From the Department of Molecular Biology, Vanderbilt University, Nashville, Tennessee 37235
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ABSTRACT |
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Replication factor C (RF-C), a complex of five
subunits, and several subassemblies of RF-C, representing intermediates
along the proposed protein assembly pathway (Podust, V. N., and
Fanning, E. (1997) J. Biol. Chem. 272, 6303-6310),
were expressed in insect cells using baculoviruses encoding individual
subunits (p140, p40, p38, p37, and p36). Purified proteins were
analyzed for ATPase activity to assess the role of individual subunits
in ATP hydrolysis. His-tagged p40 contained low ATPase activity, but
tagged p37 and p36 did not. Complexes of p40·p37·p36 bearing a His
tag on any subunit displayed DNA-stimulated ATPase activity, in
agreement with a recent report (Cai, J., Gibbs, E., Uhlmann, F.,
Philips, B., Yao, N., O'Donnell, M., and Hurwitz, J. (1997)
J. Biol. Chem. 272, 18974-18981). In contrast,
complex p38·p37·p36-his displayed no ATPase, suggesting that p40 is
essential for ATPase activity. Although p38 was not required for ATPase
activity, the activity of the p40-his·p38·p37·p36 complex was
more salt-resistant than that of the p40-his·p37·p36 complex. The
p140 subunit further increased the specific ATPase activity of RF-C
complex by enhancing its stimulation by DNA. Taken together, the data
indicate that all five RF-C subunits constitute ATPase activity,
although the contributions of the individual subunits differ. Predicted
ATP-binding domains of all five subunits were mutated to assess the
importance of multiple ATP-binding sites of RF-C. In each case, the Lys
of the conserved P-loop motif was replaced by Glu. The ATP-binding domain of p38 was found to be dispensable for the activity of the
five-subunit RF-C in polymerase DNA synthesis. In contrast, mutation of the ATP-binding domains in other RF-C subunits impaired RF-C assembly, function, or both.
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INTRODUCTION |
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DNA polymerase auxiliary factors
PCNA1 and RF-C are essential
proteins required for both replicative and repair DNA synthesis (1, 2).
PCNA is composed of three identical subunits, which form a torus-like
structure with a central cavity sufficient to accommodate
double-stranded DNA (3, 4). RF-C loads trimeric PCNA molecules onto
DNA, thereby forming a stable complex, called the sliding clamp. The
clamp specifically interacts with pol or pol
to assemble the
corresponding holoenzymes functional in DNA replication (5-8) and DNA
repair (9). The function of RF-C as a PCNA clamp loader is dependent on
its interaction with ATP, which apparently occurs in a complex way.
First, ATP stimulates the binding of RF-C to DNA (10, 11). Second, ATP is absolutely required to form an RF-C-dependent complex of
PCNA with DNA (12). Third, ATP cofactor is required to form a stable salt-resistant complex of RF-C with PCNA in the absence of DNA (13).
ATP
S, a nonhydrolyzable analog of ATP, substitutes for ATP in these
reactions (10-13). However, the RF-C·PCNA·DNA complex formed in
the presence of ATP
S is not competent to interact efficiently with
pol
or pol
to form the corresponding holoenzymes (5, 7). A
change in conformation or perhaps composition of the protein/DNA
intermediate is apparently required to yield the stable functional
clamp and depends on ATP hydrolysis. The mechanism of this conversion
remains to be elucidated.
RF-C is composed of five subunits, one large subunit and four small subunits. The subunits of human RF-C were named according their apparent molecular masses on SDS-PAGE: p140, p40, p38, p37, and p36. The cDNAs encoding all five subunits of human RF-C (14-17) and yeast RF-C (18-21) have been cloned. Each human RF-C subunit corresponds closely to its yeast counterpart (Ref. 21; reviewed in Ref. 22). All human and yeast subunits show extensive amino acid sequence homology (16, 17, 19, 21). One feature of this homology is that each RF-C subunit contains a sequence motif characteristic for NTP-binding/hydrolyzing proteins that consists of two separate units: GXXGXGKT followed by DE(A/V)D (Ref. 23 and references therein). Such a redundancy of NTP binding sites in the five-subunit RF-C complex would be consistent with several models for ATP hydrolysis by RF-C: 1) only one subunit is the putative ATPase, and the other subunits play no role in the binding/hydrolysis of ATP despite the characteristic sequence motifs; 2) each subunit is an ATPase and acts independently of the other subunits; 3) none of the subunits is able to hydrolyze ATP unless a functional subcomplex is assembled; and 4) one subunit is a minimal ATPase, but interaction of two or more subunits is required for ATP hydrolysis and the specific activity of the ATPase depends on the protein complexity.
