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J. Biol. Chem., Vol. 277, Issue 34, 31115-31123, August 23, 2002

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Direct Interaction between Mammalian DNA Polymerase  and Proliferating Cell Nuclear Antigen^*

Padmini S. Kedar, Soon-Jong Kim^§, Anthony Robertson^¶, Esther Hou, Rajendra Prasad, Julie K. Horton, and Samuel H. Wilson

From the  Laboratory of Structural Biology, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709

Received for publication, February 13, 2002, and in revised form, June 11, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEEDURES RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

Proliferating cell nuclear antigen (PCNA) plays an essential role in nucleic acid metabolism as a component of the DNA replication and DNA repair machinery. As such, PCNA interacts with many proteins that have a sequence motif termed the PCNA interacting motif (PIM) and also with proteins lacking a PIM. Three regions in human and rat DNA polymerases  (-pol) that resemble the consensus PIM were identified, and we show here that -polymerase and PCNA can form a complex both in vitro and in vivo. Immunoprecipitation experiments, yeast two-hybrid analysis, and overlay binding assays were used to examine the interaction between the two proteins. Competition experiments with synthetic PIM-containing peptides suggested the importance of a PIM in the interaction, and studies of a -polymerase PIM mutant, H222A/F223A, demonstrated that this alteration blocked the interaction with PCNA. The results indicate that at least one of the PIM-like sequences in -polymerase appears to be a functional PIM and was required in the interaction between -polymerase and PCNA.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEEDURES RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

DNA repair is vital for cell survival and maintenance of genomic stability. DNA polymerase  (-pol)¹ is known to be involved in short-gap filling DNA synthesis in mammalian cells. The enzyme plays roles in base excision repair (BER) (1) and in some cases, can function in DNA replication as well as other pathways of DNA repair (2). Base lesions in DNA arise from a variety of physical and chemical agents. These lesions are repaired in part by BER. There are at least two subpathways of BER, differentiated by the repair patch sizes and the enzymes involved, and these subpathways are classified as "single-nucleotide" and "long patch" BER, respectively (3, 4). Four purified human enzymes can reconstitute single-nucleotide BER of uracil-DNA (5-7). This repair pathway is a sequential process initiated by uracil-DNA glycosylase base removal and formation of the apurinic/apyrimidinic (AP) site, this was followed by AP endonuclease (APE) incision of the AP site (8, 9). The resulting single nucleotide gap is filled by -pol, and the enzyme also conducts another required enzymatic step, removal of the sugar phosphate from the incised AP site (10, 11). Finally, DNA ligase I or the x-ray cross-complementing factor 1-DNA ligase III complex completes this BER subpathway (5, 12-14).

It has been proposed that the various sequential steps in the single-nucleotide BER subpathway are coordinated through protein-protein interactions. A direct interaction between -pol/DNA ligase I has been described, as has interaction between -pol and x-ray cross-complementing factor 1-ligase III (5, 12-14). These interactions could have biological consequences, as cells deficient in the proteins, -pol, DNA ligase I, DNA ligase III, or x-ray cross-complementing factor 1, are hypersensitive to DNA alkylating agents (15-20), and extracts from the cells are defective in BER in vitro (21, 22). Also, an interaction between -pol and DNA-bound APE has been reported (23), yet these two proteins do not directly interact in solution. Finally, uracil-DNA glycosylase has been proposed to recruit APE to the AP site after release of uracil from uracil-DNA (24).

The long patch BER subpathway involves multiple proteins, in addition to those described above for single-nucleotide BER. These include replication factor C, PCNA, DNA polymerases / (pol /), flap endonuclease-1 (FEN-1), and poly(ADP-ribose) polymerase-1 (25-30). PCNA is known to interact with some of the BER enzymes, including FEN-1 and DNA ligase I (20). Klungland and Lindahl (31) found that PCNA enhances -pol-dependent long patch BER of AP sites by stimulating FEN-1 activity. No role, however, has been proposed for PCNA in single-nucleotide BER.

PCNA is also well known as a component of the DNA replication system in mammalian cells, and it plays roles in multiple cellular pathways in addition to DNA replication and BER, including the following: nucleotide excision repair (32, 33), mismatch repair (34), cell cycle control (35-37), apoptosis (38), and transcription (39). Thus, PCNA has been termed a "cellular communicator" by virtue of its ability to connect various cellular processes (40). Finally, PCNA is known to function as a processivity factor for DNA polymerases such as pol  and pol  in vitro (41).

Whereas evaluating the question of potential interacting partners for mammalian -pol in BER, we identified three short sequences (7-9 residues) that were similar to the PCNA interacting motif or PIM in some of the known PCNA-binding proteins, and we subsequently found, by cell extract immunoprecipitation and yeast two-hybrid experiments, that PCNA appeared to interact with -pol in vivo. A possible direct interaction between -pol and PCNA was examined using the purified samples of the two proteins and a combination of co-immunoprecipitation and overlay assay binding techniques. We found that the proteins interact directly and mapped a region of -pol responsible for its interaction with PCNA to a sequence resembling a consensus PIM. These results and possible implications of the -pol and PCNA interaction are discussed.

