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1 Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, Noda, Chiba 278-8510, Japan
2 R and D Division of Medical and Biological Laboratories, Co., Ltd, Ina, Nagano 396-0002, Japan
3 Cyclex, Co., Ltd, Ina, Nagano 396-0002, Japan
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) was recently identified as a new member of the family X of DNA polymerases. Here, we show that Pol directly binds to proliferating cell nuclear antigen (PCNA), an auxiliary protein for DNA replication and repair enzymes, both in vitro and in vivo. A pull-down assay using deletion mutants of Pol showed that the confined C-terminal region of Pol directly binds to PCNA. Furthermore, a synthetic peptide of 20-mers derived from the C-terminal region of Pol competes with full-length Pol for binding to PCNA. The residues between amino acids 518 and 537 of Pol are required for binding to PCNA, and are different from the consensus PCNA interacting motif (PIM). Pol associates with PCNA in vivo by immunoprecipitation analysis and EGFP-tagged Pol co-localizes with PCNA as spots within a nucleus using fluorescent microscopy. Through direct binding, PCNA suppressed the distributive nucleotidyltransferase activity of Pol . Pol µ, which also belongs to the family X of DNA polymerases, binds to PCNA by a pivotal amino acid residue. |
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, 
, 
, 
, 
, , µ, 
, 
and REV1, which are believed to specifically synthesize DNA using damaged DNA template (Hubscher et al. 2000; Burgers et al. 2001; Marini et al. 2003). The DNA polymerases are classified into four families based on their amino acid sequence homology and termed families A, B, X and Y (Ito & Braithwaite 1991; Braithwaite & Ito 1993; Burgers et al. 2001; Ohmori et al. 2001). Among the families, family X includes well-studied enzymes, Pol ß and terminal deoxynucleotidyltransferase (TdT) and newly identified Pol , Pol µ and Pol (Ito & Braithwaite 1991; Braithwaite & Ito 1993; Aoufouchi et al. 2000; Dominguez et al. 2000; Nagasawa et al. 2000; Wang et al. 2000). The family X of DNA polymerases (Pol X polymerases) have evolved as nucleotidyltransferases to catalyze DNA polymerization in a distributive manner (Aravind & Koonin 1999). Apart from Pol , the Pol X polymerases are composed of a nuclear localization signal (NLS), a BRCA1 C-terminal (BRCT) domain, a proline rich region (which functions as a suppressive domain for DNA polymerase activity (SDPA)) and a Pol ß like region in their C-terminal half (Shimazaki et al. 2002). However, Pol ß lacks a NLS, a BRCT domain and a proline rich region/SDPA, which are conserved in the amino-terminal regions of other Pol X polymerases. Although Pol X polymerases share a high degree of amino acid sequence homology, the presumed functions of each member are divergent. In particular, Pol ß functions in the short-patch base excision repair (BER) pathway, which is a major pathway for repairing aprinic/apyrimidinic (AP) sites and modified bases of DNA (Wilson 1998). On the other hand, TdT expands repertories of Immunoglobulin (Ig) or T-cell receptor (TcR) gene by adding extra nucleotides at the junctions between V and D or between D and J segments during V(D)J recombination, which is a specialized form of non-homologous end joining (NHEJ) of double stranded DNA breaks (DSBs) (Gilfillan et al. 1993; Komori et al. 1993). Pol was recently identified as a member of family X (Aoufouchi et al. 2000; Garcia-Diaz et al. 2000; Nagasawa et al. 2000). Pol has a high degree of amino acid sequence homology to Pol ß, and conserves all the critical residues involved in DNA binding, substrate binding and the Pol X motif, which is an active center for nucleotidyltransfer. Indeed, Pol possesses a similar enzymatic nature to Pol ß and has, apart from template preferences (Shimazaki et al. 2002) and a high affinity for dNTP (Garcia-Diaz et al. 2002), similar requirements for cations, optimal pH conditions and NaCl concentrations. Recently, the crystal structure of truncated Pol complexed with DNA was elucidated and found to be very similar to that of Pol ß (Garcia-Diaz et al. 2004). While Pol ß shows both open and closed conformations, Pol adopts only a closed conformation, indicating that Pol has low processive DNA polymerase activity.