Recombinant RF-C has been successfully expressed using several approaches. Yeast RF-C has been overexpressed in yeast by using plasmids encoding all five RF-C subunits under the control of inducible promoters (13). Human RF-C has been reconstituted using an in vitro coupled transcription/translation system (24) and a baculovirus expression system (25, 26). Analysis of physical interactions among RF-C subunits suggested two models for assembly of individual subunits into the five-subunit complex (25, 26). One of the intermediates common to the assembly of the five-subunit RF-C complex in both models, the subcomplex p40·p37·p36, has been recently characterized (27). The three-subunit subcomplex, like the five-subunit RF-C, contained DNA-stimulated ATPase. The p40·p37·p36 complex was unable to load PCNA onto DNA but did unload PCNA clamp from singly nicked circular DNA, although 1000-fold less efficiently than the five-subunit RF-C. The three-subunit complex was able to bind primed DNA and PCNA, but again more weakly than the five-subunit complex (27).
Using recombinant human RF-C expressed in the baculovirus system, we
have further investigated the role of individual RF-C subunits in ATP
hydrolysis. The in vitro DNA-stimulated ATPase activity of
various RF-C subunits and RF-C subcomplexes has been analyzed. The
ability of five-subunit RF-C complexes assembled with subunits bearing
mutations in the ATP-binding motifs to stimulate pol holoenzyme DNA
synthesis has also been characterized.
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MATERIALS AND METHODS |
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Proteins and Nucleic Acid
Calf thymus pol and recombinant human PCNA have been
described (8, 11). Monoclonal antibody raised against RFC140 subunit (monoclonal antibody 19; Ref. 16) was kindly provided by B. Stillman
(Cold Spring Harbor). Polyclonal antibody specific to the His tag
sequence MRGSH6 was from Santa Cruz Biotechnology, Inc.
(Santa Cruz, CA). Pwo DNA polymerase was from Boehringer Mannheim; restriction endonucleases and T4 DNA ligase were from Promega. Prestained molecular mass standard protein mixture (195, 112, 84, 63, 52.5, 35, and 32 kDa) was from Sigma. M13(mp19) ssDNA and
singly primed ssDNA was prepared as described (26).
PCNA-Agarose
Recombinant human PCNA was bound to Affi-Gel 10 (Bio-Rad) as described for yeast PCNA (13). Prepared PCNA-agarose contained 3.5 mg of bound PCNA per ml of resin.
Recombinant WT Baculoviruses
Recombinant WT baculoviruses encoding nonfused RF-C subunits (called v140, v40, v38, v37, v36) and N-terminal His tag-fused subunits (v40-his, v37-his and v36-his) have been described (26). Control virus was prepared from unmodified baculovirus transfer vector pBacHisC and encoded a 54-amino acid peptide with a His tag at the N terminus (26).
Construction of Mutant Baculovirus Transfer Vectors
pVL1393/RFC37(K84E)-- The mutation of Lys84 to Glu84 was generated by mismatch PCR using the T7 promoter primer (Promega), the backward primer dGCTGCTGCCAAAATAGTGGATGTTTCTCCAGTTCC, and modified pET/RFC37 plasmid, encoding full-length RFC37 cDNA (26) as a template. The PCR product was digested by BamHI and BstXI. The fragment containing the 5'-end of RFC37(K84E) cDNA was used to replace the BamHI/BstXI fragment in the pET/RFC37. The full-length RFC37(K84E) cDNA was transferred as a BamHI/EcoRI fragment into pVL1393 (Invitrogen).