	EXPERIMENTAL PROCEEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEEDURES RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

Materials-- Dulbecco's modified Eagle's medium and GlutaMAX-1 were from Invitrogen. Fetal bovine serum was from Summit Biotechnology (Ft. Collins, CO) and hygromycin was from Roche Molecular Biochemicals (Indianapolis, IN). Anti--pol affinity purified polyclonal antibody has been described previously (14); anti-PCNA polyclonal antibody (Ab-5) and anti-PCNA monoclonal antibody (Ab-2) were from Oncogene Research Products (Boston, MA); anti-PCNA monoclonal antibody (SC-56) was from Santa Cruz Biotechnology (Santa Cruz, CA). Anti--pol mouse monoclonal antibody (SJK132-20) and rabbit monoclonal antibody (DPN) were gifts from Dr. W. C. Copeland, NIEHS, National Institutes of Health. Anti-FEN-1 monoclonal antibody (FEN-1-4EP) was from Genetex (San Antonio, TX). Matchmaker two-hybrid systems were from CLONTECH (Palo Alto, CA). The mouse IgG secondary antibody used was goat anti-mouse IgG (H+L) binding grade affinity purified horseradish peroxidase conjugate, and the rabbit IgG secondary antibody used was goat anti-rabbit IgG (H+L)-horseradish peroxidase conjugate, both from Bio-Rad. Protein A-Sepharose CL-4B and SP-Sepharose (fast flow) were from Amersham Biosciences. Protein G-agarose and the protease inhibitor complete (EDTA-free) were from Roche Molecular Diagnostics. Leupeptin, aprotinin, and phenylmethylsulfonyl fluoride were from Calbiochem (La Jolla, CA). Normal goat serum was from Vector Laboratories (Burlingame, CA).

Proteins and Peptides-- Human -pol, rat -pol, and human PCNA were purified as described previously (42-44). Special care was taken to remove DNA from the PCNA preparation, because some DNA persisted in co-elution with purified PCNA. DNA was removed by chromatography on phenyl-Sepharose, Resource S, and Superdex S200 columns (Amersham Biosciences) in buffer containing 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM dithiothreitol, and 100 mM KCl. UV spectra were measured before and after each column step. The final preparation was free of DNA as measured by spectral analysis and by ethidium bromide staining after native gel electrophoresis. The peptide derivative of p21, KRRQTSMTDFYHSKRRLIFS (amino acids 141-160 of p21) contains the site for p21^WAF1 and PCNA interaction (34, 45). The negative control or "jumbled" peptide, QDKTRYFHRTMSRSKSIRLF, had the same amino acid composition as the p21 peptide. The peptide derivative of human MSH6, MSRQSTLYSFFPKSPALSDA, contains the site for MSH6 and PCNA interaction (46). A peptide containing the PIM-like sequence found in human -pol (Fig. 2, region II), HQVVEQLQKVHFITDTLSKGE, was obtained. All peptides were from Research Genetics Inc. (Huntsville, AL); purity was found to be greater than 75% by high performance liquid chromatography. Peptides were weighed and dissolved in water at 5-10 mg/ml and stored at 80 °C.

Cell Lines-- The cell lines used were a clone of the wild-type (WT) mouse embryonic fibroblast cell line M16tsA, a clone of the isogenic -pol null line M19tsA described previously (16), and a -pol null cell line (termed 19HB3) stably transfected with a FLAG--pol vector and expressing a high level of the protein (26). Cells were routinely grown at 34 °C in a 10% CO₂ incubator in Dulbecco's modified Eagle's medium supplemented with GlutaMAX-1, 10% fetal bovine serum, and hygromycin (80 µg/ml). All cells were routinely tested and found to be free of mycoplasma contamination.

Lysate Preparation, Co-immunoprecipitation, and Western Blotting-- The WT, -pol null, and 19HB3 cells were harvested and washed two times in phosphate-buffered saline. Cell lysates were prepared in a lysis buffer (47) (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 25 mM NaF, 0.1 mM sodium orthovanadate, 0.2% Triton X-100, 0.3% Nonidet P-40) containing protease inhibitors, 0.1 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, and 5 µg/ml leupeptin. Cells in the lysis buffer were incubated on ice for 30 min. The lysates were centrifuged at 14,000 rpm for 30 min at 4 °C and the supernatant fraction was transferred to another tube. The protein concentration in the extract was determined using the Bio-Rad protein assay, with bovine serum albumin (BSA) as standard. For co-immunoprecipitations, equal amounts (1 mg of protein) of cell lysate were mixed with 0.7 µg of affinity purified anti--pol polyclonal antibody or rabbit nonimmune IgG. The mixture was incubated with rotation for 4 h at 4 °C. The immunocomplex was adsorbed onto protein A-Sepharose and protein G-agarose beads by incubating the mixture overnight at 4 °C. The beads were washed four times with lysis buffer containing protease inhibitors. Finally, the beads were resuspended in SDS sample buffer, heated for 5 min, and the soluble proteins were separated by 4-12% SDS-PAGE. The proteins were then transferred onto a nitrocellulose membrane in a transblot apparatus for 3 h at 25 V. The membrane was incubated with 5% nonfat dry milk in Tris-buffered saline (TBS) containing 0.1% (v/v) Tween 20 (TBS-T) and eventually probed with the anti-PCNA monoclonal antibody (1:1,000 dilution). Goat anti-rabbit IgG conjugated to horseradish peroxidase (1:10,000 dilution) was used as secondary antibody and immobilized horseradish peroxidase activity was detected by enhanced chemiluminescence (ECL). The same blot was stripped by incubating with buffer containing 62.5 mM Tris-HCl, pH 6.8, 100 mM -mercaptoethanol, and 1% SDS for 30 min at 50 °C, followed by two washes with TBS-T at room temperature. The presence of -pol was confirmed by incubating the membrane with mouse anti--pol monoclonal antibody 18S (48). Similarly, the cell lysate was immunoprecipitated with anti-PCNA polyclonal antibody, Ab-5, as described above. The blot was developed with anti--pol monoclonal antibody 18S to detect -pol. After stripping the blot, the presence of PCNA was confirmed using the anti-PCNA monoclonal antibody SC-56. The same method was used for immunoprecipitation and probing with anti--pol and anti-FEN-1 antibodies.