Pol ß possesses 5'-deoxyribose phosphate (dRP) lyase activity for removing dRP from AP sites and DNA polymerase activity to fill in the short gaps in the N-terminal 8 kDa and C-terminal 31 kDa domains, respectively (Matsumoto & Kim 1995; Piersen et al. 1996). These activities are required for processing in short-patch BER. Pol also carries dRP lyase activity in vitro (Garcia-Diaz et al. 2001), suggesting that Pol might also function in BER. Pol -deficient mice show a defect of inner dynein arms in the cilia of ependymal and respiratory epithelium, resembling the phenotype of immotile cilia syndrome (Kobayashi et al. 2002). The functional relationship between DNA polymerase activity of Pol and the development of inner dynein arms has not, however, been clarified. In addition, since mouse embryonic stem (ES) cells defective in Pol seem to have no sensitivity to various DNA damaging agents, the function of Pol could be rescued by redundancy of other DNA polymerases in ES cells. Despite these observations, recent biochemical studies have revealed that Pol is required for gap-filling and end-joining in NHEJ of DSBs together with XRCC4-LigIV complex, which is a core component of NHEJ, as demonstrated by immunodepletion analysis using HeLa cell extracts (Lee et al. 2004) and a biochemically defined in vitro NHEJ system (Ma et al. 2004).
We first reported that Pol directly binds to PCNA in vitro (Shimazaki et al. 2002), which was originally characterized as an accessory protein for DNA polymerase and functions as a DNA sliding clamp. Processivity of Pol 
, which is an essential component for eukaryotic chromosomal DNA replication, entirely depends on the PCNA by stabilizing the enzyme on a template-primer end of the replicative DNA end by specific protein–protein interactions (Einolf & Guengerich 2000). PCNA also associates with the family Y DNA polymerases Pol , Pol and Pol (Haracska et al. 2001a, b,c, 2002). Efficiency of nucleotide incorporation with the family Y DNA polymerases is stimulated by PCNA, together with a clamp loader, replication factor C (RFC) and a single-stranded DNA binding protein, replication protein A (RPA). In contrast, we recently demonstrated an inhibitory effect of PCNA on TdT activity via direct binding through the Pol ß like region of TdT (Ibe et al. 2001).
Here, we demonstrate that a confined C-terminal region of Pol is required for direct binding to PCNA in vitro. We also show that Pol associates with PCNA in vivo. This direct binding of Pol to PCNA consequently results in negative regulation for the distributive nucleotidyltransferase activity of Pol .
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contains a confined PCNA binding region in its C-terminus
During biochemical and structural studies of human Pol , we found that Pol directly binds to PCNA through its catalytic domain in vitro (Shimazaki et al. 2002). To further confine the region in Pol that directly binds to PCNA, we constructed deletion mutants of the Pol C-terminal region. Figure 1A shows the schematic representation of the deletion mutants of Pol . E. coli lysates expressing GST or GST fused proteins were incubated with purified recombinant PCNA and subsequently reacted with glutathione-Sepharose beads. Bound proteins were analyzed by Western blotting using anti-GST or anti-PCNA antibodies. As shown in Fig. 1B, the region composed of 56 residues (C4; residues 508–563) in the C-terminal region of Pol was found to be the specific region for PCNA binding. The proteins that bind to PCNA usually possess a consensus PCNA interacting motif (PIM; QXX[I/L]XXF[F/Y]) (Tsurimoto 1999; Warbrick 2000). However, Pol does not contain the typical PIM in the region between residues 508–563. The results of C3, C4 and C5 in Fig. 1 show that, despite the positive binding ability of C4, no binding was detected to C3 and C5. We therefore suspected that the residues around the C3 C-terminal or C5 N-terminal regions are crucial for binding between Pol and PCNA. Then, we designed three kinds of peptides (LP508, 513 and 518) that were composed of 20 amino acid residues derived from the Pol C-terminal region (Fig. 2A). To examine whether the synthetic peptides compete with the full-length Pol for binding to PCNA, the peptides were added to the reaction mixture containing GST fused Pol and PCNA, and a GST pull-down assay was performed. Bound proteins were analyzed by Western blotting using anti-PCNA antibodies. As shown in Fig. 2B, binding between Pol and PCNA was inhibited in the presence of LP518 but not with LP508 and LP513. From these findings, we can deduce that the amino acid sequence from residues 518–537 is required for binding to PCNA. In particular, although 15 residues overlap between LP513 and LP518, only LP 518 was able to effectively compete with full-length Pol for binding to PCNA. These results strongly suggest that the residues 533STAVV537 in LP 518 are crucial for binding to PCNA.