pBacHis/RFC40(K82E)-- The mutation of Lys82 to Glu82 was generated by mismatch PCR using the forward primer dTCAAGGATCCATATGGAGGTGGAGGCCG, the backward primer dCAGGGCCCGGGCCAAGCACAGAATGCTTGTGGTCTCGCCGGTTC, and the modified pET/RFC40 described in Ref. 26 as a template. The PCR product was digested by BamHI and XmaI. The fragment containing the 5'-end of RFC40(K82E) cDNA was used to replace the WT BamHI/XmaI fragment in the pBacHis/RFC40.
pVL1393/RFC140(K657E)-- The mutation of Lys657 to Glu657 was generated by mismatch PCR using the forward primer dGAATCTCAGCAACATTCC, the backward primer dGACACACCAGGGAAGCTGTGGTGGTTTCGCCAAC, and pSK/RFC140 as a template. The PCR product was digested by SacI and XcmI. The resulting fragment was used to replace the SacI/XcmI fragment in a pKS plasmid carrying the SacI/PstI fragment of RFC140 cDNA. The new plasmid carrying the SacI/PstI fragment mutated at Lys657 was digested with SacI and EcoRI. The corresponding fragment was used to replace the WT SacI/EcoRI fragment in pVL1393/RFC140.
pVL1393/RFC36(K66E)-- The mutation of Lys66 to Glu66 was generated by PCR overlap extension (28) using the primers dCCGGATTATTCATACCGTCC (first forward primer), dGGTAGATGTCTCGCCTGTCC (first backward), dGGACAGGCGAGACATCTACC (second forward), and dGATCGGTCCTCGAATGATGTC (second backward) and pVL1393/RFC36 as a template. The PCR product was digested first with BamHI and then with NcoI. The resulting fragment was used to replace the WT BamHI/NcoI fragment in the pVL1393/RFC36.
pVL1393/RFC38(K48E)-- The mutation of Lys48 to Glu48 was generated by PCR overlap extension (28) using T7 promoter primer (first forward primer), dCTTGTCTTTTCTCCAGCAC (first backward), dGTGCTGGAGAAAAGACAAG (second forward), and dGATGTAGAATTGCAGCAC (second backward) and pET19b/His38 as a template. The PCR product was digested with BamHI and NcoI. The resulting fragment was used to replace the WT BamHI/NcoI fragment in the pVL1393/RFC38.
Growth and maintenance of Sf9 and High Five insect cells (ITC Biotechnology GmbH) in adherent cultures, preparation of recombinant baculoviruses, and infection of the cells were performed as described (26). Infected cells were incubated for 48 h prior to harvesting the recombinant proteins.Purification of RF-C Subunits and Complexes
Purification of Individual Subunits p40-his, p37-his, and p36-his-- 7.6 × 107 cells infected by the corresponding baculoviruses were lysed in 3 ml of buffer A (20 mM Tris-HCl (pH 7.5), 0.2% (v/v) Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml concentration each of aprotinin, leupeptin, and pepstatin) containing 0.1 M NaCl. Cell debris was removed by centrifugation. The supernatant was passed over 0.8 ml of DEAE-Sephacel (Amersham Pharmacia Biotech) equilibrated in lysis buffer, and the column was then washed with 1 ml of the same buffer. The flow through and wash were combined and gently mixed with 50 µl of Ni-NTA resin (QIAGEN) for 2 h at 4 °C. The resin was washed three times in batch with 0.5 ml of buffer B (20 mM Tris-HCl (pH 7.5), 2 mM imidazole-HCl, 300 mM NaCl, 0.02% (v/v) Nonidet P-40), and proteins were eluted with 200 µl of buffer C (300 mM imidazole-HCl (pH 7.2), 0.3 M NaCl, 10% (v/v) glycerol).
