGGAGGCCGTCTGTG-3
and C terminus
5
-GATTTCCATGGTTCTCCTCAAGGC-3
; p40(156-354), N terminus
5
-GAGGAGAACCATGGAAATCTACTC-3
and C terminus
5
-CGCGGATCCTAAGGCGCATTTTCCCCCATC-3
. The PCR products formed
were cleaved with restriction endonucleases NdeI and
NcoI (p40(156-354)) or NcoI and BamHI
(p40(156-354)). The processed fragments were used to replace parts of
the coding DNA sequence in pET3cp37 that were excised from this vector
using the same restriction endonucleases. NdeI and
NcoI were used to remove the fragment encoding amino acids
1-164 of p37, and NcoI and BamHI were employed
to remove the fragment encoding amino acid 165 through the C terminus
of p37. The resulting vectors were designated pET3p40N/37C, encoding
the chimeric subunit containing the p40 N terminus and the p37 C
terminus (p40N/37C), and pET3p37N/40C, encoding the chimeric subunit
containing the p37 N terminus and the p40 C terminus (p37N/40C). All
constructs were validated by DNA sequencing.
, and RFC (24).
. Reactions were incubated for 60 min at 37 °C
with frequent shaking and stopped with 10 mM EDTA. An
aliquot (15 µl) of the reaction mixture was added to 0.1 ml of a
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 GF/C) and the acid-insoluble
material formed was quantitated by liquid scintillation counting.
Regions in the Small Subunits Required for RFC Complex
Formation
Fig. 1.
Influence of deletions within the small
subunits on formation of the RFC complex. A, deletions
within the p40 subunit. Full-length p40 and deletion mutants, labeled
with [35S]methionine after in vitro
transcription/translation, were separated on a 7.5-20%
SDS-polyacrylamide gradient gel, and are shown in lanes
1-5. Complex formation of p40 with the other 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 the p40 subunit 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 p40 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. B, deletions within the p38 subunit.
Full-length p38 and the respective deletion mutants, after in
vitro translation, were analyzed on a 10% SDS-polyacrylamide gel
(lanes 1-4). The assay for complex formation was carried
out as described in A. Reactions containing 10% of the
input material and the immunoprecipitates are shown for experiments
with p38, deleted p38 variants, and without p38, as indicated
(lanes 5-15). C, deletions within the p37
subunit. The p37 subunit and various deletion mutants are shown in
lanes 1-5. Complex formation was studied by
immunoprecipitation of mixtures containing the respective p37 variant
and the other four RFC subunits. Reactions containing 10% of the input
material (lanes 6, 9, 11,
13, and 15) and the immunoprecipitates
(lanes 7, 10, 12, 14, and
16) were separated on a 7.5-20% SDS-polyacrylamide gradient gel. Controls using preimmune serum (lane 8) and
omitting p37 (lanes 17 and 18) are shown.
D, deletions within the p36 subunit. Full-length p36 and
deletion mutants of this subunit were resolved on a 7.5-20%
SDS-polyacrylamide gradient gel (lanes 1-5). Complex formation was assayed in immunoprecipitation experiments as described. Reactions containing 10% of the input material and the
immunoprecipitates are shown (lanes 6-18).
[View Larger Version of this Image (57K GIF file)]
using poly(dA)4000·oligo(dT)12-18 as the
template. For this purpose, RFC complexes (40 fmol) formed from
in vitro translation reactions were isolated on immunobeads using antibodies against the p37 subunit and used directly in the
assay. DNA synthesis carried out with RFC immobilized on immunobeads was dependent on RFC, PCNA, pol
, and HSSB (31). An in
vitro translation reaction not expressing RFC did not support
nucleotide incorporation (Fig. 2 and data not shown).
All RFC complexes isolated from in vitro translation
reactions were formed with a C-terminal fragment of the p140 large
subunit (spanning amino acids 555-1148 of p140) in place of the
full-length large subunit. RFC complexes formed with this p140 fragment
were approximately 5 times more active than the complex formed with
full-length p140 (31). Qualitatively, similar results were obtained
when full-length p140 was used to assemble RFC complexes in the
experiments described below (data not presented).
Fig. 2.
Replication activity of RFC complexes
containing various deleted small subunits. RFC complexes
containing deleted small subunits, as shown in Fig. 1 but containing
the p140 C-terminal fragment instead of full-length p140, were isolated
on immunobeads and assayed for their ability to support
RFC-dependent replication as described under "Materials
and Methods." The amount of replication products formed in each
reaction after 60 min is presented. At this time point all replication
reactions were still proceeding, and results qualitatively similar to
those presented above were obtained at shorter incubation periods.
