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1 Institute for Virus Research, Kyoto University, 53 Shogoin-Kawaracho, Sakyo-ku, Kyoto 606-8507, Japan
2 Graduate School of Medicine, Nagoya University, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan
3 Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan
4 RIKEN, DRI and CREST, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
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Abstract |
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is one of many DNA polymerases involved in translesion DNA synthesis (TLS). It belongs to the Y-family of polymerases along with Pol
, Pol
and hREV1. Unlike Pol encoded by the xeroderma pigmentosum variant (XPV) gene, Pol is unable to bypass UV-induced DNA damage in vitro, but it is able to bypass benzo[a]pyrene (B[a]P)-adducted guanines accurately and efficiently. In an attempt to identify factor(s) targeting Pol to its cognate DNA lesion(s), we searched for Pol-interacting proteins by using the yeast two-hybrid assay. We found that Pol interacts with a C-terminal region of hREV1. Pol and Pol were also found to interact with the same region of hREV1. The interaction between Pol and hREV1 was confirmed by pull-down and co-immunoprecipitation assays. The C-terminal region of hREV1 is known to interact with hREV7, a non-catalytic subunit of Pol
that is another structurally unrelated TLS enzyme, and we show that Pol and hREV7 bind to the same C-terminal region of hREV1. Thus, our results suggest that hREV1 plays a pivotal role in the multi-enzyme, multi-step process of translesion DNA synthesis. |
Introduction |
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, Pol, Pol and REV1) (Ohmori et al. 2001) and one B-family DNA polymerase (Pol). In Saccharomyces cerevisiae, Pol consists of two subunits: REV3, the catalytic subunit with a high similarity to the catalytic subunit of Pol
and an accessory subunit, REV7, of undefined function (Nelson et al. 1996b). Recently, a human homologue of yeast REV7 was identified (designated hREV7) (Murakumo et al. 2000) and shown to interact with a C-terminal region of hREV1, as well as with hREV3, but it did not form a stable REV3-REV7-REV1 triple complex (Murakumo et al. 2001). The C-terminal sequences are not conserved between the yeast and human REV1 proteins, suggesting that the interaction between hREV1 and hREV7 has been acquired during evolution.
The presence of multiple TLS enzymes in human cells raises a question of what function each of them exerts within the cells. There is an accumulating body of evidence from in vitro experiments that they have different, but partially overlapping, specificity for lesion-bypass. For example, Pol, which is deficient in xeroderma pigmentosum variant-type patients that are predisposed to sunlight-induced skin cancer (Masutani et al. 1999a, 1999b; Johnson et al. 1999), can bypass a thymine-thymine (T-T) cyclobutane pyrimidine dimer (CPD) in vitro with the same efficiency and accuracy with which it replicates undamaged T-T (Prakash & Prakash 2002). While Pol does not bypass the more structurally distorting T-T (6–4) photoproduct, it can bypass a variety of other DNA lesions with varying degrees of efficiency and fidelity (Prakash & Prakash 2002). In contrast, Pol does not bypass T-T CPDs or (6–4) photoproducts (Gerlach et al. 2001; Johnson et al. 2000a; Ohashi et al. 2000b). It is, however, able to bypass benzo[a]pyrene (B[a]P)-adducted guanine (dG-N2-BPDE adducts) efficiently and accurately by inserting dC opposite the bulky lesion (Rechkoblit et al. 2002; Suzuki et al. 2002; Zhang et al. 2000a, 2002a). Pol-defective mutant cells isolated from a mouse embryonic stem (ES) cell line showed a hypersensitivity to B[a]P treatment and accumulated mutations, especially G-to-T substitutions, upon B[a]P treatment at a frequency that was 10-fold higher than the parental ES cells (Ogi et al. 2002). The enhanced level of G-to-T mutations in the Pol-defective mutant cells was presumably caused by insertion of dA opposite B[a]P-adducted guanines by Pol (Chiapperino et al. 2002; Rechkoblit et al. 2002; Zhang et al. 2000b).