Co-immunoprecipitation of purified PCNA and -pol was performed in the presence of binding buffer (25 mM Tris, pH 8, 10% glycerol, 100 mM NaCl, 0.01% Nonidet P-40) containing protease inhibitors (0.1 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, and 5 µg/ml leupeptin). To the mixture of 1.5 µM -pol and 1.5 µM PCNA in a final volume of 50 µl either anti--pol or anti-PCNA antibody were added, and the mixture was incubated with rotation for 4 h at 4 °C. The protein complex was adsorbed onto protein A-Sepharose and protein G-agarose beads by incubating the mixture overnight at 4 °C. The beads were washed four times with binding buffer containing protease inhibitors. The beads were suspended in SDS sample buffer, heated for 5 min, and the soluble proteins were separated by 4-12% SDS-PAGE. After transferring the proteins to nitrocellulose membrane, the membrane was blocked in 5% milk in TBS-T. Immunoblotting was performed with the appropriate antibody as described above.

Two-hybrid Constructs-- The -pol two-hybrid constructs used in this study were prepared from a full-length human -pol cDNA as a restriction fragment. Adapters were used as needed for in-frame insertion relative to the GAL4 activation domain encoded in the pACT2 yeast two-hybrid vector plasmid (CLONTECH). The -pol 31-kDa domain construct codes for Arg¹⁰² to Glu³³⁵ and was prepared by insertion of a Xhol restriction fragment of -pol. The -pol-(1-251) construct codes for Met¹ to Asp²⁵¹ and was prepared by insertion of a NcoI-EcoRV restriction fragment of -pol and contains a vector encoded stop codon. The -pol-(251-335) construct codes for Asp²⁵¹ to Glu³³⁵ and was prepared by insertion of an EcoRV-XhoI restriction fragment of -pol. Full-length -pol and the N-terminal 8-kDa domain were also prepared as in-frame inserts into pACT2. The 8-kDa domain construct codes for Met¹ to Arg¹⁰². The full-length PCNA two-hybrid construct used in this study was prepared from a full-length human cDNA as a restriction fragment inserted in-frame relative to the GAL4-binding domain in the pAS2-1 yeast two-hybrid vector plasmid (CLONTECH). All of the constructs were confirmed by sequencing.

Two-hybrid Analysis-- The yeast media used to determine nutritional requirements in the directed two-hybrid selections were prepared following established recipes. Chemical reagents for transformation were obtained from Sigma, and the yeast plasmid vectors and host cells were obtained from CLONTECH. Before testing for protein interactions, each construct was first checked for background His expression on defined medium without histidine. No histidine expression or colony formation were observed for any of the constructs tested, where transformation was always confirmed by reversion to the Trp⁺ or Leu⁺ phenotype for the PCNA-binding domain or -pol activation domain constructs, respectively.

Protein interactions were tested by selection for his+ revertants following co-transformation of yeast strain CG1945, carrying the his3 and lacZ reporter genes under control of the GAL4 responsive element, with the PCNA-binding domain and -pol activation domain constructs. Co-transformed cells were plated on dropout medium containing 2.5 mM His3 inhibitor, 3-amino-1,2,4-triazole, and lacking Trp, Leu, and His (DO3). This was compared with an equal volume of transformants plated on dropout medium lacking Trp and Leu (DO2). The preparation of competent cells and transformations were performed by the LiCl method as described in the Matchmaker GAL4 two-hybrid user manual (CLONTECH PT3061-1). The transformation reactions were split and added in equal amounts to the DO2 and DO3 selection plates, which were grown at 30 °C and photographed after 5 days. All protein interactions detected by nutritional selection were confirmed by -galactosidase assays performed using the Gal-Screen^TM protocol with detection on a TR717^TM Microplate Luminometer (Applera Corp.).

Overlay Binding Assay-- The overlay protein binding assay was performed as described previously (49). Briefly, purified -pol (24 µg) was digested with trypsin (substrate to trypsin ratio, 10:1, w/w) in a final volume of 105 µl in 25 mM Tris-HCl, pH 7.5, 25 mM NaCl, 4 mM MgCl₂, and 1 mM EDTA (44, 48). The reaction was carried out at room temperature. Aliquots were withdrawn at 0, 1, 5, 15, 30, 60, and 120 min, mixed with SDS sample buffer, boiled for 5 min, and proteins were separated by 12% SDS-PAGE. The proteins were transferred to a nitrocellulose membrane. The membrane was incubated in a buffer containing 10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM MgCl₂, 0.1% (v/v) Tween 20, and 5% nonfat dry milk at 4 °C for 16 h. Membranes were then incubated at room temperature for 4 h in the same buffer with 0.1 µM PCNA, either with or without competing peptide, with buffer alone, or with IgG as a control (in a buffer containing protease inhibitors and 0.1% Tween 20, 1% BSA, and 0.5% Triton X-100). After incubation, the membrane was washed five times with the same buffer and subjected to immunoblot analysis using anti-PCNA monoclonal antibody diluted 1:1000 in TBS-T. Blots were incubated in 5% normal goat serum in TBS prior to secondary antibody (goat anti-mouse IgG, 1:10,000) incubation, followed by ECL.