Purification of Complexes of Small RF-C Subunits-- To prepare p40-his·p38·p37·p36 and p40-his·p37·p36 complexes, 3 × 108 insect cells infected by corresponding viruses were lysed in 10 ml of buffer A, 0.1 M NaCl. Cell debris was removed by centrifugation, and the supernatant was mixed with 200 µl of Ni-NTA resin. The suspension was mixed for 2 h at 4 °C, and the resin was pelleted by centrifugation, packed into a column, and washed with 4 ml of buffer B. The proteins were eluted from the resin with 2 ml of buffer C. The eluate was diluted 5-fold with buffer Q (20 mM Tris-HCl (pH 8.0), 10% glycerol, 1 mM DTT, 0.5 mM EDTA, 0.01% Nonidet P-40) and loaded onto a 1-ml Mono Q column (Amersham Pharmacia Biotech). Proteins were eluted with a 20-ml gradient of NaCl from 0 to 350 mM in buffer Q and collected in 0.4-ml fractions.
p40·p37-his·p36 and p40·p37·p36-his were expressed using the corresponding viruses and purified analogously to p40-his·p37·p36. Regardless of which of the three subunits was His-tagged, the trimeric complex eluted from the Mono Q column in the same fractions (fractions 33 and 34). To prepare the p38·p37·p36-his complex, 1.5 × 108 insect cells infected by v38, v37, and v36-his were lysed in 5 ml of buffer A, 0.1 M NaCl, and proteins were bound to 100 µl of Ni-NTA resin. The resin was washed three times in batch with 1 ml of buffer B, and proteins were eluted with 150 µl of buffer C.Purification of Five-subunit RF-C-- 4.2 × 108 insect cells infected with viruses encoding all five RF-C subunits (v140, v40-his, v38, v37, and v36-his) were lysed in 12 ml of buffer A, 0.35 M NaCl. Cell debris was removed by centrifugation, and the supernatant fraction bound to 300 µl of Ni-NTA resin. The suspension was mixed for 2 h at 4 °C, and the resin was pelleted by centrifugation, packed into a column, and washed with 4 ml of buffer B. The proteins were eluted from the resin with 2 ml of buffer C. The eluate was diluted with an equal volume of buffer D (50 mM Tris-HCl (pH 7.5), 10% glycerol, 1 mM DTT). MgCl2 and ATP were added to final concentrations of 10 mM and 1 mM, respectively. The solution was mixed with 200 µl of PCNA-agarose for 2 h at 4 °C. The resin was then packed into a column and washed with 4 ml of buffer D containing 10 mM MgCl2, 1 mM ATP and 300 mM NaCl. Protein was eluted with buffer containing 30 mM Tris-HCl (pH 7.5), 10% glycerol, 400 mM NaCl, 1 mM DTT, and 2 mM EDTA.
Pol Holoenzyme Assay
Reaction mixtures (final volume of 25 µl) contained 40 mM Tris-HCl (pH 7.5); 0.2 mg/ml bovine serum albumin; 1 mM DTT; 10 mM MgCl2; 1 mM ATP; a 50 µM concentration each of dATP,
dGTP, and dTTP; 20 µM [-32P]dCTP (500 cpm/pmol); 100 ng of primed ssDNA; 100 ng of PCNA; 1.2 µg of
Escherichia coli single-stranded DNA-binding protein; 0.25 units of pol
; and RF-C as indicated in the figures. Samples were
incubated for 30 min at 37 °C, the reactions were terminated by
adding 1 ml of ice-cold 10% (w/v) trichloroacetic acid, and acid-insoluble material was analyzed by scintillation counting. One
unit of RF-C activity was defined as the incorporation of 1 nmol of
total dNMP into singly primed ssDNA in the presence of pol
, PCNA,
and E. coli single-stranded DNA-binding protein in 30 min at
37 °C. For product length analysis, the reactions were terminated by
treating them with proteinase K (60 µg/ml) for 30 min at 37 °C in
the presence of 1% (w/v) SDS and 20 mM EDTA (pH 8.0). The
DNA was then precipitated with ethanol, and the products analyzed on an
alkaline 1.5% agarose gel as described (29).