The yeast and mammalian REV1 proteins insert dC opposite abasic sites in vitro (Lin et al. 1999; Masuda et al. 2001; Nelson et al. 1996a) and experiments with antisense RNAs to hREV1 and hREV3 revealed that reduction in hREV1 or hREV3 expression results in a decreased level of UV-induced mutagenesis (Gibbs et al. 1998, 2000). Interestingly, a S. cerevisiaerev1 mutant (rev1-1) that retains its dCMP transferase activity, exhibited decreased levels of UV-induced mutagenesis, suggesting that yeast REV1 has a second function in UV-induced mutagenesis, in addition to its dCMP transferase activity (Nelson et al. 2000). Pol inserts one or two dAs opposite (6–4) T-T photoproducts, and the inserted bases can then be extended by Pol (Johnson et al. 2000b; Tissier et al. 2000). Pol also inserts T opposite dA-N6-BPDE adducts, but is unable to bypass the lesion without the assistance of another polymerase, such as Pol (Frank et al. 2002).
Another important question relates to how each of such TLS enzymes is recruited to its cognate lesion. For example, for error-free bypass of T-T CPD or dG-N2-BPDE, Pol or Pol, respectively, needs to be recruited to the lesion. Furthermore, due to the intrinsic low-fidelity of the TLS enzymes, once bypass has occurred the TLS enzyme needs to be quickly replaced by the highly processive and accurate replicative enzymes (Pol and/or Pol
) to avoid gratuitous mutations in the non-damaged region of template DNA. How such a switching from one DNA polymerase to another occurs at a DNA lesion is largely unknown. Recent data suggest that a key step may be the post-translational modification of PCNA (Hoege et al. 2002; Steler & Ulrich 2003). A study on the localization of Pol has suggested that the enzyme localizes in replication foci in 10–15% of undamaged cells and these foci accumulate in most of UV-irradiated cells (Kannouche et al. 2001). More recently, Pol was found to co-localize with Pol, with or without DNA damaging treatment (Kannouche et al. 2003). In this paper, we present evidence indicating that Pol, Pol and Pol, as well as hREV7, interact with the C-terminal portion of hREV1. These results imply that TLS enzymes form a transient multiprotein complex, in which hREV1 seems to play a central role.
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Results |
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We assumed that there might be one or more factor(s) that help recruit TLS enzymes to a particular DNA lesion site. To search for such proteins, and especially those interacting with Pol, we employed yeast two-hybrid screening with the full-length Pol as bait. The coding region of hPOLK-cDNA was fused in frame with the E. coli LexA DNA binding domain in pLexA (HIS3, AmpR) so as to construct pLexA-POLK. A yeast strain (EGY48; MATa, his3, trp1, ura3, LexAop(x6)-LEU2) carrying a reporter plasmid, p8op-lacZ (URA3, AmpR) was transformed with pLexA-POLK and a human testis cDNA library constructed in pB42AD (TRP1, AmpR). Approximately 5 x 106 independent Ura+ His+ Trp+ transformants were screened on the SD/–Ura, -His, -Trp, -Leu, +X-gal plates. One hundred and eight clones with blue colour were obtained on the selective plates, all of which were subjected to DNA sequence analysis. Sequencing revealed that among 108 positive clones, 34 contained a C-terminal portion of hREV1. Furthermore, these clones exhibited a darker blue colour than the other positive clones not containing an hREV1 fragment. These results led us to pursue the possibility that Pol may interact with the C-terminus of hREV1.
To further localize the region of hREV1 that interacts with Pol, we made use of a series of hREV1 deletion derivatives that were previously used to study the interaction between hREV1 and hREV7 (Murakumo et al. 2001). In that study, hREV7 was shown to bind to 1130–1251 amino acids at the very C-terminus of hREV1 (Murakumo et al. 2001). The results shown in Fig. 1A indicate that Pol also interacts with the same region, since a small deletion of the 33 residues (1219–1251) from the C-terminus of hREV1 abolished the binding to both Pol and hREV7 (Fig. 1A) (Murakumo et al. 2001).
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to identify which portion of the polymerase is required for the interaction with hREV1. The N-terminal region (1–560 amino acids) of Pol contains multiple sequence motifs conserved among the Y-family DNA polymerases, and is sufficient for bypass activity (Ohashi et al. 2000b), while the C-terminal region (561–870 amino acids) is considered necessary for exhibiting a higher processivity (Ohashi et al. 2000a). Sequences similar to a bipartite nuclear localization signal (NLS) and a consensus PCNA binding site are found at the extreme C-terminus of Pol (Gerlach et al. 1999; Haracska et al. 2002). As shown in Fig. 1B, a central region (560–615 amino acids) of Pol is necessary and sufficient for binding to hREV1. We hereafter designate this central region as the hREV1-intereacting domain of Pol.