-Pol SP-Sepharose Pull-down Assay-- Equimolar amounts (1.5 µM) of -pol and PCNA were incubated in binding buffer (100 mM NaCl, 50 mM Tris-HCl, pH 7.5, 0.1% Nonidet P-40, and the protease inhibitors) in a final volume of 50 µl. A suspension of SP-Sepharose (30 µl) pre-equilibrated with binding buffer was added and the mixture was incubated overnight at 4 °C on a rotating shaker. Increasing amounts of peptide II or jumbled peptide were added to the incubation mixture as indicated in the figure legends. Protein-bound Sepharose beads were washed three times with binding buffer; SDS sample buffer was added, the mixture was heated for 5 min at 95 °C, and soluble proteins were resolved by 4-12% SDS-PAGE. Proteins were transferred onto a nitrocellulose membrane and the blots were developed as described above.

Mutant -Pol-- The mutant -pol (H222A/F223A) expression construct was prepared with the assistance of Dr. T. G. Wood, University of Texas Medical Branch, as described (50). The -pol mutant protein was overexpressed and purified, as described (42).

In Vitro BER Assay-- A partial BER reaction was reconstituted with purified proteins under the following conditions: the reaction mixture (10 µl) contained 50 mM Hepes, pH 7.5, 10 mM MgCl₂, 2 mM dithiothreitol, 20 mM KCl, 100 µg/ml BSA, 4 mM ATP, 250 nM 34-base pair DNA substrate with a uracil residue at position 16, 20 µM each of dATP, dGTP, and dTTP, and 2.3 µM [-³²P]dCTP (specific activity: 1 × 10⁶ dpm/pmol). Uracil-containing DNA substrate was pretreated with uracil-DNA glycosylase as described previously (30). The reaction mixture was assembled on ice by mixing 10 nM APE and 2.5 nM wild-type -pol or H222A/F223A mutant -pol. Incubation was for 20 min at 37 °C and was within a linear range for product formation as a function of time and enzyme concentration. The reaction products were separated by electrophoresis in a 15% denaturing polyacrylamide gel as described previously (30).

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEEDURES RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

Co-immunoprecipitation of PCNA and -Pol from Mouse Embryonic Fibroblast Extract-- Because PCNA is involved in many DNA repair functions, we considered it to be a candidate for interaction with -pol. To explore this possibility, we conducted co-immunoprecipitation experiments with anti--pol antibody and extracts from WT and -pol null mouse cells and also with extract from null cells (19HB3) stably transfected with a -pol expression vector. With WT cell extract the anti--pol antibody immunoprecipitated PCNA (Fig 1A, panel 1, lanes 2 and 3). A protein of similar size was not detected in the immunoprecipitate prepared with preimmune IgG and WT extract (Fig. 1A, panel 1, lane 1) or with immunoprecipitates of -pol null cell extract prepared with anti--pol antibody (Fig. 1A, panel 1, lanes 4 and 5). Extract of null cells supplemented with purified -pol or extract from null cells stably transfected with a -pol expression vector also showed immunoprecipitation of PCNA (Fig. 1A, lanes 7 and 8). To verify that -pol had been immunoprecipitated, the filter was stripped and immunoblotted with anti--pol antibody (Fig. 1A, panel 2). As expected, -pol was immunoprecipitated from WT cell extract and null cell extract supplemented with purified -pol, and also from 19HB3 cell extract (Fig. 1A, panel 2, lanes 2, 3, 7, and 8), but not from -pol null cell extract. We also conducted control experiments to verify that our anti--pol antibody did not immunoprecipitate miscellaneous DNA-binding proteins. After immunoprecipitation, membranes were immunoblotted with anti-FEN-1 or anti--pol antibody. As these proteins do not interact with -pol (Fig. 1A, panel 3 or 4, respectively), no immunoprecipitation of these DNA-binding proteins was detected.

View larger version (31K): [in this window] [in a new window]   Fig. 1.   Interaction of -pol and PCNA revealed by co-immunoprecipitation. Experiments were conducted as described under "Experimental Procedures." Photographs of ECL-stained immunoblots are shown. A, immunoprecipitation with anti--pol antibody. Panel 1, or top, immunoblotting to detect PCNA; lanes 2 and 3 and 4 and 5, respectively, are duplicate experiments. Lanes 1-3, immunoprecipitation with WT cell extract: lane 1, preimmune IgG control; lanes 2 and 3, anti-PCNA antibody. Lanes 4 and 5, immunoprecipitation with null cell extract. Lane 6, a positive control with one-twentieth of the WT cell extract (50 µg) processed directly in SDS-PAGE. Lane 7, null cell extract supplemented with purified -pol (3 µg/mg of extract). Lane 8, 19HB3 cell extract. Panel 2, the blot was stripped and re-probed with anti--pol antibody. Panel 3, a stripped blot was re-probed with anti-FEN-1 antibody. Panel 4, or bottom, a stripped blot was re-probed with anti--pol antibody. B, controls and cellular extracts as in A; immunoprecipitation was with anti-PCNA antibody. C, immunoprecipitation with a mixture of 1.5 µM each purified -pol and PCNA. Panel 1, lane 1, the mixture was immunoprecipitated with nonimmune IgG; lane 2, anti--pol antibody; lane 3, anti--pol antibody, except -pol was omitted from the mixture; lane 4, anti--pol antibody, except PCNA was omitted from the mixture; lane 5, purified PCNA added directly to the SDS-PAGE. Panel 2, the blot was stripped and probed with anti--pol antibody. D, immunoprecipitation with purified proteins as in C, and anti-PCNA antibody. Experiments were as in C. Lane 5 had -pol added directly to the SDS-PAGE. IP and IB indicate immunoprecipitate and immunoblot, respectively.

