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Originally published In Press as doi:10.1074/jbc.M200065200 on May 1, 2002

J. Biol. Chem., Vol. 277, Issue 27, 24340-24345, July 5, 2002
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Direct Interaction of Proliferating Cell Nuclear Antigen with the Small Subunit of DNA Polymerase delta *

Xiaoqing LuDagger , Cheng-Keat TanDagger , Jin-Qiu ZhouDagger §, Min You, L. Michael Carastro||, Kathleen M. DowneyDagger , and Antero G. SoDagger **

From the Departments of Dagger  Medicine and  Biochemistry and Molecular Biology, University of Miami School of Medicine, Miami, Florida 33101

Received for publication, January 3, 2002, and in revised form, April 8, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The interaction between proliferating cell nuclear antigen (PCNA) and DNA polymerase delta  is essential for processive DNA synthesis during DNA replication/repair; however, the identity of the subunit of DNA polymerase delta  that directly interacts with PCNA has not been resolved until now. In the present study we have used reciprocal co-immunoprecipitation experiments to determine which of the two subunits of core DNA polymerase delta , the 125-kDa catalytic subunit or the 50-kDa small subunit, directly interacts with PCNA. We found that PCNA co-immunoprecipitated with human p50, as well as calf thymus DNA polymerase delta  heterodimer, but not with p125 alone, suggesting that PCNA directly interacts with p50 but not with p125. A PCNA-binding motif, similar to the sliding clamp-binding motif of bacteriophage RB69 DNA polymerase, was identified in the N terminus of p50. A 22-amino acid oligopeptide containing this sequence (MRPFL) was shown to bind PCNA by far Western analysis and to compete with p50 for binding to PCNA in co-immunoprecipitation experiments. The binding of p50 to PCNA was inhibited by p21, suggesting that the two proteins compete for the same binding site on PCNA. These results establish that the interaction of PCNA with DNA polymerase delta  is mediated through the small subunit of the enzyme.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

DNA polymerase delta  (pol delta ),1 the principal DNA replicase in eukaryotes, also participates in several DNA repair pathways, including nucleotide excision repair, mismatch repair, and long patch base excision repair (1, 2). For both replication and repair functions, pol delta  requires an accessory protein, the proliferating cell nuclear antigen (PCNA), to carry out highly processive DNA synthesis (3, 4). Crystallographic studies have demonstrated that PCNA forms a homotrimeric ring that encircles double-stranded DNA and functions to tether the DNA polymerase to its template/primer, thus dramatically increasing the processivity of the enzyme (5, 6).

In addition to functioning as a processivity factor for pol delta  (7, 8), PCNA also interacts with other replication/repair proteins such as DNA ligase 1 (9), the structural flap endonuclease 1 (10), and replication factor C (11), as well as with several repair proteins, e.g. the mismatch repair proteins MSH3 and MSH6 (12, 13) and the nucleotide excision repair endonuclease XPG (14). PCNA also interacts with proteins that are involved in postreplication processes such as DNA (cytosine-5) methyl transferase (15) and chromatin assembly factor 1 (CAF1) (16, 17), as well as with several proteins that are involved in cell cycle control, e.g. the cyclin-dependent kinase inhibitors p21WAF1,CIP1,SDI (18-20) and p57 (21) and the DNA damage response protein GADD45 (22). Many of these proteins have been shown to contain a consensus PCNA-binding motif initially identified in p21 (23) that specifically binds at the interdomain connector loop of PCNA (6), suggesting that PCNA may coordinate DNA replication with DNA repair as well as with cell cycle progression by functioning as a regulatory target (1, 24).

Despite the fact that PCNA was identified as a processivity factor for pol delta  and a replication protein essential for in vitro SV40 DNA replication nearly 15 years ago (8, 25), the site on pol delta  that interacts with PCNA is still unresolved, as is the quaternary structure of the pol delta  species that interacts with PCNA. Purified pol delta  from calf thymus tissue has been shown to be a heterodimer comprised of a catalytic subunit of 125 kDa containing the DNA polymerase and 3' to 5' exonuclease active sites, and a 50-kDa subunit of unknown function (26, 27). The heterodimeric core enzyme (p125/p50), which is essentially distributive, can be transformed into a highly processive DNA polymerase by PCNA (8, 28), suggesting that functional interaction of pol delta  with PCNA is mediated through interaction with either the 125- or the 50-kDa subunit of the enzyme or with both. In contrast, highly purified pol delta  from Schizosaccharomyces pombe is composed of four subunits: the homologues of p125 (Pol3/Cdc6) and p50 (Cdc1) and two additional subunits, Cdc27 and Cdm 1 (29). In Saccharomyces cerevisiae, active pol delta  can be isolated either as a heterodimer composed of the homologues of mammalian p125 and p50, i.e. Pol3 and Pol31, or as a heterotrimer with, in addition to Pol3 and Pol31, Pol32, the homologue of S. pombe Cdc27 (30). Interestingly, although cdc27+ is an essential gene in S. pombe (31), POl32 is not an essential gene in S. cerevisiae (30). Recently, it was reported that a homologue of S. pombe Cdc27 or S. cerevisiae Pol32 is present in preparations of mammalian pol delta , i.e. p66 (32).