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PIM has been considered to bind to a hydrophobic pocket on the surface of PCNA and the interdomain-connecting loop (ICL) that joins the two structural domains of PCNA (Gulbis et al. 1996; Jonsson et al. 1998). In the present work, we elucidated a novel PCNA binding region in Pol . We then asked whether or not Pol binds to a hydrophobic pocket in PCNA. After confirmation of binding between Pol and PCNA in yeast cells (Fig. 3A), we constructed a series of deletion mutants of human PCNA and tested whether the deletion mutants of PCNA bind to Pol . Figure 3B shows the schematic diagram of the deletion mutants for PCNA. From the results of a yeast two-hybrid assay, Pol was shown to bind to the full-length PCNA, del1 and del4 but not to del2 and del3, suggesting that the central region containing ICL of PCNA is essential for binding to Pol .
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From the results showing that TdT and Pol directly bind to PCNA (Ibe et al. 2001; present study) we expected that Pol µ, which also belongs to the family X, would also directly bind to PCNA. To prove this we attempted to examine their binding ability to PCNA by a GST pull-down assay. As shown in Fig. 4, as expected, Pol µ also directly bound to PCNA. Based on these findings, we further tested whether TdT and Pol µ also bind to PCNA through their C-terminal regions as well as Pol . From these studies, using a GST pull-down assay, we found that the C-terminal 89 residues of TdT and the C-terminal 91 residues of Pol µ are responsible for PCNA binding (data not shown). We surveyed the amino acid sequences of TdT and Pol µ corresponding to the PCNA binding region in Pol and found PIM like sequences, QRELRRFS at residues 441–448 in Pol µ and also ERDLRRYA at residues 456–463 in TdT (in which the conserved residues are indicated in bold type). Figure 5A shows the amino acid sequence alignment of the PIM-like sequences in TdT and Pol µ, the PCNA binding region in Pol and the corresponding region of Pol ß. The hydropathy of the residues that are important for binding to PCNA is well conserved, especially the 7th aromatic residue that is critical within the PIM (Nakanishi et al. 1995; Warbrick et al. 1995). TdT and Pol µ conserve the 7th aromatic residue in the PIM like sequences as Y462 in TdT and F447 in Pol µ, whereas Pol has L519, which is hydrophobic and not aromatic side chain, and Pol ß has H285 at the corresponding site. We then examined whether these regions in TdT and Pol µ actually work as the PCNA binding motifs by constructing mutants, in which Y462 in TdT and F447 in Pol µ were replaced with an A. As shown in Fig. 5B, lanes 2–5, their binding to PCNA was greatly reduced compared with that of the wild-type, strongly suggesting that TdT and Pol µ bind to PCNA through conserved motifs. It was also noticeable that the Pol L519A mutant did not change its binding for PCNA (Fig. 5B, lanes 6 and 7), suggesting that Pol binds to PCNA through different sequences with a conserved motif.