Consistent with the results above, -pol was reciprocally co-immunoprecipitated with anti-PCNA antibody (Fig. 1B), whereas no -pol was observed in the immunoprecipitate prepared with preimmune IgG or with -pol null extract, as expected (Fig. 1B, panel 1). Fig. 1B illustrates that PCNA was immunoprecipitated, as expected, with anti-PCNA antibody (panel 2, lanes 2-5 and 7 and 8). These results indicate that PCNA and -pol can be co-immunoprecipitated from extracts of WT cells, -pol null cells supplemented with -pol, and -pol expression vector transformed null cells, 19HB3. A similar immunoblot probed with anti-FEN-1 antibody revealed immunoprecipitation of FEN-1 (Fig. 1B, panel 3), whereas probing with anti--pol antibody was negative (Fig. 1B, panel 4). These control experiments indicated that our anti-PCNA antibody did not immunoprecipitate a non-PCNA-interacting DNA-binding protein, -pol, yet did immunoprecipitate the known PCNA-binding protein FEN-1.

These results with cellular extracts were consistent with a direct interaction between -pol and PCNA, in addition to other possibilities. To test for a direct interaction, a mixture of purified -pol and PCNA was subjected to immunoprecipitation with anti--pol or anti-PCNA antibody; the results illustrate that these purified proteins were co-immunoprecipitated with the antibodies (Fig. 1, C, lane 2, and D, lane 2). Control experiments illustrated that the co-immunoprecipitation signals required both proteins, as expected, and that anti-PCNA antibody did not immunoprecipitate -pol alone, whereas anti--pol antibody did not immunoprecipitate PCNA alone. We concluded from these experiments that purified -pol and PCNA do interact.

-Pol Contains Sequences Resembling the PCNA Interacting Motif-- It is known that some DNA enzymes and other proteins bind to PCNA through a conserved sequence motif termed the PCNA interacting motif or PIM. To evaluate the presence of a PIM in -pol, the human and rat -pol sequences were aligned with the consensus 8-amino acid PIM sequence. We observed three sequences in human -pol with some degree of similarity to the PIM consensus sequence; these sequences are in -helix I, -helix L, and -helix M and are designated I, II, and III, respectively (Fig. 2). The sequences are identical in the human and rat enzymes, except for codon 222 in -helix L, which is His in human and Arg in rat. None of these sequences precisely matches the PIM consensus. To examine which of these sequences, if any, is involved in the interaction between PCNA and -pol, we used several experimental approaches: co-immunoprecipitation experiments, yeast two-hybrid analysis, and an overlay binding assay. Reference to -pol crystal structures (51) indicated that sequences I and II are solvent exposed, and distal to the DNA-binding surface, whereas sequence III is partially buried and in the vicinity of the DNA (Fig. 2B).

View larger version (27K): [in this window] [in a new window]   Fig. 2.   Sequence alignments illustrating the presence of three sequences in -pol resembling the consensus PCNA interacting motif or PIM. A, three PIM-like sequences were identified in -pol that resemble the consensus PIM, consisting of the sequence QXX(h)XX(a)(a), where h is a moderately hydrophobic side chain, a is a hydrophobic side chain, and X is any residue. The figure illustrates alignments of the PIM consensus sequence (top) with three PIM-like sequences in human and rat -pol. The PIM-like sequences are designated I, II, and III, and are aligned within the 39-kDa 335-residue -pol sequence. These PIM-like sequences are found in -helices I, L, and M (51). B, crystal structure surface image of a -pol·gapped DNA·dNTP complex (51) illustrating the locations of PIM-like sequences I, II, and III.

Yeast Two-hybrid Analysis of -Pol Constructs in pACT2 with Full-length PCNA in pAS2-1-- To confirm the -pol and PCNA interaction and also to map possible functional PIM sequences in -pol, we performed two-hybrid analysis. The full-length PCNA/GAL4-binding domain in pAS2-1 was tested for interaction with full-length human -pol and four -pol segments cloned into the pACT2 two-hybrid vector (Fig. 3A). Full-length FEN-1 was also included in place of -pol as a positive control. Co-transformation of the full-length PCNA construct with -pol-(1-335), -pol-(1-251), or -pol-(102-335) resulted in the appearance of yeast colonies on the DO3 plates. In contrast, the -pol-(1-80) and -pol-(251-335) constructs showed only minimal (background) colonies on the DO3 plates (Fig. 3B). These observations confirmed that -pol and PCNA can interact in vivo and were also consistent with the idea that PIM-like sequences I and II (Fig. 2) of -pol could be functional PIMs; all three of the positive constructs contained these sequences, whereas the construct with region III alone was negative. Furthermore, the construct with -pol-(1-102) lacks the region III PIM-like sequence, yet it still binds PCNA; the constructs -pol-(1-80) and -pol-(251-335) did not show binding with PCNA suggesting that these regions of -pol are not sufficient to confer binding. As noted, these results of two-hybrid analysis were consistent with the idea that PIM-like sequences I and II could be functional PIMs, although the negative results with other regions of -pol could have been because of misfolding of these proteins in vivo.

View larger version (52K): [in this window] [in a new window]   Fig. 3.   Mapping of -pol and PCNA interaction by yeast two-hybrid analysis. The yeast two-hybrid constructs are described under "Experimental Procedures." A, schematic representation of the -pol constructs made in the GAL4 activation domain in the vector pACT2. The -pol constructs are designated 1-5. B, yeast CG 1945 cells co-transformed with the indicated -pol-GAL4 activation domain construct and with the PCNA construct and grown on DO3 plates. Some background colonies were observed in negative controls and with -pol constructs 2 and 5. For a positive control, human FEN-1 and PCNA constructs were used, as the strong interaction between these proteins is well known (60).