Similarly, we carried out two-hybrid assay experiments to examine whether Pol and Pol might interact with hREV1. Interestingly, it turned out that Pol and Pol also interact with the same C-terminal portion of hREV1. The 1125–1251 amino acids of hREV1 was sufficient for the interaction with Pol, and a further deletion mutant carrying the 1165–1251 amino acids lost the activity to interact with Pol (data not shown). Also, as shown for the interaction with Pol in Fig. 1A, the pLexA derivative carrying the 1085–1251 or 1130–1251 amino acids of hREV1 was positive for the interaction with Pol, but the derivative carrying the 1085–1218 amino acids was negative (data not shown). As indicated in Fig. 1, different vector systems were used to examine the interaction between hREV1 and Pol or Pol, the LexA-based system constantly exhibited higher ß-galactosidase activities. In fact, the full-length of Pol exhibited an activity similar to that of Pol when cloned in pLexA; however, it showed little activity when cloned in GAL4-based pGBKT7 vector probably due to the masking of the interaction domain. Nevertheless, an internal fragment of Pol (449–589 amino acids) gave a clear positive phenotype in pGBKT7 (Fig. 1D). Thus, we conclude that Pol 509–557 amino acids and Pol 449–589 amino acids are essentially required for the interaction with hREV1. Both of them are located downstream of the catalytic domains, as in the case of Pol. However, we do not find any obvious consensus sequence in the hREV1-interacting domains of Pol, Pol, Pol and hREV7.
Physical interaction between Pol and hREV1
To study the physical interaction between Pol and hREV1, we carried out an in vitro pull-down assay with the purified proteins. GST (Glutathione S transferase) was fused in frame to the N-terminus of hREV1 fragment containing a central (387–825 amino acids) or C-terminal portion (826–1251 amino acids). The GST-fusion proteins were overproduced in E. coli cells and immobilized on Glutathione-Sepharose beads. The beads were subsequently incubated with His-tagged Pol (1–560 or 1–615 amino acids), which were also overproduced in and purified from E. coli cells. The total (T) proteins and bound (B) faction (loaded in 5-fold excess compared with the total proteins) were resolved by SDS-PAGE and probed with anti-His antibody. As shown in Fig. 2, an interaction between Pol and hREV1 was detected when Pol contained the hREV1-interacting domain (561–615 amino acids) and hREV1 contained the C-terminal region, but not when either one lacked their respective binding domain. We therefore conclude that Pol and hREV1 interact with each other directly.
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or Pol
To verify the interaction between Pol and hREV1 in vivo, we performed a co-immunoprecipitation assay. Since endogenous level of hREV1 in human cells was undetectable by anti-hREV1 antibodies, we over-expressed a tagged recombinant protein. The full-length hREV1 or its truncated form hREV1
C lacking the C-terminal portion (731–1251 amino acids), was fused in frame with GFP (Green Fluorescent Protein) at its N-terminus and co-expressed in 293T cells with the full-length Pol or its deletion mutant Pol (561–614) that was also tagged with FLAG at the N-terminus. Cell extracts were prepared and incubated with anti-FLAG antibody, and the immunoprecipitated fractions (P) were resolved by SDS-PAGE and then probed with anti-GFP antibody, in parallel with the total (T) and supernatant (S) fractions. The results shown in Fig. 3A indicate that co-immunoprecipitation of hREV1 and Pol was observed only when the two proteins contained their interacting domains, but not with constructs of Pol lacking the central hREV1-interacting domain or hREV1 lacking the C-terminal region. In addition to the interaction between Pol and hREV1, we were also able to detect the co-immunoprecipitation of GFP-hREV1 with FLAG-Pol (Fig. 3B). Thus, we conclude that REV1 physically interacts with Pol and Pol, and probably with Pol, in vivo.
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and hREV7 simultaneously. Cell extracts from 293T cells overexpressing GFP-hREV1 and FLAG-Pol were immunoprecipitated with either anti-FLAG or anti-GFP antibody, and the precipitates were then immunoblotted with anti-GFP, anti-FLAG or anti-hREV7 antibody. When GFP-hREV1 was precipitated with anti-GFP antibody, we could detect hREV7, as well as FLAG-Pol, in the pellet fraction, indicating that a fraction of the GFP-hREV1 proteins formed a complex both with hREV7 and Pol. In contrast, when FLAG-Pol was precipitated with anti-FLAG antibody, we could detect GFP-hREV1, but not hREV7, in the pellet fraction, while hREV7 was detected in the supernatant. This result implies that GFP-hREV1 does not form a triple complex with Pol and hREV7.