ATPase Assay
Reaction mixtures (final volume of 10 µl) contained 20 mM Tris-HCl (pH 7.5), 0.1 mg/ml bovine serum albumin, 0.5 mM DTT, 10 mM MgCl2, 50 µM [-32P]ATP (1 Ci/mmol), 200 ng of
M13(mp19) ssDNA, and protein to be tested. Samples were incubated for
10 min at 37 °C, and reactions were terminated with 10 µl of
ice-cold 50 mM EDTA. 2 µl of reaction mixtures were
spotted on a polyethyleneimine-cellulose thin layer plate, which was
developed in 1 M LiCl, 0.5 M formic acid. The amounts of [
-32P]-ATP hydrolyzed to
[32P]orthophosphate were quantified using a
PhosphorImager (Molecular Dynamics, Inc.). The rates of ATP hydrolysis
were determined in the linear range of reaction time and protein
concentration dependence.
Stability of RF-C Subunits and Complexes
To test the stability of the three- and four-subunit RF-C subcomplexes under the conditions employed to measure the ATPase activity, 10-20 µg of Mono Q-purified p40-his·p37·p36 or p40-his·p38·p37·p36 complexes were bound to 25 µl of Ni-NTA resin. Beads were washed with 1 ml of buffer E (20 mM Tris-HCl (pH 7.5), 0.02% (v/v) Nonidet P-40) containing 300 mM NaCl and then resuspended in 1 ml of buffer E and divided into two equal parts. Supernatant was removed by aspiration, and the beads were resuspended in 200 µl of ATPase reaction mixture (DTT was omitted; ATP was nonradioactive). The suspensions were incubated for 10 min on ice or at 37 °C with periodical gentle agitation. Then the resin was pelleted by centrifugation at 0 or 37 °C, respectively, the supernatant was quickly aspirated, and the beads were resuspended in 30 µl of elution buffer C. Eluted proteins were analyzed by SDS-PAGE followed by Coomassie staining.
To test the stability of the five-subunit RF-C, 3.8 × 107 insect cells were infected by the corresponding baculoviruses, and expressed proteins were bound to 25 µl of Ni-NTA resin. The beads were washed with buffer B, resuspended in buffer E, and divided into two equal parts. The resin with bound proteins was incubated for 10 min at 0 or 37 °C with 200 µl of mixture containing 40 mM Tris-HCl (pH 7.5), 0.2 mg/ml bovine serum albumin, 10 mM MgCl2, 1 mM ATP, and 4 µg/ml PCNA. Then the resin was pelleted by centrifugation at 0 or 37 °C, respectively, the supernatant was aspirated, and the beads were resuspended in 30 µl of elution buffer C. Eluted proteins were analyzed by SDS-PAGE. The lower half of the gel was stained with Coomassie to visualize small RF-C subunits. The upper half of the gel was blotted to nitrocellulose membrane and analyzed by immunoblotting with antibody against p140 (monoclonal antibody 19).
Other Methods
Western blot analysis was performed as described (26). Protein concentration was determined by densitometric scanning of Coomassie-stained protein bands in denaturing polyacrylamide gels using Image Store 7500 (Ultra-Violet Products, Inc.). As protein standards, known amounts of bovine serum albumin were loaded onto the same gel.
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RESULTS |
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Characterization of a Complex of Four Small RF-C Subunits--
We
have recently analyzed the pathway of assembly of RF-C from individual
subunits and proposed that in the last step, the large subunit p140
binds to a preformed complex of four small subunits (26).
Characterization of this four-subunit complex showed that it lacked
most of the properties of RF-C. It was inactive in the PCNA loading and
pol DNA synthesis reactions. Like the three-subunit complex
p40·p37·p36 (27), the four-subunit complex interacted very weakly
with DNA and PCNA (data not shown). The results suggest that p140 is
required for stable interaction of RF-C with DNA and PCNA.