Studies aimed at identifying the subunit of pol delta  that interacts with PCNA have produced conflicting answers. Binding studies using recombinant human p125 and PCNA have demonstrated direct interaction between these two polypeptides by protein overlay experiments using biotinylated PCNA and by biochemical cross-linking experiments (33); however, pull-down assays using PCNA-linked beads have yielded both positive (34) and negative (35) results with respect to the binding of PCNA and p125. Similar studies with S. cerevisiae (30, 36) and S. pombe (37, 38) pol delta  failed to detect any interaction between PCNA and the catalytic subunits of the yeast enzymes. The small subunits of pol delta  from both budding yeast (Pol31) and mammalian (p50) sources were found not to directly bind PCNA (30, 33, 34, 36).

A third subunit of pol delta , initially identified in S. pombe (Cdc27), was found to interact with Cdc1 (homologue of mammalian p50 and S. cerevisiae Pol31) and to bind PCNA through a consensus PCNA-binding motif, suggesting that the third subunit mediates the interaction between pol delta  and PCNA (31, 38). Homologues of Cdc27 in S. cerevisiae (Pol32) and mammalian cells (p66) also contain a consensus PCNA-binding motif (30, 32), and Pol32 has also been found to interact with both Pol31 and PCNA (30).

Functional studies with recombinant p125 from human (28, 39, 40), mouse (41), and S. pombe (42) sources have shown that DNA synthesis catalyzed by p125 alone is not significantly stimulated by PCNA. On the other hand, PCNA was shown to stimulate the activity and processivity of the recombinant human heterodimer to the same extent as the native two-subunit enzyme isolated from calf thymus (28), suggesting the possibility that the interaction of PCNA with pol delta  is mediated through the small subunit. Similarly, heterodimeric pol delta  from S. cerevisiae (Pol3/Pol31) was found to be highly processive in the presence of PCNA (43). In fact, heterodimeric pol delta  was found to be as processive as heterotrimeric pol delta  (Pol3/Pol31/Pol32), the only difference being that the latter enzyme required considerably lower concentrations of PCNA for highly processive synthesis (43). In contrast, recent functional studies in which recombinant human enzymes containing either two subunits (p125/p50) or three subunits (p125/p50/p66) were directly compared demonstrated that only the three-subunit enzyme could be stimulated by PCNA (34, 35). The reasons for these discrepancies are not clear. In the present study we have used antibodies to the individual subunits of mammalian pol delta  and PCNA to examine the interactions among these polypeptides by co-immunoprecipitation to identify the PCNA-binding site on pol delta .

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Plasmids-- The plasmids pT7/hPCNA and pETp21His were the kind gifts of Drs. Bruce Stillman and Shou Waga (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). The plasmid pET16b-p50 was constructed by subcloning the human p50 cDNA (44) into the NdeI site of pET16b (Novagen).

Proteins-- Calf thymus pol delta  was purified through step 7 as described by Downey and So (45). Recombinant human p125 and p50 were overexpressed in Sf9 cells using the baculoviruses AcN-p125-14 and AcN-p50-1 and purified as described previously (28, 39). Recombinant human PCNA was produced in Escherichia coli using the plasmid pT7/hPCNA and purified as described in Brush et al. (46). Polyhistidine-tagged p21 (His-p21) was expressed in E. coli using the plasmid pETp21His and purified according to Waga et al. (19). Polyhistidine-tagged p50 (His-p50) was expressed in E. coli using the plasmid pET16b-p50. The fusion protein was purified by binding to a nickel-chelate-nitrilotriacetic acid column (Qiagen) in 50 mM Tris-HCl, pH 7.8, 7.5% glycerol, and 2 mM phenylmethylsulfonyl fluoride. After washing with the same buffer containing 40 mM imidazole, His-p50 was eluted with the same buffer containing 400 mM imidazole and dialyzed against phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.5) containing 2 mM dithiothreitol.