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associates with PCNA in vivo
Next, to demonstrate association between Pol and PCNA in vivo, we transiently over-expressed HA-tagged Pol in HeLa cells and performed immunoprecipitation analysis with the cell extracts using specific antibodies against HA-tag bound to protein A Sepharose beads. The reaction mixture contained 100 µg/mL of ethidium bromide to inhibit DNA dependent protein–protein association (Lai & Herr 1992). Precipitated proteins were analyzed by Western blotting using specific antibodies against Pol or PCNA. As shown in Fig. 6, PCNA was selectively co-precipitated with HA-Pol . We also performed immunoprecipitation using intact HepG2 cell extracts with an anti-Pol antibody. A faint protein band of PCNA was observed after Western blotting as the co-precipitants (data not shown) since the endogenous expression level of Pol was very low. These results indicate that Pol stably associates with PCNA in vivo as well as in vitro.
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and PCNA
From our findings that Pol directly binds to PCNA in vivo as shown by immunoprecipitation analysis, we next asked whether Pol co-localizes with PCNA in a cell. Initially, to clarify the cellular localization of Pol , the cDNA-encoding enhanced green fluorescence protein (EGFP) was fused in-frame to the N-terminus of Pol (EGFP-Pol ), and the construct was transiently transfected into HeLa cells. As shown in Fig. 7B, EGFP-Pol was diffusely located within the nucleus with some brightly fluorescent spots. To exclude the possibility that the distribution of EGFP-Pol was caused by artifacts derived from the EGFP-tag, we examined the distribution of untagged Pol in a HeLa cell using a pcDNA-Pol construct and monoclonal antibodies against Pol . Since similar localizations of Pol to those of EGFP-Pol were observed (Fig. 7C), we concluded that this localization reflects the nuclear distribution of wild-type Pol .
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co-localizes with PCNA in the nucleus. Staining of endogenous PCNA using specific antibodies against PCNA showed partial co-localization with EGFP-Pol (Fig. 7D). During DNA replication, PCNA has been observed to form replication foci at the S phase nuclei (Nakamura et al. 1986; Leonhardt et al. 2000). Therefore, we asked whether Pol localizes at the replication foci through association with PCNA. HeLa cells transiently expressing Pol were pulse labeled with BrdU and replication foci were visualized using antibodies against BrdU. However, the spots of Pol were not co-localized with those of BrdU, suggesting that Pol does not participate in DNA replication at the S phase nuclei under normal conditions (data not shown).
PCNA negatively affects distributive nucleotidyltransferase activity of Pol
Whereas the DNA polymerase activity of Pol dramatically increases by direct binding to PCNA, Pol results in reduced dTMP incorporation into poly(dA)/oligo(dT) when used as a template-primer in the presence of PCNA (Shimazaki et al. 2002). As shown in Fig. 8A, Pol showed distributive nucleotidyltransferase activity with 19-mer oligonucleotides primed to a 60-mer DNA, since DNA synthesis unlimitedly increased with increasing amounts of the enzyme. Biochemical studies have shown that PCNA promotes the stability of Pol /template-primer complexes without dissociation from the DNA end (Einolf & Guengerich 2000). Therefore, we suspected that Pol is also stabilized on the template-primer by direct binding to PCNA, as seen in the case of Pol , and thus the Pol transferase activity might ultimately be inhibited. We then examined the effect of PCNA on the Pol transferase activity. As shown in Fig. 8B, DNA products synthesized were shortened with increasing amounts of PCNA, strongly suggesting that PCNA stabilizes Pol onto the template-primer.