ATPase Activity of Individual RF-C Subunits, Subcomplexes, and Five-subunit RF-C-- His-tagged small RF-C subunits p40-his, p37-his, and p36-his can be expressed as soluble proteins and purified in ample amounts (26) (Fig. 1A). As a control to distinguish the putative ATPase activity of expressed RF-C subunits from possible host protein contaminants, the same amount of cells used to prepare individual RF-C subunits was infected with control virus, and cellular extract was purified according to the protocol for p40-his, p37-his, and p36-his (Fig. 1A, control preparation lane). Analysis of individual subunit preparations for ATPase activity showed that only p40-his displayed very weak ATPase activity (0.017 mol ATP hydrolyzed per mol of p40-his in 1 min), and this activity was not dependent on ssDNA (Table I).
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Rationale for Mutation of ATP-binding Motifs--
The results
above suggested that each of the subunits of RF-C may contribute to its
ATPase activity to some extent. To confirm this interpretation and to
assess the importance of the multiple ATP-binding motifs of the RF-C
subunits, we created mutations in the P-loop of each subunit. The
invariant Lys of the P-loop motif is thought to bind - and
-phosphates of the ATP molecule (Refs. 23, 30, and 31 and references
therein). To inactivate the P-loop, this essential amino acid was
replaced by Glu.
Analysis of the p37 ATP-binding Site Mutant-- p40-his·p38·p37(K84E)·p36 was expressed and purified in the same way as the WT four-subunit complex (Fig. 2A). Four-subunit complexes formed with p37WT and p37(K84E) were indistinguishable in polypeptide composition (Fig. 2B). Therefore, the K84E mutation in the ATP-binding motif of p37 did not impair the ability of this subunit to assemble with other RF-C subunits. The four-subunit complexes containing WT or mutant p37 subunits were both stable and showed no dissociation under the conditions used to measure ATPase activity (data not shown).
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Analysis of the p40 ATP-binding Site Mutant-- To analyze the role of the p40 ATP-binding site in the activities of RF-C, Lys82, of p40 (analogous to Lys84 of p37) was replaced by Glu. Expression and purification of p40(K82E)-his or complexes containing this subunit resulted in a very low yield of purified proteins in comparison with p40WT-his (not shown). In an effort to solve this problem, we tested whether the low yield of soluble mutant protein was due to poor expression or decreased solubility of the expressed subunit. Insect cells were infected with v40-his or v40(K82E)-his, and soluble and insoluble cellular fractions were analyzed by SDS-PAGE followed by Western blot using polyclonal antibody specific for the His tag sequence. p40WT-his was found to be largely soluble. In contrast, p40(K82E)-his, although well expressed, was mostly insoluble. To minimize the possible structural distortions caused by the mutation, a mutant bearing Ala instead of Lys in the P-loop of p40-his was also constructed, but most of this protein was also insoluble (data not shown).
To test whether the minor fraction of soluble p40(K82E)-his could be assembled into five-subunit RF-C complex, insect cells were co-infected with v140, v38, v37, and v36-his and either v40WT-his or v40(K82E)-his. RF-C complexes were isolated using Ni-NTA resin and analyzed by Western blot using antibody against p140. Omission of either p40-his or p36-his during the co-infection resulted in the complete absence of p140 (Fig. 4A, lanes 1 and 2), as expected (26). Infection of the cells with five baculoviruses, including v40(K82E)-his, yielded a protein preparation containing p140, suggesting that the soluble fraction of p40(K82E)-his was able to assemble in the five-subunit complex. However, the amount of isolated 5xRF-C (p40 K82E) was estimated to be about one-tenth of the amount of the 5xRF-C WT (Fig. 4A, lanes 3 and 4).
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Analysis of the p140 ATP-binding Site Mutant--
Insect cells
were infected with v40-his, v38, v37, v36-his, and v140WT or
v140(K657E). Replacement of v140WT with v140(K657E) did not affect
expression and co-purification of the four small subunit complex, but
much lower amounts of p140(K657E) were detected by Western blot in the
purified preparations in comparison with p140WT (Table II; data not
shown). As with 5xRF-C (p40 K82E), 5xRF-C (p140 K657E) did not
detectably stimulate pol DNA synthesis (Table II).
Analysis of the p36 ATP-binding Site Mutant--
Insect cells were
infected with v140, v40-his, v38, v37, and v36WT or v36(K66E).