Antibodies for Immunoprecipitation-- Human autoantiserum to PCNA was a generous gift of Dr. Irving Kushner (Cleveland Metropolitan General Hospital, Cleveland, OH). Rabbit antibodies to the N-terminal one-third of human p125, expressed as a glutathione S-transferase fusion protein, were prepared as described previously (28). Polyclonal antibodies to His-p50 were raised in rabbits. IgGs from human anti-PCNA, rabbit anti-Hisp50, and rabbit anti-glutathione S-transferase p125 were purified on protein A-Sepharose 4B (Sigma) and then coupled to protein A-Sepharose 4B beads using dimethyl pimelidate (Pierce) according to Harlow and Lane (47). Control beads were prepared by cross-linking preimmune rabbit IgG or normal human IgG to protein A-Sepharose beads.

Antibodies for Immunoblotting-- Monoclonal anti-PCNA antibody (PC-10) was from Sigma. Monoclonal anti-p125 antibody (clone 22) was from BD Bioscience. Chicken anti-p50 antibodies were prepared by Alpha Diagnostic International (San Antonio, TX). Peroxidase-conjugated rabbit anti-mouse IgG and peroxidase-conjugated donkey anti-chicken IgY were from Jackson Immunoresearch (West Grove, PA).

Immunoprecipitation and Immunoblot Analysis-- Purified recombinant proteins were mixed with antibody-linked protein A-Sepharose beads at 4 °C for 3 h in phosphate-buffered saline containing 0.2% Nonidet P-40 (200 µl of total volume). After extensive washing with the same buffer, the beads were mixed with an equal volume of 1× Laemmli SDS sample buffer and denatured at 90 °C for 10 min. The proteins in the supernatants were separated by 10% SDS-PAGE and electroblotted onto a nitrocellulose membrane (Bio-Rad). After incubation for 2 h at room temperature with blocking buffer, Tris-buffered saline (25 mM Tris-HCl, pH 7.5, 150 mM NaCl) containing 5% nonfat dry milk, the membrane was incubated with primary antibody for 2 h at room temperature in the same buffer, washed extensively with Tris-buffered saline containing 0.05% Tween 20 (TBST), and incubated with peroxidase-conjugated secondary antibody for 1 h. After extensive washing with TBST, the proteins were detected by SuperSignal enhanced chemiluminescent substrate (Pierce).

Far Western Analysis-- Peptides or proteins were slot-blotted onto a polyvinylidene difluoride membrane (Bio-Rad) and processed as described by He et al. (48).

Peptides-- Wild type p50 peptide (amino acids 51-72) (AHIYATRLIQMRPFLENRAQQH), mutated p50 peptide (F64A,L65A) (AHIYATRLIQMRPAAENRAQQH), an unrelated peptide (AGSYIVPEDKREMWMACIKEAA), and p21 peptide (amino acids 139-160) (GRKRRQTSMTDFYHSKRRLIFS) were synthesized by ResGen (Huntsville, AL).

Protein Determination-- The protein concentration was determined using the Bio-Rad DC protein assay kit with bovine serum albumin as a standard.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

PCNA Interact with p50 and Pol delta  but Not with p125-- To identify the subunit(s) of pol delta  that interact(s) with PCNA, we have used purified recombinant human p125, p50, and PCNA and polyclonal antibodies to each of these polypeptides linked to protein A-Sepharose beads to detect co-immunoprecipitation of PCNA with one or both of the subunits of the pol delta  core enzyme. As shown in Fig. 1A, anti-PCNA beads co-immunoprecipitated p50 in the presence but not in the absence of PCNA, and the amount of p50 co-immunoprecipitated increased with increasing amounts of PCNA in the reaction mixture. To confirm the association of PCNA with p50, reciprocal immunoprecipitation experiments were carried out using anti-p50 polyclonal antibodies. As shown in Fig. 1B, anti-p50 beads co-immunoprecipitated PCNA in the presence but not in the absence of p50. These experiments provide strong evidence for a direct interaction of p50 and PCNA.