Co-localization of hREV1 and Pol
To study complex formation between Pol and hREV1 in vivo by a different approach, we examined whether the two proteins co-localize within human cells. GFP-Pol and FLAG-hREV1 were transiently overexpressed in normal MRC5 fibroblasts. After fixation, FLAG-hREV1 was stained using an anti-FLAG antibody and a Rhodamine-conjugated secondary antibody, while GFP-Pol was detected by green fluorescence. Without any DNA damaging treatment, a small percent of the cells expressing Pol and hREV1 exhibited focus formation, while the remaining majority of the cells showed a homogeneous distribution of the proteins or sometimes with a small numbers of speckles. Figure 4A shows almost complete co-localization of GFP-Pol and FLAG-hREV1. Similar co-localizations were observed when GFP-hREV1 and FLAG-Pol were used (Fig. 4B). The frequency of foci formation increased in cells expressing both GFP-Pol and FLAG-hREV1 compared with cells expressing either GFP-Pol or FLAG-hREV1 alone (for example, 17.5% vs. 4.5% or 6%, respectively, when measured in one experiment). When we used GFP-Pol (561–614) lacking the hREV1 interacting domain, co-localizations with FLAG-hREV1 were also observed, although at a reduced frequency (data now shown).
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Discussion |
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(561–615 amino acids), Pol (509–557 amino acids) and Pol (449–589 amino acids), as well as hREV7, the non-catalytic subunit of Pol. The hREV1-interacting domains in Pol, Pol and Pol are located in the region downstream of the catalytic domain in each of the three enzymes. The interaction between Pol and hREV1 was further confirmed through an in vitro pull-down assay and co-immunoprecipitation of the two proteins overexpressed in human cells. Since the co-immunoprecipitation of hREV1 with Pol was also observed, it seems very likely that similar interactions of hREV1 with Pol and Pol occur in human cells. As shown in Fig 4, Pol and hREV1 co-localize within human cells without any DNA damaging treatment. However, because the hREV1-interacting domain of Pol is not required for its focus formation, such co-localization does not depend on the direct interaction between Pol and hREV1, suggesting that another factor(s) mediates localization into replication machineries. A previous study with Pol (Kannouche et al. 2001) showed that the C-terminal 119 residues of Pol (595–713 amino acids) were necessary and sufficient for its localization into replication foci. This domain contains a putative C2H2 zinc finger, a bipartite NLS and a potential PCNA-binding site, the latter two of which are well conserved in the extreme C-terminal region of Pol. Clearly, the hREV1-interacting domain in Pol is also well separated from the region required for its focus formation.
Although experimental evidence indicating that the interaction with hREV1 is essential for in vivo TLS is still lacking, we may well discuss the following two possibilities as to how such interactions might be involved in multi-step TLS events, that are not necessarily mutually exclusive. One model is that TLS occurs sequentially, as originally suggested for the bypass of abasic site by yeast REV1 and Pol (Nelson et al. 1996a). Human REV1 is also known to insert dC opposite an abasic site, but cannot extend further (Masuda et al. 2001). Another enzyme, for example Pol or Pol, might continue elongation. To test this possibility, we examined whether hREV1 and Pol co-operate to enhance bypass synthesis past an abasic site in vitro in the absence of other replication factors such as PCNA, RPA and RFC. In these assays, hREV1 and Pol inserted dCMP and dAMP, respectively, opposite the abasic site and at a very similar efficiency. While some additive effects were observed by adding the two enzymes compared with either polymerase alone, the same effects were, however, observed when we used a truncated one of Pol (1–560 amino acids) lacking the hREV1-interacting domain instead of the intact form (1–870 amino acids) (data not shown). It therefore seems very likely that the in vitro system does not reflect the in vivo TLS events and further experiments are required. In the case of dG-N2-BPDE, Pol was shown to continue synthesis after hREV1 inserts dCMP opposite the bulky adduct (Zhang et al. 2002b). But, the results did not indicate any effect due to the direct interaction between the two enzymes. The other model is that hREV1 plays a second role other than dCMP transferase (Nelson et al. 2000; Haracska et al. 2001). Both yeast and human REV1 proteins have a BRCT domain near their N-terminus. The BRCT domain is present in many DNA repair proteins and functions as an interface for interactions with other protein(s), especially those that are phosphorylated (Manke et al. 2003; Yu et al. 2003). In fact, in the case of the yeast REV1, a mutation in the BRCT domain abolished mutagenesis after UV-irradiation, while retaining its dCMP transferase activity (Nelson et al. 2000). hREV1 might therefore help mediate one of the proteins, such as Pol, Pol, and Pol, bound to its C-terminus to interact with a protein bound to the BRCT domain, thereby guiding a TLS enzyme to a DNA lesion site. It is tempting to speculate that a protein may interact with hREV1 at the BRCT domain after phosphorylation by a DNA damage checkpoint mechanism so as to transfer a signal for the presence of DNA damage.