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has newly been identified as a member of the Pol X polymerases in mammalian cells (Aoufouchi et al. 2000; Garcia-Diaz et al. 2000; Nagasawa et al. 2000). Pol has the most homologous amino acid sequence to that of Pol ß among the Pol X members and possesses both DNA-directed DNA polymerase and dRP lyase activities, but its biological function remains unclear. The direct binding of Pol to PCNA in vitro (Shimazaki et al. 2002) led to us to investigate the functional role of the Pol -PCNA complex. In this study, we have established that Pol associates with PCNA in vivo, as well as in vitro. Pol and PCNA form a stable complex in cells under physiological salt conditions as determined by immunoprecipitation analysis, and EGFP-Pol shows partial co-localization with PCNA as spots in a HeLa cell nuclei. However, the spots of Pol do not co-localize with those of BrdU, suggesting that Pol does not participate in DNA replication at the S phase nuclei under normal conditions. Pol is a low processive and error-prone DNA polymerase, with low fidelity and high frame-shift infidelity (Bebenek et al. 2003). Therefore, it is conceivable that Pol does not participate in processive DNA replication, which relies on DNA polymerases with high processivity and high fidelity (e.g. Pol , Pol 
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The proteins that directly bind to PCNA usually possess a consensus PCNA interacting motif PIM; [Q1X2X3(L/I/M)4X5X6F7(F/Y)8] at the N- or C-terminus (Tsurimoto 1999; Warbrick 2000). In particular, M4 and F7 of the PIM in the regulatory protein of the cell-cycle dependent kinase p21 are crucial for recognition of PCNA (Nakanishi et al. 1995; Warbrick et al. 1995). Recently, Xu et al. (2001) identified a novel PCNA binding motif, termed the KA-box [KA-(A/L/I)-(A/L/Q)-x-x-(L/V)], using a proteomics approach with a random peptide library. This motif is also present in several PCNA binding proteins (e.g. Pol , Pol , RFC). However, the other proteins without these consensus motifs (e.g. CAF-1 p150, GADD45) also directly bind to PCNA (Warbrick 2000). In the present study, Pol has been revealed to have a novel PCNA binding region in its C-terminal region, which is related to neither the PIM nor the KA-box. According to the crystal structure of the C-terminal half of the human Pol -gapped DNA complex (Garcia-Diaz et al. 2004), the region corresponding to the PCNA binding region in Pol (residues 508–563) contains a long loop protruding on the surface of Pol (Fig. 9). Furthermore, the region corresponding to the Pol peptide LP518 (residues 518–537) contains ß-strands 6, 7 and the N-terminus of the long loop structure, which is buried in the vicinity of the DNA. Interestingly, the loop in Pol is larger than that of Pol ß and includes a ß-strand 8 that is not present in other Pol X members. Therefore, we could expect that these unique structures of Pol are crucial for binding to PCNA.
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, the results of the yeast two-hybrid assay strongly suggest that the central region of PCNA containing the ICL is involved in binding to Pol . Since human PCNA forms a homo-trimeric ring, it is possible that three different molecules could bind to the surface of PCNA. Then, we examined the stoichiometry of the Pol -PCNA complex using a BMH cross-linker. We detected three forms of PCNA (monomer; 32 kDa) by SDS-PAGE after BMH cross-linking analysis as the control. When Pol (monomer; 68 kDa) was added to the PCNA solution, a clear band was observed at a position of 160 kDa after cross-linking, which coincides with the stoichiometry of one molecule of a Pol /a PCNA trimer (date not shown).
PCNA has been well characterized as an auxiliary protein for Pol , which functions as an essential component for chromosomal DNA replication. Processivity of Pol depends entirely on PCNA, by holding enzymes on a template-primer end of replicative DNA without dissociation. Recent studies have shown that PCNA is involved in many aspects of DNA transaction, forming a sliding platform that can mediate the protein–protein interaction (Tsurimoto 1999; Warbrick 2000). The family Y DNA polymerases Pol , and , which are believed to function in translesion DNA synthesis, have also been revealed to bind directly to PCNA. PCNA slightly promotes family Y DNA polymerase activity by reduction of the Km value together with replication factor C (RFC) and replication protein A (RPA) (Haracska et al. 2001a,b,c, 2002). In the case of Pol , Pol transferase activity was decreased in the presence of PCNA without other associated components. PCNA fundamentally functions as DNA sliding clamp and stabilizes the DNA polymerase/template-primer complex to enhance DNA polymerase activity, such as Pol , when it works in a processive manner. On the other hand, we hypothesize that the same function of PCNA, which stabilizes the DNA polymerase/template-primer complex, offers negative effects on DNA polymerase when it works in a distributive manner; namely, after addition of a nucleotide to the primer end, the DNA polymerase is released from the site without being held at the template-primer end.