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  		Fig. 1.   PCNA interacts with p50 and pol delta  but not with p125. A, anti-PCNA beads were mixed with 200 ng of p50 and varying amounts of PCNA (lanes 2-4). The samples were processed as described under "Experimental Procedures." Lane 1, input p50 (100 ng); lane 2, 50 ng of PCNA; lane 3, 100 ng of PCNA; lane 4, 150 ng of PCNA; lane 5, anti-PCNA beads and 200 ng of p50 (no PCNA). p50 was detected with chicken anti-p50 antibody. B, anti-p50 beads were mixed with 150 ng of PCNA and varying amounts of p50 (lanes 2-4). The samples were processed as described under "Experimental Procedures." Lane 1, input PCNA (75 ng); lane 2, 50 ng of p50; lane 3, 100 ng of p50; lane 4, 200 ng of p50; lane 5, anti-p50 beads and 150 ng of PCNA (no p50). PCNA was detected with monoclonal antibody PC10. C, anti-p125 beads were mixed with PCNA and either p125 (lane 2) or pol delta  (lane 3) and processed as described under "Experimental Procedures." Lane 1, input PCNA (100 ng); lane 2, 300 ng of PCNA and 250 ng of p125; lane 3, 300 ng of PCNA and 340 ng of pol delta ; lane 4, anti-p125 beads and 300 ng of PCNA (no p125, no pol delta ). PCNA was detected with monoclonal antibody PC10. D, anti-PCNA beads were mixed with PCNA and either p125 (lane 3) or pol delta  (lane 4) and processed as described under "Experimental Procedures." Lane 1, input p125 (50 ng); lane 2, input pol delta  (80 ng); lane 3, 300 ng of PCNA and 250 ng of p125; lane 4, 300 ng of PCNA and 340 ng of pol delta ; lane 5, anti-PCNA beads and 250 ng of p125 (no PCNA); lane 6, anti-PCNA beads and 340 ng of pol delta  (no PCNA). p125 was detected with monoclonal anti-p125 (clone 22).

As shown in Fig. 1C, anti-p125 antibodies failed to co-immunoprecipitate PCNA in the presence of purified p125; however, anti-p125 antibodies effectively co-immunoprecipitated PCNA in the presence of calf thymus pol delta , i.e. when p125 is complexed with p50 in the heterodimeric core enzyme. Reciprocal experiments were carried out to further substantiate that the interaction of PCNA with p125 is mediated through p50. As shown in Fig. 1D, anti-PCNA beads failed to co-immunoprecipitate recombinant p125 in the presence of PCNA but efficiently co-immunoprecipitated p125 when it was complexed with p50 in native calf thymus pol delta . These experiments suggest that the interaction of PCNA with p125 is mediated through p50.

Interaction of p50 with PCNA Is Inhibited by p21-- It has been shown that p21 and human pol delta  both bind to the interdomain connector loop of PCNA (6, 49) and that p21 inhibits pol delta -catalyzed DNA synthesis in vitro (19, 20). If the interaction of PCNA with pol delta  is indeed mediated through p50, one would expect that the binding of p50 to PCNA would be disrupted by the addition of p21. That this is the case is shown in Fig. 2. The addition of molar ratios of p21 to PCNA monomer greater than 1:1 resulted in inhibition of binding of p50 to PCNA, and complete disruption was achieved when the molar ratio of p21 to PCNA exceeded 3:1 (Fig. 2A). The addition of an oligopeptide containing the p21 PCNA-binding motif also inhibited the binding of PCNA to p50 (Fig. 2B), and the p21 peptide was nearly as efficient as the p21 protein in inhibiting PCNA binding to p50, i.e. binding was ~50% inhibited at 22 nM p21 protein and 35 nM p21 peptide (Fig. 2C).