Replacement of v36WT with v36(K66E) decreased the yield of the
five-subunit RF-C complex (Table II; data not shown). However, the
5xRF-C (p36 K66E) displayed weak but detectable activity in pol DNA
synthesis. After correction for the relative yield of mutant RF-C of
10%, the activity of 5xRF-C (p36 K66E) was measured to be 3% of the
activity determined for 5xRF-C WT in parallel (Table II).
Analysis of the p38 ATP-binding Site Mutant--
When insect cells
were infected with v40-his, v38 (K48E), v37, and v36, only the trimer
p40-his·p37·p36 could be isolated, while infection using v38WT
instead of the mutant v38 resulted in stoichiometric tetramer (Fig.
5A). In contrast, when the
cells were infected with five viruses including v140, the yield of
mutant 5xRF-C (p38 K48E) was close to that observed for 5xRF-C WT (Fig. 5B). p140 thus appeared to stabilize the defective p38
subunit within the five-subunit complex. Analysis of
RF-C-dependent pol DNA synthesis revealed that 5xRF-C
(p38 K48E) was functionally active (Fig. 5C). The initial
slope of the dependence of DNA synthesis on concentration of 5xRF-C
(p38 K48E) was 75% of that for 5xRF-C WT, while at saturating amounts
of 5xRF-C, DNA synthesis was the same with both proteins. The results
demonstrate that the ATP-binding motif of the p38 subunit was not
required for RF-C activity. Salt sensitivity of pol
DNA synthesis
catalyzed by 5xRF-C WT and 5xRF-C (p38 K48E) was essentially the same
(data not shown), indicating that the ability of p38 to increase salt
resistance of ATPase activity did not require an intact P-loop
motif.
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DISCUSSION |
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The function of RF-C in DNA replication is to catalyze loading of
the PCNA trimer onto DNA to form a so-called sliding clamp, which
tethers pol and pol
to the site of DNA synthesis (reviewed in
Refs. 22 and 32). During assembly of the clamp, RF-C interacts with
DNA, PCNA, and ATP. Elucidation of the mechanism of clamp loading will
require an understanding of the roles of RF-C subunits in these
interactions. In this study, we have measured the ATPase activity of
RF-C subunits and complexes and its DNA dependence and probed the
functional role of the ATP-binding motif in each subunit by
mutagenesis.
To facilitate purification of the individual subunits and different
RF-C (sub)complexes, His-tagged subunits have been used. The
possibility that the His tags influenced ATPase measurements seems to
be unlikely for the following reasons. First, the five-subunit RF-C
complex assembled with WT His-tagged subunits showed no defects in
RF-C-dependent pol DNA synthesis (Figs. 3-5 and data
not shown; Ref. 25). Second, the ATPase activity of the
p40-his·p37·p36 complex and DNA stimulation of its ATPase
determined in this study (Table I) are essentially the same as those
determined for untagged p40·p37·p36 complex (27). Third, the
DNA-stimulated ATPase activity of p40·p37·p36 was not dependent on
which subunit was His-tagged (Table I). Therefore, no significant
effect of the His-tags on the biochemical properties of any RF-C
complexes has been detected so far, suggesting that it is likely to be
minimal.
The p40 subunit appears to play an important role in ATP hydrolysis. Among the p40, p37, and p36 subunits, only p40 possessed weak ATPase activity, and only subcomplexes containing p40 were active as an ATPase (Table I). Consistent with these results, only p40 was reported to be cross-linked with ATP (11, 14). We also tested for ATP cross-linking to tetrameric complex p40-his·p38·p37·p36 and individual subunits p40-his, p37-his, and p36-his. The results confirmed that only p40 could be labeled with [32P]ATP (data not shown).