We showed that Pol µ, which has also recently been identified as a Pol X polymerase and has a highly homologous amino acid sequence to TdT, also directly binds to PCNA. TdT and Pol µ have their own PCNA binding regions and are different to that of Pol . When the residues with an aromatic side chain in the PIM like sequence within Pol µ and TdT, which are considered to be essential for binding to PCNA, were mutated, binding of TdT and Pol µ to PCNA greatly reduced. Ibe et al. reported that TdT directly binds to PCNA through the entire Pol ß-like region as determined by a yeast two-hybrid assay after construction of a series of TdT deletion mutants (Ibe et al. 2001). In our findings, however, the confined region containing the PIM in TdT directly bound to PCNA, and when the amino acid in the PIM was mutated direct binding between them was reduced, as determined by a pull-down assay, indicating that binding is through the conserved PIM. The difference in binding regions is considered to be due to the different analysis methods used, namely a yeast two-hybrid assay in vivo and a pull-down assay in vitro. Based on our findings showing that TdT, Pol and Pol µ directly bind to PCNA, we tested whether the Pol peptide also competes with TdT and Pol µ for binding to PCNA. As a result, no inhibitory effect was observed with regards to their binding to PCNA (data not shown). By yeast two-hybrid screening using TdT as bait, the C-terminal region of PCNA was isolated, but this did not include the ICL of PCNA. In the present work, we showed that the central region containing the ICL of PCNA is involved in binding to Pol by the yeast two-hybrid assay. Therefore, it is reasonable to assume that TdT and Pol µ do not compete with Pol for binding to PCNA. These facts raise the possibility that Pol X polymerases could bind to PCNA individually without competing with each other and might form a large complex through the PCNA.
Three members of Pol X polymerase, TdT, Pol and Pol µ, are candidates for processing in NHEJ of repairing DSBs. TdT is a lymphoid specific enzyme and its expression is restricted to immature lymphocytes. TdT can synthesize DNA in a template independent manner and specifically add extra nucleotides at the junctions between V and D or between D and J segments during V(D)J recombination, which is a specialized form of NHEJ (Gilfillan et al. 1993; Komori et al. 1993). Pol µ has been reported to associate with Ku, the XRCC4-LigIV complex, which are core NHEJ components (Mahajan et al. 2002). Moreover, a genetic knockout study of Pol µ revealed that Pol µ is involved in immunoglobulin light chain gene rearrangement, indicating that Pol µ functions in V(D)J recombination (Bertocci et al. 2003). From other approaches used to investigate which Pols are involved in the NHEJ, Pol has been shown to be required for gap-filling and end-joining in NHEJ together with the XRCC4-LigIV complex, as determined by immunodepletion analysis using HeLa cell extracts (Lee et al. 2004). Very recently, Ma et al. (2004) demonstrated that three members of Pol X polymerase, TdT, Pol and Pol µ, specifically contribute to the processing of NHEJ together with Ku and the XRCC4-LigIV complex using a biochemically defined in vitro NHEJ system. Therefore, based on these facts and our findings showing that Pol , TdT and Pol µ directly bind to PCNA, we suspect that PCNA regulates the polymerization activities of these Pols at the DSB junction. This hypothesis is supported by evidence that DNA-PKcs and Ku70/80, core NHEJ components, were recovered from eluted nuclear extracts of a human cell after PCNA fixed resin affinity chromatography (Ohta et al. 2002). These observations strongly suggest that PCNA plays a significant role in NHEJ of DSBs.
In conclusion, we have shown that human Pol directly binds to PCNA through its C-terminal region both in vitro and in vivo. The consequence of the binding is negative regulation of its nucleotidyltransferase activity. We also found that the three members of Pol X polymerase, TdT, Pol and Pol µ, directly bind to PCNA.