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  		Fig. 2.   p21 inhibits the interaction of PCNA and p50. A, anti-PCNA beads were mixed with PCNA, p50, and varying amounts of His-p21 and processed as described under "Experimental Procedures." Lane 1, input p50 (100 ng); lane 2, 200 ng of p50 and 84 ng of His-p21 (no PCNA); lane 3, 120 ng of PCNA and 200 ng of p50; lane 4, 120 ng of PCNA, 200 ng of p50, and 42 ng of His-p21; lane 5, 120 ng of PCNA, 200 ng of p50, and 84 ng of His-p21; lane 6, 120 ng of PCNA, 200 ng of p50, and 126 ng of His-p21; lane 7, 120 ng of PCNA, 200 ng of p50, and 168 ng of His-p21; lane 8, 120 ng of PCNA, 200 ng of p50, and 210 ng of His-p21. p50 was detected with chicken anti-p50 antibody. B, anti-PCNA beads were mixed with PCNA, p50, and varying amounts of p21 peptide and processed as described under "Experimental Procedures." Lane 1, input p50 (100 ng); lane 2, 120 ng of PCNA and 200 ng of p50; lane 3, 120 ng of PCNA, 200 ng of p50, and 10 ng of p21 peptide; lane 4, 120 ng of PCNA, 200 ng of p50, and 20 ng of p21 peptide; lane 5, 120 ng of PCNA, 200 ng of p50, and 40 ng of p21 peptide; lane 6, 120 ng of PCNA, 200 ng of p50, and 60 ng of p21 peptide; lane 7, 120 ng of PCNA, 200 ng of p50, and 120 ng of p21 peptide; lane 8, 120 ng of PCNA, 200 ng of p50, and 120 ng of unrelated peptide; lane 9, 200 ng of p50 (no PCNA). p50 was detected by chicken anti-p50 antibody. C, graphical representation of the data in A and B. The amount of p50 co-immunoprecipitated with PCNA via anti-PCNA beads was quantitated using National Institutes of Health ImageJ version 1.26t software. The amounts of p50 in lane 3 of A and in lane 2 of B (without competition by p21 peptide) were considered to be 100% bound p50 remaining. The inhibition of p50-PCNA interaction by His-p21 is shown with solid squares, and by p21 peptide is shown with solid triangles.

It has been demonstrated that p21 effectively competes with a number of replication/repair proteins such as flap endonuclease 1 (50) and DNA ligase 1 (9), repair proteins such as XPG (14), as well as the postreplication processing protein DNA (cytosine-5) methyl transferase (15), all of which share a consensus p21-like PCNA-binding motif, for binding to PCNA at the interdomain connector loop. The observation that p21 can dissociate the p50-PCNA complex suggests that the PCNA-binding motif of p50 targets the same site on PCNA as p21.

Identification of a Putative PCNA-binding Motif in the N-terminal Region of p50-- Examination of the amino acid sequence of human p50 (44) did not identify the consensus eight-amino acid PCNA-binding motif (Q1XX(I/L/M)4XX(F/H)7(F/Y)8) originally identified in p21 and found in most proteins that interact with PCNA (24, 51). However, a hydrophobic five-amino acid sequence (MRPFL) that is homologous to a recently identified motif in the C termini of RB69 DNA polymerase (LFDMF) and T4 DNA polymerase (LDFLF) and that is necessary for interaction of these polymerases with their respective sliding clamps (52) was identified in human p50. However, the motif was found at the N terminus (residues 61-65) rather than the C terminus of p50. Similar to the sliding clamp-binding sites of p21 and RB69 DNA polymerase, the p50 sequence is also in a helical conformation.

To determine whether the MRPFL sequence in human p50 indeed represents a PCNA-binding motif, far Western analysis was carried out. A 22-amino acid peptide containing the putative PCNA-binding motif was synthesized, as was a mutated peptide, an unrelated peptide, and the p21 peptide as a positive control. The peptides, along with p50 protein and p21 protein, were slot-blotted to a polyvinylidene difluoride membrane, overlaid with PCNA, and probed with a monoclonal antibody to PCNA. The results (Fig. 3) demonstrated that the peptide containing MRPFL did interact with PCNA, whereas the mutated peptide and the unrelated peptide did not. Although the p50 peptide, the p21 peptide, and p21 protein bound PCNA, p50 protein did not, possibly because of denaturation or improper folding of the protein on the membrane.


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  		Fig. 3.   Far Western analysis of the interaction of PCNA with a p50 peptide. The proteins or peptides were bound to a polyvinylidene difluoride membrane and overlaid with PCNA as described under "Experimental Procedures." Lane 1, p50 protein (30 µg); lane 2, wild type p50 peptide (30 µg); lane 3, mutated p50 peptide (30 µg); lane 4, unrelated peptide (30 µg); lane 5, p21 peptide (30 µg); lane 6, His-p21 protein (500 ng); lane 7, PCNA protein (24 ng). Bound PCNA was detected with PC10 monoclonal antibody.