Reactions were carried out with RFC isolated from HeLa cells
(hRFC control), as well as in the presence of immunobeads
without RFC (no RFC). RFC complexes (40 fmol), isolated from
in vitro translation reactions containing either all
full-length small subunits (no deletion) or the various deleted variants (as indicated), were assayed for replication activity.
The experiment was repeated several times and yielded similar
results.
[View Larger Version of this Image (20K GIF file)]
-catalyzed DNA synthesis, while in the loading assay
as many as 10 molecules of trimeric PCNA on average were loaded onto
each DNA molecule by RFC containing the full-length small subunits.
This may explain the quantitative differences observed using these two
assays.
Fig. 3.
Loading of PCNA onto nicked circular DNA by
RFC containing N-terminally deleted small subunits. RFC complexes
(40 fmol) containing all full-length small subunits (RFC) or one of the N-terminally deleted small subunits p40N56, p37N54, or p36N40 were
isolated from in vitro translation reactions using
immunobeads. Loading 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 (20K GIF file)]
Fig. 4.
RFC complexes formed with p37/p40 chimeric
subunits. A, RFC complex formation with the chimeric
subunits. The chimeric subunits p40N/37C (lane 2) and
p37N/40C (lane 3), after in vitro translation,
were analyzed by SDS-PAGE and compared with the wild-type p40 and p37
subunits (lanes 1 and 4, respectively). RFC
complex formation was detected by immunoprecipitation of a mixture of all five RFC subunits with antibodies against the p36 subunit (lane 5, 10% of the input material; lane 6,
immunoprecipitate with antibodies against p36; lane 7,
immunoprecipitate using preimmune serum). Complex formation with the
chimeric subunits was studied by replacing the p40 subunit with
p37N/40C (lanes 8-10), replacing the p37 subunit with
p40N/37C (lanes 11-13), or replacing both wild-type
subunits with the two chimeras (lanes 14-16). B,
replication activity of RFC complexes containing chimeric subunits. RFC
complexes (40 fmol) containing chimeric or N-terminally deleted
subunits (as indicated) were isolated from in vitro
translation reactions and assayed for activity in an
RFC-dependent elongation reaction as described. The amount
of replication product formed is presented and compared with a reaction
with RFC containing the full-length small subunits (no
deletion).
[View Larger Version of this Image (29K GIF file)]
Fig. 5.
Summary of the small subunit deletions and
their effects on complex formation and RFC replication activity.
The names given to the deleted small subunit variants indicate the
first or last amino acid present at the N or C terminus, respectively, and the number of amino acids in the full-length subunit is indicated in parentheses. The RFC homology boxes are indicated by
Roman numerals. The relative activities given refer to the
replication activity of an RFC complex containing the respective
subunit variant and are compared with a complex assembled with
full-length small subunits (indicated as 1). The
abbreviation N/A indicates not applicable since these
fragments did not form RFC complexes.
[View Larger Version of this Image (19K GIF file)]
in
Escherichia coli, gp45 of bacteriophage T4) and a clamp
loader (RFC,
/
complex in E. coli, gp44/62 complex of T4) (reviewed in Ref. 27). Each of these clamp loaders possesses a
five-subunit structure that is heteropentameric in eukaryotes and in
E. coli. The T4 complex, however, consists of one gp62 subunit and four identical gp44 subunits (28), a striking example of
redundancy of the small subunits. DNA replication proteins in archaea
are related to their eukaryotic counterparts (29). In the genome of the
archaeon Methanococcus jannaschii, whose entire nucleotide
sequence has been determined (29), the genes for PCNA and RFC subunits
have been identified that show a high degree of homology to the
eukaryotic proteins. Interestingly, only two different genes for RFC
subunits are present in the M. jannaschii genome, one with
high homology to the human RFC p140 subunit (37% identity/57%
similarity) and the other most similar to the human small subunit p40
(45% identity/65% similarity). This raises the possibility that the
putative M. jannaschii RFC complex might be formed from one
large subunit homolog and four identical small subunit homologues, a
situation similar to bacteriophage T4, although it involves subunits
closely related to human RFC. Diversification of the four small
subunits may have occurred during evolution in increasingly complex
organisms where additional functions were distributed among the
different single subunits. Evidence has been presented, for example,
that the Saccharomyces cerevisiae Rfc5 gene product (the
homologue of the human RFC p38) is involved in linking DNA replication
and cell cycle control (30). Further functions for the eukaryotic RFC
subunits in signaling, DNA repair, or recombination may be revealed by
future studies.
*
These studies were supported in part 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: pol, polymerase;
RFC, replication factor C; PCNA, proliferating cell nuclear antigen;
HSSB, human single-stranded DNA-binding protein (also called RPA); BSA,
bovine serum albumin; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; 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.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.