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Construction of Pol and PCNA expression vectors, and expression and purification of the protein have all been previously described (Ibe et al. 2001; Shimazaki et al. 2002). Cloning of full-length cDNA for human TdT has also been previously described (Ibe et al. 2001).
Full-length cDNA for human Pol µ was amplified by the polymerase chain reaction (PCR) using the primers M-F1 (5'-GAAGAATTCATGCTCCCCAAACGGCGGCGAGCG-3') and M-R1 (5'-GCACTCGAGGGCGTTTCTCTGCTCTGGAGGAAG-3') from a human placenta cDNA library (Clontech). M-F1 contains a EcoRI site and M-R1 contains a XhoI site, respectively (underlined). PCR was performed following a standard procedure.
Site-directed mutantagenesis of Pol and TdT was performed by PCR using a Quick Change site-directed mutagenesis kit from Stratagene. A pair of mutagenic primers was used for the Pol mutant L519A (5'-CGCTCCATGCGAGCGGCGGCCAAAACCAAGGG-3' and 5'-CCCTTGGTTTTGGCCGCGGCTCGCATGGAGCG-3') and for the TdT mutant Y462A the primers 5'-AGAGACCTCCGGCGCGCTGCCACACATGAGCG-3' and 5'-CGCTCATGTGTGGCAG CGCGCCGGAGGTCTCT-3' were used. Pol µ mutant F477A was generated by PCR using a pair of mutagenic primers (5'-AAGCTTTTCCAGCGGGAGCTGCGCCGCGCCAGCC-3' and 5'-GCACTCGAGGGCGTTTCTCTGCTCTGGAGGAAG-3'). A cDNA fragment of the Pol µ mutant was substituted with the corresponding region of wild-type cDNA for Pol µ. The nucleotide sequences were determined by the dideoxy termination method. The cDNA fragments containing wild-types or each mutation were subcloned into pGEX vectors (Amersham Bioscience).
Full-length cDNA for Pol was subcloned into EcoRI and SalI sites of pEGFP-C2 vector (Clontech) to produce EGFP tagged protein and a pcDNA vector (Invitrogen) to produce untagged protein.
Antibodies
Rabbit polyclonal and mouse monoclonal antibodies against Pol were produced by immunization with the recombinant Pol using a standard procedure. Monoclonal anti-PCNA antibody (PC10) was purchased from Dako, monoclonal anti-HA tag antibody from MBL and rabbit polyclonal anti-GST antibody was from ABR Inc. The secondary antibodies for immunoblotting, namely horseradish peroxidase (HRP) conjugated goat anti-mouse IgG antibody and HRP conjugated horse anti-mouse IgG antibody, were purchased from New England Biolabs. The secondary antibodies used for immunofluorescence, namely Alexa Fluor 546 F(ab')2 fragment of goat anti-mouse IgG (H+L), was purchased from Molecular Probe.
GST pull-down assay
A GST pull-down assay was performed according to previously described methods (Shimazaki et al. 2002). E. coli cells expressing GST or GST fused proteins were lyzed and the crude cell extracts were mixed with 0.5 µg of purified PCNA in binding buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM DTT, 0.1% Nonidet P-40, 10% glycerol, 0.1% BSA, 1 mM PMSF, 1 mM benzamidine, 10 µg/mL aprotinin, 5 µg/mL leupeptin and 2 µg/mL pepstatin A). The protein complex was coupled to Glutathione Sepharose beads (Amersham Bioscience) and binding proteins were separated by SDS-PAGE. Following transfer onto nitrocellulose membrane, immunoblotting was performed with both anti-GST and anti-PCNA antibodies.
For the competition experiments, 20-mer peptides (LP508, LP513 and LP518) were synthesized to cover the C-terminus of Pol (QIAGEN). Each peptide was dissolved in water to a final concentration of 2–10 mg/mL and stored at –80 °C. To examine whether the full-length Pol and synthetic peptides compete for binding to PCNA, a GST pull-down assay was performe with full-length Pol and PCNA in the presence of increasing amounts of the peptides. Binding proteins were separated by SDS-PAGE and analyzed by immunoblotting with both anti-GST and anti-PCNA antibodies.