Overlay Binding Assay for Interaction between -Pol and PCNA-- Blots containing trypsin-digested -pol (Fig. 4A) were overlaid with PCNA, washed, and subsequently incubated with anti-PCNA antibody. As seen in Fig. 4B, PCNA bound to full-length 39-kDa -pol and to the 31- and 27-kDa fragments of -pol. However, there was no binding to the 12-, 10-, or 8-kDa fragments (Fig. 4B). To confirm that the PCNA preparation was free of DNA contamination that might interfere with protein binding, ethidium bromide was added to the overlay solution along with PCNA, to chelate any DNA present. There was no difference in results obtained in the presence or absence of ethidium bromide (data not shown).

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Interaction of PCNA with -pol and its tryptic fragments revealed by an overlay binding assay. A, photograph of a Coomassie Blue-stained gel showing the products of trypsin digestion of human -pol. Purified -pol (24 µg) was digested with trypsin (substrate to enzyme ratio, 10:1, w/w) at room temperature. Aliquots were withdrawn at 0, 1, 5, 15, 30, 60, and 120 min (lanes 1-7, respectively) and separated by 12% SDS-PAGE as described under "Experimental Procedures." B, mapping of PCNA interaction with -pol using the overlay assay. Proteins as in A were transferred to a membrane and incubated with PCNA as described under "Experimental Procedures." The blot was probed with anti-PCNA antibody. The positive signals correspond to full-length -pol (lane 1), 31-kDa domain (lane 2), and the 27-kDa fragment (lanes 3-7). The 12- and 10-kDa fragments showed no binding to PCNA. C, various controls were: "protein controls" (lanes 1-4), "overlay (OL) solution controls" (lanes 5 and 6), and "antibody controls" (lanes 7 and 8). Lane 1 is the same as lane 1 in B. D, schematic representation of the -pol tryptic fragments and their binding to PCNA.

Fig. 4D summarizes the -pol tryptic fragments (48) and the results of PCNA overlay binding. As seen in the diagram, the 27-kDa fragment has PIM-like sequences II and III, but not I (Fig. 2); thus, these results indicate that even when sequence I was not present, PCNA could bind to the tryptic fragment. Extending the trypsin digestion for a longer period decreased the 27-kDa fragment and increased 10- and 12-kDa fragments (Fig. 4A, lanes 5-7). These two fragments are formed by trypsin digestion at Lys²²⁰ and Lys²³⁰ (52). Lys²²⁰ is the middle residue in PIM-like sequence II (Fig. 2). As this motif was disrupted by the digestion, binding of either of these fragments to PCNA by virtue of PIM-like sequence II would not be expected. The 12-kDa fragment failed to show binding to PCNA (Fig. 4B, lanes 5-7), despite the fact that it had intact PIM-like sequence III. The N-terminal 8-kDa fragment of -pol lacks a PIM and failed to show binding.

Finally, several controls for these overlay experiments were evaluated (Fig. 4C): PCNA did not bind to BSA, the 8-kDa domain of -pol, or the blank lane (lanes 2-4); for overlay solution controls, BSA was overlaid on -pol and probed with anti-PCNA antibody, and the buffer alone was overlaid on -pol and probed with anti-PCNA antibody; both of these lanes were negative (lanes 5 and 6); antibody negative controls (lanes 7 and 8) were anti-glutathione S-transferase and preimmune mouse IgG, both of which failed to recognize PCNA overlaid on -pol.

Association between PCNA and -Pol and Its Domains-- To further examine the association between -pol and PCNA, we used full-length -pol and large amounts of purified samples of recombinant 31-, 22-, 14-, and 8-kDa domain proteins of -pol (53). FEN-1 was included as a positive control. Fig. 5A is a photograph of the stained gel and Fig. 5C shows a diagram of -pol and the domain proteins, as well as a summary of the binding results (Fig. 5B). The blot was overlaid with PCNA and washed, and bound PCNA was then detected with anti-PCNA antibody. The results confirmed that -pol binds PCNA (Fig. 5B, lanes 1 and 6), and the 31- and 22-kDa proteins also showed PCNA binding (Fig. 5B, lanes 2 and 5); these proteins have PIM-like sequences II and III. The 14-kDa protein, which does not have a complete PIM sequence, did not bind PCNA (Fig. 5B, lane 3), and the 8-kDa protein was similarly negative (Fig. 5B, lane 4). As expected, FEN-1 was positive for PCNA binding (Fig. 5B, lane 7).

View larger version (21K): [in this window] [in a new window]   Fig. 5.   Interaction of PCNA with full-length -pol and its recombinant domain proteins in the overlay binding assay. A, photograph of a Coomassie Blue-stained gel showing full-length -pol, its domain proteins, and FEN-1. B, proteins as in A separated by SDS-PAGE were transferred onto a membrane and incubated with PCNA as described under "Experimental Procedures." The blot was washed and probed with anti-PCNA antibody. Full-length -pol (lanes 1 and 6, human and rat, respectively), 31-kDa protein (lane 2), 22-kDa protein (lane 5), and FEN-1 showed interaction with PCNA; the 14- and 8-kDa proteins (lane 3 and 4, respectively) did not interact with PCNA. C, schematic representation of human and rat -pols and summary of PCNA binding results in B.