The p50 Peptide Competes with p50 for Binding to PCNA-- To further examine whether the interaction of p50 with PCNA is mediated through the sequence motif MRPFL, we investigated the effects of the addition of increasing amounts of the oligopeptide containing the identified PCNA-binding motif on the interaction of p50 with PCNA in co-immunoprecipitation experiments (Fig. 4). As shown in Fig. 4A, the p50 peptide, but not the mutated peptide in which residues Phe and Leu are substituted by alanines, effectively blocked the binding of p50 to PCNA. A 50% inhibition of binding was achieved at 200 nM peptide (Fig. 4B), corresponding to a molar ratio of p50 peptide to p50 protein of 10:1. These results establish that the MRPFL motif is responsible for the binding of p50 to PCNA.


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  		Fig. 4.   p50 peptide inhibits the interaction of PCNA and p50. A, anti-PCNA beads were mixed with PCNA, p50, and varying amounts of p50 peptide and processed as described under "Experimental Procedures." Lane 1, input p50 (100 ng); lane 2, 120 ng of PCNA and 200 ng of p50; lane 3, 120 ng of PCNA, 200 ng of p50, and 40 ng of p50 peptide; lane 4, 120 ng of PCNA, 200 ng of p50, and 80 ng of p50 peptide; lane 5, 120 ng of PCNA, 200 ng of p50, and 120 ng of p50 peptide; lane 6, 120 ng of PCNA, 200 ng of p50, and 160 ng of p50 peptide; lane 7, 120 ng of PCNA, 200 ng of p50, and 200 ng of p50 peptide; lane 8, 120 ng of PCNA, 200 ng of p50, and 200 ng of mutated p50 peptide; lane 9, 200 ng of p50 (no PCNA). p50 was detected by chicken anti-50 antibody. B, graphical representation of the data shown in A. The amount of p50 co-immunoprecipitated with PCNA via anti-PCNA-linked beads was quantitated using National Institutes of Health ImageJ version 1.26t software. The amount of p50 in lane 2 of A (without competition by p50 peptide) was considered to be 100% bound p50 remaining.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this report we have demonstrated that PCNA interacts directly with the small subunit of mammalian pol delta  (p50) by co-immunoprecipitation of these two proteins using polyclonal antibodies to each of them. We have further demonstrated, using polyclonal antibodies to either p125 or PCNA, that the catalytic subunit can only be co-immunoprecipitated with PCNA when it is complexed with p50 in heterodimeric pol delta  and not when it exists as a single subunit. These results suggest that PCNA does not directly interact with the catalytic subunit and that the interaction between pol delta  and its sliding clamp is mediated through the small subunit.

The demonstration of a direct interaction between PCNA and p50 in the present study differs from previous reports of a lack of interaction between these two proteins from both S. cerevisiae (30, 36) and human sources (33, 34). Studies on the interactions between PCNA and the subunits of S. cerevisiae pol delta , i.e. Pol3 (the catalytic subunit), Pol31 (the small subunit), and Pol32 (the third subunit) by protein overlay analysis using radioactively labeled PCNA as probe detected a direct interaction only between PCNA and Pol32. No interactions could be detected between PCNA and either Pol3 or Pol31, although it was pointed out by the authors that failure to detect interactions with the other pol delta  subunits might be the result of binding conditions or the failure of the other subunits to properly refold on the membrane (36). Similar reasons could also account for our inability to detect an interaction of PCNA with p50 by far Western analysis in the present study.

Our demonstration of a direct interaction between human PCNA and p50, as well as a lack of interaction between PCNA and p125 also differs from the results of recent studies on the interaction of PCNA with human pol delta . Zhang et al. (33), using PCNA overlay analysis to identify the subunits of pol delta  that interact with PCNA, failed to detect any interaction between PCNA and recombinant human p50 expressed in E. coli but did demonstrate a direct interaction between PCNA and recombinant human p125 expressed in insect cells. In that study the p125-PCNA interaction was confirmed by co-immunoprecipitation of PCNA and p125 co-expressed in insect cells by monoclonal antibody against PCNA, and by biochemical cross-linking studies. Reciprocal experiments using anti-p125 antibodies to co-immunoprecipitate PCNA and p125 were not reported. Shikata et al. (34) studied the interactions between recombinant human PCNA and either p125, p50, or p66 by pull-down assays with PCNA-linked beads. In that study, PCNA was found to interact weakly with p125, strongly with p66, and not at all with p50. The reasons for the discrepancies between these studies and the results of the present study are not apparent. Possible explanations include differences in the methods used in the expression and/or purification of the recombinant proteins, differences in the assays used, or a combination of these two factors.