Yeast two-hybrid assay
Yeast Y190 cells were co-transformed with bait plasmids pAS2-1 or pAS2-1-Pol and prey plasmids pACT2, pACT2-PCNA or pACT2-PCNA deletion mutants and grown on selective DO2 plates. Protein–protein interactions were determined by a ß-galactosidase assay according to the manufacturer's protocol (MATCH MAKER II from Clontech).
Cell culture and transfection
HeLa cells were grown at 37 °C under a humidified atmosphere containing 5% CO2 with Dalbecco's modified Eagle's medium (Gibco BRL) supplemented with 10% foetal bovine serum. Transfection of plasmid DNA was carried out using Lipofect AMINE plus (Invitrogen) according to the manufacturer's protocol. All operations were carried out at room temperature.
Immunoprecipitation
HeLa cells transfected with HA-Pol expressing plasmids were harvested and washed with PBS. Cells were lyzed on ice with lysis buffer (25 mM HEPES-KOH, pH 7.4, 200 mM NaCl, 1% NP40, 10% glycerol, 1 mM DTT, 1 mM PMSF, 1 mM benzamidine, 10 µg/mL aprotinin, 5 µg/mL leupeptin and 2 µg/mL pepstatin A) and centrifuged. Anti-HA tag antibody or normal mouse IgG that bound to protein A Sepharose beads (Amersham Bioscience) were added to the soluble cell extract, and binding was allowed to proceed for 2 h at 4 °C. After washing extensively with the same buffer, the immunobeads were denatured in SDS sample buffer, fractionated on a 10% SDS-PAGE gel, and then analyzed by immunoblotting with both anti-Pol and anti-PCNA antibodies.
Immunofluorescence microscopy
HeLa cells were grown on coverslips and transfected with plasmids encoding EGFP or untagged Pol . All operations were carried out at room temperature unless otherwise indicated. For visualization of EGFP-Pol , the cells transfected with EGFP-Pol were rinsed twice in PBS and fixed with cold methanol, then mounted with PBS containing 4,6-diamidio-2-phenylindole (DAPI). For detection of untagged Pol , fixed cells with cold methanol were incubated with 1% BSA in PBS for 30 min and subsequently reacted with monoclonal Pol antibody, followed by Alexa Fluor 546 conjugated goat anti-mouse IgG. For detection of PCNA, fixed cells with cold methanol and acetone were blocked with 1% BSA in PBS and subsequently reacted with PC10, followed by Alexa Fluor 546 conjugated goat anti-mouse IgG. The cells were imaged on an Axiovert 200 microscope with an AxioCam HRc (Carl Zeiss).
In vitro primer extension assay
5'-IRD800 dATP labeled M13 forward (-29) primer 5'-CACGACGTTGTAAAACGAC-3' (LI-COR) was annealed to a 60 mer template oligonucleotide 5'-CTCTAGAGTCGACCTGCAGGCATGCATGCAAGCTTGGCACTGGCCGTCGTTTTACAACGTCGTG-3' in a stoichiometric ratio of 1 : 1. The reaction mixture (10 µL) contained 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 2 mM MgCl2, 1 mM DTT, 200 µg/mL BSA, 5 µM dTTP, 1 pmol of template-primer and proteins as indicated in Fig. 8. The reaction mixture was incubated at 37 °C for 30 min and then stopped by adding formamide gel loading solution. The products were resolved on a 9% Long ranger (FMC) gel containing 8 M urea and visualized using a dNA Analyzer Gene Reader 4200 (ALOKA).
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Acknowledgements |
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* Correspondence: E-mail: snoriko{at}rs.noda.tus.ac.jp
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References |
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Received: 28 February 2005
Accepted: 29 March 2005
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