Synthetic Peptides Inhibit the -Pol and PCNA Interaction-- The studies described thus far are consistent with the idea that the PIM-like sequence II in -helix L (Fig. 2) is involved in the binding between PCNA and -pol. We performed overlay binding experiments to evaluate whether a synthetic peptide corresponding to this region could inhibit binding. Different blots containing the 27-kDa fragment of tryptic digestion of -pol were used (Fig. 6). PCNA binding was observed as usual (Fig. 6, panel A), whereas negative controls with nonimmune IgG or incubation without PCNA revealed only minor background signals (Fig. 6, B and E, respectively). The synthetic peptide corresponding to the -pol PIM-like II sequence inhibited PCNA binding, whereas a negative control "jumbled peptide" did not (Fig. 6, D and C, respectively). Next, we examined the effect of two synthetic peptides corresponding to the PIM sequences of either p21 (34) or MSH6 (46) (Fig. 7). It is known that these PIM containing synthetic peptides can bind to PCNA and block its binding to the respective partner protein. PCNA did not bind to -pol in the presence of either of these synthetic peptides (Fig. 7, C and D). Finally, as -pol binds tightly to SP-Sepharose beads (42), we made use of this property to immobilize -pol in an alternate test for peptide inhibition of PCNA binding. Binding of PCNA to -pol was readily detected in this assay (Fig. 8A, lane 2). As the concentration of -pol PIM-like sequence II synthetic peptide was increased, binding of PCNA decreased to a negative level (Fig. 8A, lanes 4 and 5). No inhibition of PCNA binding was detected in experiments with the negative control jumbled peptide (Fig. 8B). Overall, these results indicate that binding between PCNA and -pol can be inhibited by synthetic peptides corresponding to PIM sequences; the results implicate a PIM-like sequence in -pol in the PCNA binding.

View larger version (36K): [in this window] [in a new window]   Fig. 6.   Synthetic peptide competition of PCNA and -pol binding in the overlay assay. The 27-kDa tryptic fragment of purified -pol was produced by digestion with trypsin for different periods, as in Fig. 4A, and immunoblotted onto a membrane. In A and B, blots were overlaid with PCNA and then with anti-PCNA antibody (A) and preimmune IgG (i.e. an antibody control) (B), respectively. In C and D, respectively, the blots were overlaid with PCNA plus a negative control jumbled peptide, and PCNA plus a synthetic peptide corresponding to the PIM-like sequence II in -pol. In E, the blot was overlaid with buffer alone (without PCNA) and then with anti-PCNA antibody (i.e. an antibody control). The position of the 27-kDa fragment of -pol is indicated by an arrow at the right and the position of a 21-kDa marker protein is indicated by an arrow at the left.

View larger version (36K): [in this window] [in a new window]   Fig. 7.   Competition of binding between PCNA and -pol by synthetic peptides corresponding to PIM sequences in two other PCNA-binding proteins, p21 and MSH6. Experiments were conducted as described in the legend to Fig. 6. The blots were overlaid with PCNA (A), PCNA plus jumbled peptide (B), PCNA plus p21 peptide (C), or PCNA plus MSH6 peptide (D). The blots were probed with anti-PCNA antibody.

View larger version (17K): [in this window] [in a new window]   Fig. 8.   Synthetic -pol PIM-like sequence II peptide competition of PCNA and -pol binding in solution. Experiments were conducted as described under "Experimental Procedures." A mixture of -pol and PCNA (equal molar ratio) was incubated with either synthetic peptide II (A) or jumbled peptide (B). Lanes 1 and 6, -pol was omitted; lanes 2 and 7, -pol and PCNA (without synthetic peptide); lanes 3-5 and 8-10, -pol, PCNA, and synthetic peptide II and -pol, PCNA, and jumbled peptide. Peptides were added to the binding mixture in 10-, 100-, or 1,000-fold molar excess over -pol (1.5 µM). After incubation with SP-Sepharose beads, the beads were washed three times with binding buffer, and PCNA bound to the beads was then assayed by immunoblotting. Blots were probed with anti-PCNA antibody and developed by ECL. The position of PCNA is indicated by the arrow.

Mutation of Residues in the PIM-like Sequence II of -Pol-- To further evaluate the role of PIM-like sequence II in the binding between -pol and PCNA, we made a double mutant of -pol in which His²²² and Phe²²³ in sequence II were changed to alanine. This strategy was chosen in view of previous results indicating that mutation of the corresponding residues in another PIM containing PCNA-binding protein ablated PCNA binding (54). Co-immunoprecipitation experiments with purified -pol and increasing concentrations of PCNA were conducted. With WT -pol, more PCNA binding was observed with increasing concentrations of PCNA (Fig. 9A), whereas with the mutant -pol, negligible binding was observed (Fig. 9B). Lane 1 is a control with preimmune IgG and lane 9 had purified protein alone, either -pol or PCNA. These results suggest that the -pol PIM-like sequence II, ²¹⁷QLQKVHF²²³ (Fig. 2), is indeed a functional PIM and that His²²² and/or Phe²²³ are essential for the interaction between -pol and PCNA. The other PIM-like sequences in this mutant -pol were not sufficient to confer binding.

View larger version (37K): [in this window] [in a new window]   Fig. 9.   Interaction between PCNA and wild type -pol or mutant -pol. A constant amount (1.7 µM) of wild-type -pol (A) or H222A/F223A mutant -pol (B) was incubated with increasing amounts of PCNA (0.4, 0.7, 1.0, 1.4, 1.7, 2.4, and 3.5 µM for lanes 2-8, respectively). The mixtures were then subjected to co-immunoprecipitation and immunoblotting as shown in the figure and as described in the legend to Fig. 1. The blots were probed with anti-PCNA antibody (panel 1 in A and B) or anti--pol antibody (panel 2 in A and B). Lane 1 represents immunoprecipitation with preimmune IgG. Lane 9 is a control where purified -pol or PCNA was subjected directly to SDS-PAGE. IP and IB indicate immunoprecipitate and immunoblot, respectively.

An alternate explanation for the lack of binding between this mutant -pol and PCNA is that the mutant protein might be misfolded. This did not appear to be the case, however, because the mutant enzyme had DNA repair DNA polymerase activity similar to that of WT -pol (Fig. 10).