The specific ATPase activity of the three-subunit complex p40-his·p37·p36 (0.15 and 4.2 mol of ATP hydrolyzed per mol of complex per min in the absence and presence of DNA, respectively) was significantly higher than that of p40-his, indicating that interaction among RF-C subunits is essential for ATPase activity, confirming the report of Cai et al. (27). However, our comparison of the ATPase activities of p40-his·p38·p37·p36 and p40-his·p37·p36 complexes has also revealed a possible role of the p38 subunit, which was not apparent when the DNA-stimulated ATPase activity of the complexes was measured; p38 enhanced ATPase activity 8.7-fold in the absence of DNA. Moreover, p38 increased the salt resistance of ATPase activity, arguing that this subunit contributes to the RF-C ATPase and stimulates its function.
Inclusion of p140 in the complex of the four small subunits did not increase the ATPase specific activity in the absence of DNA. In contrast, DNA-stimulated ATPase activity was further enhanced by the presence of p140, such that the resulting 8.6-fold DNA stimulation of the ATPase activity of five-subunit recombinant complex was equivalent to that of natural RF-C (8, 10). Taken together, the biochemical analysis of ATP hydrolysis indicates that all five RF-C subunits constitute ATPase activity, although the contribution of the subunits is different.
To further substantiate this conclusion, we mutated the predicted ATP-binding sites in all five subunits. Mutation of the invariant Lys in the P-loops of p140, p40, and p36 strongly impaired their ability to assemble into the five-subunit complex, arguing that intact ATP-binding sites are essential not only for function but also for the structure of these proteins. In contrast, the K84E mutation in p37 had no significant effect on the assembly of five-subunit RF-C, although its P-loop motif is very similar to those of the other four subunits. Curiously, although the P-loop mutation in p38 did not significantly impair assembly of the five-subunit complex, a stable four-subunit complex with mutant p38 could not be isolated. We suggest that the mutant p38 subunit, in contrast to the WT subunit, binds too weakly to p40·p37·p36 complex and is lost upon protein purification. However, the unstable mutant four-subunit complex may be sufficient to serve as a platform for binding of p140, which in turn stabilizes the mutant five-subunit complex.
Mutation of the ATP-binding sites in p140, p40, p37, and p36 resulted in the inactivation of RF-C activity in DNA synthesis, suggesting that multiple ATP binding/hydrolysis events are required for RF-C function. The only subunit whose ATP-binding motif was not essential for RF-C activity was p38 (Table II). The requirement of four ATP-binding motifs for the activity of the five-subunit clamp loader is reminiscent of the bacteriophage T4 counterpart of human RF-C. The ATPase motifs of the gp62/44 complex reside on the four gp44 subunits of the complex, and indeed, four ATP molecules bind and are hydrolyzed in each gp45 clamp loading event (33, 34).
In summary, we have shown that multiple interactions among the five RF-C subunits potentiate and modulate its ability to hydrolyze ATP. Mutational analysis of the P-loop motifs in each subunit has demonstrated that the predicted ATP-binding sites of all subunits except for p38 are essential for RF-C function.
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ACKNOWLEDGEMENTS |
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We thank Lynda O'Rear for excellent technical assistance and Bruce Stillman for monoclonal antibody against p140.
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FOOTNOTES |
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* This work was supported by Vanderbilt University, National Institutes of Health Grant GM 52948, and a shared equipment grant from the National Science Foundation (BIR-9419667).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.
To whom correspondence should be addressed: Dept. of Molecular
Biology, Vanderbilt University, Box 1820, Station B, Nashville, TN
37235. Tel.: 615-343-5677; Fax: 615-343-6707; E-mail:
fannine{at}ctrvax.vanderbilt.edu.
1
The abbreviations used are: PCNA, proliferating
cell nuclear antigen; RF-C, replication factor C; pol, DNA polymerase;
ssDNA, single-stranded DNA; DTT, dithiothreitol; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; Ni-NTA,
nickel-nitrilotriacetic acid; p140, p40, p38, p37, and p36, recombinant
human RFC140, RFC40, RFC38, RFC37, and RFC36 subunit, respectively;
v140, v40, v38, v37, and v36, recombinant baculovirus encoding p140,
p40, p38, p37, and p36 subunit, respectively; -his, His-tag N-terminal fusion; WT, wild type; ATPS, adenosine
5'-O-(thiotriphosphate).
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REFERENCES |
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