The present results are in full agreement with the report of a lack of interaction between S. cerevisiae PCNA and p125 (37). In those studies co-immunoprecipitation of yeast PCNA and p125 co-expressed in insect cells was initially investigated by using an antibody specific to either PCNA or p125 and confirmed by reciprocal experiments using the antibodies against the partner. Our observations are also consistent with reports that the human, S. pombe or S. cerevisiae catalytic subunit by itself is not significantly stimulated by PCNA (39, 40, 42, 53). That the interaction between PCNA and p125 is indirect and mediated through p50 also provides a plausible explanation for the requirement of the small subunit for the stimulation of the activity and processivity of pol delta  by PCNA.

The results of recent studies demonstrating that only the recombinant three-subunit complex and not the two-subunit form of human pol delta  co-expressed in baculovirus-infected insect cells could be stimulated by PCNA (34, 35) differ from previous studies in which the two-subunit recombinant human (28, 40) or S. cerevisiae pol delta  (43), as well as the native calf thymus heterodimeric pol delta  (28), were found to be fully responsive to PCNA stimulation. The reasons for the discrepancies are not clear at present. However, in the studies of Ducoux et al. (35), the stimulation observed was only severalfold, and there was no indication that the stimulation was due to an increase in processivity. Thus, the increase could possibly be due to the stabilization of pol delta -PCNA interaction by p66, as suggested by Shikata et al. (34) in their studies on human recombinant pol delta , as well as by the studies of Gerik et al. (30) in budding yeast, which demonstrated that the third subunit was not absolutely required for interaction of pol delta  with PCNA but that it increased the affinity of the enzyme for PCNA. A further argument against the third subunit being the mediator of the interaction between pol delta  and PCNA is the observation that deletion of the PCNA-binding motif of Pol32 in S. cerevisiae did not significantly affect the processivity of PCNA-dependent DNA synthesis by pol delta  (54).

The results of the present study establish that the interaction between PCNA and pol delta  is mediated via interaction with the small subunit and not the third subunit of the enzyme. It is unlikely that the PCNA-dependent processivity of heterodimeric calf thymus pol delta  is due to contamination of the preparations with p66, as suggested by Ducoux et al. (35), because the enzyme used in our studies was purified by heparin-agarose chromatography (45), which completely separates the third subunit from the PCNA-dependent polymerase activity and from the 125- and 50-kDa subunits, as analyzed by Western blot analysis (55).

Bacteriophage RB69 DNA polymerase is a member of the pol alpha  family of DNA polymerases and is responsible for processive DNA synthesis on both the leading and lagging strands in vivo. Recent crystallographic studies of the RB69 sliding clamp complexed with an 11-residue C-terminal peptide from RB69 DNA polymerase revealed that the five-amino acid residues 897-901 (LFDMF) at the C terminus of RB69 DNA polymerase bind to the RB69 sliding clamp at a position identical to that of the binding of p21, residues 147-151 (MTDFY), to PCNA (52). It was further demonstrated that the C-terminal peptide of RB69 DNA polymerase binds at a hydrophobic pocket of the sliding clamp through residues Leu-897, Met-900, and Phe-901, whereas as previously shown p21 is anchored in a hydrophobic cavity in the interdomain connector loop of PCNA through Met-147, Phe-150, and Tyr-151 (6).

The structural similarity between the C-terminal peptide of the replicative DNA polymerase of bacteriophage RB69 and the C terminus of the cell cycle inhibitor p21, as well as the parallel manner by which these peptides bind at the hydrophobic pockets of their respective sliding clamps, combined with the ability of the p21 peptide to effectively inhibit DNA synthesis by competing with pol delta  for binding to PCNA (23, 56) led to the proposal that the eukaryotic DNA polymerase binds its sliding clamp at the same position and likely in the same manner as the bacteriophage enzyme (51, 57). Our demonstration that the binding of p50 to PCNA is mediated through a PCNA-binding sequence homologous to that of RB69 DNA polymerase and p21 and that the interaction of p50 and PCNA is effectively inhibited by p21 and its PCNA-binding oligopeptide as well as by the PCNA-binding p50 oligopeptide strongly supports this hypothesis. Our observations further support the suggestion that the T4-related bacteriophage RB69 DNA replisome is a good model system for understanding the mechanism by which DNA polymerases interact with their sliding clamps at the molecular level (56). Although the mode of interaction of RB69 DNA polymerase, T4 DNA polymerase, and pol delta  with their respective sliding clamps appears to be highly conserved, there are distinct differences. For example, the binding sites on RB69 and T4 DNA polymerases for their sliding clamps are located at the C termini of the proteins, whereas the PCNA-binding site of the 50-kDa subunit of mammalian DNA polymerase delta  is located in the N-terminal region of the protein, within a conserved region (region I) identified by multiple sequence alignment of the small subunits of a number of eukaryotic species (58). The PCNA-binding sites of a number of PCNA-interacting proteins, e.g. DNA ligase 1 (59), CAF1 (17), the mismatch repair proteins MSH3 and MSH6 (12, 13), and DNA (cytosine-5)-methyltransferase (15) are also located at the N termini of the proteins. The significance of this difference in the location of the sliding clamp-binding motifs in different proteins is not known at present. The observation that the binding of p50 to PCNA is inhibited by p21 as well as by the p21 oligopeptide suggests that the mechanism by which p21 inhibits pol delta  activity is by competition with the small subunit of the enzyme for binding to PCNA.