View larger version (17K): [in this window] [in a new window]   Fig. 10.   Reconstitution of uracil-initiated DNA BER using purified proteins, including mutant -pol. A, diagram of the DNA substrate (34-base pair) containing uracil and the BER product resulting from replacement of dUMP with 32P-labeled dCMP. B, photograph of an autoradiogram after denaturing PAGE of the reaction products of in vitro BER. The BER reaction was conducted as described under "Experimental Procedures." Reaction mixtures in lanes 1 and 2 contained wild-type -pol and H222A/F223A mutant of -pol, respectively. The position of the BER reaction product (16-mer) is shown by the arrow at the left-hand side of the photograph. nt, nucleotide.

	CONCLUSIONS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEEDURES RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

In recent years, it has become clear that PCNA is involved in many aspects of DNA metabolism by mediating interactions of proteins with DNA (55-58). Thus, PCNA is a key factor in the life of the cell, playing a role in DNA replication, as well as several forms of DNA repair, including nucleotide excision repair, BER, and mismatch repair (59, 60). In mediating its roles, PCNA binds directly to various other proteins, as well as to DNA. An observation from studies of these PCNA/protein interactions is that some of the PCNA-binding proteins contain a conserved PCNA interacting motif or PIM. Yet, it is well known that several proteins, including GADD45, MyD118, and CR6 (61, 62), can interact with PCNA without the benefit of a PIM. In this study, three sequences resembling the consensus PIM were identified in -pol by sequence alignments (Fig. 2A). None of these sequences are a perfect match to the PIM consensus, but two of them are at the surface and distal to the DNA-binding groove (Fig. 2B).

We chose to first examine whether PCNA and -pol are in a complex in extracts from mouse fibroblasts. Co-immunoprecipitation of the proteins was readily demonstrated, and co-immunoprecipitation was also observed with a mixture of purified human PCNA and -pol. We then conducted yeast two-hybrid analysis of deletion mutants of human -pol and demonstrated that of the three PIM-like sequences in -pol, all peptides with PIM-like sequence II, spanning residues 217-223, were able to bind to human PCNA. The region(s) within -pol required for interaction with PCNA was further characterized using an overlay binding assay. PCNA binding by various -pol fragments appeared to require the intact PIM-like sequence II, and inhibition studies with synthetic peptides showed that peptides with a PIM sequence could inhibit binding. Finally, a mutant of the PIM-like sequence II in -pol was prepared by replacing histidine and phenylalanine residues at codons 222 and 223 with alanine. This H222A/F223A form of human -pol showed little or no affinity for human PCNA in immunoprecipitation assays with purified proteins. We concluded that PCNA and -pol binding requires the presence of the PIM-like sequence II in -pol. Characterization of this mutant form of -pol revealed that it has wild-type enzyme-like repair synthesis activity on a single-nucleotide gapped DNA substrate (Fig. 10), indicating that the protein was properly folded. A role of the PIM-like sequence II in -pol interaction with PCNA appears to be consistent with the solvent-exposed, surface location of this region of the enzyme (Fig. 2B) (51).

Further studies will be required to evaluate the biological significance of the PCNA and -pol interaction using, among other approaches, the synthetic peptides and the -pol mutant described here. One can imagine that in the microenvironment of a BER intermediate-BER protein complex in the cell, the PCNA/-pol interaction could play an important role in the efficiency of BER by assisting in coordination among the various proteins in the complex multiprotein process of BER. PCNA, for example, also interacts with other key BER proteins, such as FEN-1 and DNA ligase I. PCNA is also known to anchor various partner proteins onto DNA and in this way PCNA can serve as a processivity factor for DNA polymerases. For DNA polymerases, one of the implications of this anchoring effect is a lower K_m for dNTP substrates, because the rate constant for polymerase-DNA dissociation is altered by PCNA. The DNA polymerase kinetic mechanism explaining this off-rate related decrease in K_m is well understood, in the cases of some well characterized DNA polymerases (63).

	ACKNOWLEDGEMENTS

We thank Drs. William A. Beard and Robert W. Sobol for discussion and critical reading of this manuscript, Donna Joyce-Gray for technical assistance, and Jennifer Myers for assistance with preparation of the manuscript. We thank Alan Clark for synthetic peptides and Dr. William C. Copeland for -pol and its antibodies.

	FOOTNOTES

^* 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.

^§ On sabbatical leave from the Dept. of Chemistry, Mokpo National University, Muan, Korea.

^¶ Present address: Stowers Inst. for Medical Research, Kansas City, MO 64110.

To whom correspondence should be addressed: NIEHS, National Institutes of Health, 111 T.W. Alexander Dr., Research Triangle Park, NC 27709. Tel.: 919-541-3267; Fax: 919-541-3592; E-mail: wilson5@niehs.nih.gov.

Published, JBC Papers in Press, June 12, 2002, DOI 10.1074/jbc.M201497200

	ABBREVIATIONS

The abbreviations used are: -pol, DNA polymerase ; BER, base excision repair; -pol, DNA polymerase ; pol , DNA polymerase ; pol , DNA polymerase ; AP, apurinic/apyrimidinic; APE, AP endonuclease; PCNA, proliferating cell nuclear antigen; FEN-1, flap endonuclease-1; PIM, PCNA interacting motif; TBS, Tris-buffered saline; TBS-T, Tris-buffered saline-Tween 20; ECL, enhanced chemiluminescence; DO3, dropout medium lacking Trp, Leu, and His; BSA, bovine serum albumin; WT, wild-type.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEEDURES RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

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