Recently, it was demonstrated that PCNA interacts with the 150-kDa subunit of CAF1, a protein that specifically targets newly replicated DNA for nucleosome assembly (16, 17), thus providing a molecular link between nucleosome assembly and DNA replication. In vitro studies have shown that PCNA functions to mark recently replicated DNA for chromatin assembly mediated via CAF1 (16); however, the mechanism by which chromatin assembly is coupled to DNA replication is not known. As shown in Fig. 5, the sequence of the PCNA-binding site on human CAF1 is very similar to that of human p50, suggesting the possibility that coupling of replication and nucleosome assembly may involve a competition between CAF1 and p50 for binding to PCNA. This extended binding site, although conserved between p50 and CAF1, is distinct from that of most PCNA-binding proteins, which share the eight-amino acid p21-like PCNA-binding motif (QXX(L/M/I)XX(F/H)(F/Y)), suggesting that regulation of the interaction between PCNA and p50 or CAF1 may be different from that between PCNA and the proteins sharing the classical PCNA-binding motif.


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  		Fig. 5.   Alignment of the PCNA-binding regions of p50 and CAF1. Identical amino acids are in bold type, and homologous residues are underlined.

The demonstration that the physical interaction of PCNA with pol delta  is mediated through the small subunit of the enzyme, in conjunction with the previous observation that the small subunit is required for functional interaction of pol delta  with PCNA (28), suggests that this interaction is primarily responsible for processive DNA synthesis by pol delta . The identification of a PCNA-binding motif in the N-terminal region of p50 that is similar to the PCNA-binding motif found in CAF1 (17) but distinct from the canonical PCNA-binding motif found in the third subunit of pol delta  (Cdc27 in S. pombe, Pol32 in S. cerevisiae, and p66 in mammalian species) and other proteins that also bind to both the small subunit and PCNA, e.g. Werner Syndrome protein (60, 61) and polymerase delta -interacting protein 1 (48), suggests that there are at least two levels of regulation of the processivity of DNA pol delta  by PCNA: a primary level of regulation through the binding of PCNA to the small subunit and a secondary level of regulation mediated through the interaction of PCNA with proteins that directly interact with the small subunit of pol delta , e.g. the third subunit, polymerase delta -interacting protein 1, and Werner Syndrome protein. Consistent with this hypothesis is the observation that Pol32 is not essential for the processivity of pol delta  in S. cerevisiae; rather it further stabilizes the interaction of the heterodimer, i.e. Pol3 and Pol31 with PCNA (43, 54).

    FOOTNOTES

* This work was supported by Grant DK26206 from the National Institutes of Health and in part by funds from the Sylvester Comprehensive Cancer Center.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Present address: Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai, China.

|| Present address: Salk Inst. for Biological Studies, Gene Expression Laboratories, La Jolla, CA 92037.

** To whom correspondence should be addressed: Dept. of Medicine (R99), University of Miami School of Medicine, P.O. Box 016960, Miami, FL 33101. Tel.: 305-243-6304; Fax: 305-243-4519; E-mail: aso@med.miami.edu.

Published, JBC Papers in Press, May 1, 2002, DOI 10.1074/jbc.M200065200

    ABBREVIATIONS

The abbreviations used are: pol delta , DNA polymerase delta ; PCNA, proliferating cell nuclear antigen; CAF1, chromatin assembly factor 1.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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