Lastly, we wish to discuss the evolutionary meaning of the interactions among TLS enzymes in higher eukaryotes. As mentioned above, a functional interaction between the yeast REV1 and Pol composed of REV3 and REV7 subunits, was originally suggested for bypass of an abasic site (Nelson et al. 1996b); however, a physical interaction between yeast REV1 and Pol has not been demonstrated thus far. Such an interaction was shown to occur between hREV7 and the C-terminal region of hREV1, which is not conserved in yeast REV1 (Murakumo et al. 2001). Our present study showed that the same C-terminal region of hREV1 is able to interact with other TLS enzymes such as Pol, Pol and Pol. One might well consider such interactions have been acquired during evolution to increase the efficiency of TLS in higher eukaryotes (Lehmann 2000). TLS events, error-free or error-prone, could be a more effective response to cope with DNA damage than more accurate, but less efficient, DNA recombination repair mechanism, especially in higher eukaryotes with more non-coding sequences than in lower eukaryotes with protein-coding sequences dominant in the genomes.
Most recently, we have learned that similar results on mouse Y-family enzymes were obtained by Friedberg and his coworkers (Guo et al. 2003). This work was done independently.
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Experimental procedures |
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For purification of His-tagged truncated Pol proteins, POLK-cDNA fragment carrying its 1–560 or 1–615 amino acids region was inserted into pQE9 (Qiagen). For in vitro binding assay, hREV1-cDNA fragment carrying its 387–825 or 826–1251 amino acids region was inserted into pGEX4T-2 (Amersham Biosciences) to produce a glutathione S-transferase (GST) fusion protein. For expression of hREV1 and Pol in human cells, the full-length or truncated fragment of hREV1- or POLK-cDNA sequence was inserted into pEGFP-C2 (CLONTECH) to produce a GFP-fusion protein. POLK- or hREV1-cDNA tagged with the FLAG sequence at the N-terminus was cloned into vector pcDNA3.1(+) (Invitrogen) to produce FLAG-tagged proteins.
Cell culture and reagents
Human 293T cells and MRC5 cells were grown in D-MEM medium supplemented with 10% foetal bovine serum. For transient expression, cells were transfected by using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. Cells were harvested 48 h after transfection for further analysis.
Yeast two-hybrid assay
First, pLexA-POLK carrying the full-length sequence of the POLK coding region, was used as bait for yeast two-hybrid library screening. The screening was performed in the yeast strain, EGY48, using the LexA-based two-hybrid system (Clontech) according to the manufacturer's protocol. Approximately 5 x 106 independent clones from a human testis cDNA library constructed in pB42AD were screened for an interaction with pLexA-POLK. Plasmid DNAs from the positive clones were extracted and subjected to DNA sequence analysis using the Big Dye Terminator cycle sequencing Kit (Applied Biosystems). To examine the interaction between hREV1 and Pol we used pPC86 and pDBLeu (PROQUEST two-hybrid system from Gibco BRL). To examine the interaction between hREV1 and Pol we used the pGBKT7 and pGADT7 plasmid system (Clontech). Quantitative ß-galactosidase assays were performed according to the respective manufacturer's protocol.
Purification of His-tagged Pol(1–560) or Pol(1–615)
E. coli JM109 cells carrying the plasmids derived from BL21 codonplus (DE3)-RIL (Stratagene) were transformed with the expression plasmid overproducing a truncated Pol protein and the transformants were grown at 37 °C in 1.5 L of L-broth. When the OD600 reached 0.6, isopropyl-1-thio-ß-D-galactopyranoside (IPTG) was added to a final concentration of 1 mM. After 3 h further incubation, cells were harvested by centrifugation at 4 °C and stored at –80 °C. Purification of the truncated pol proteins was performed at 4 °C. Frozen cells were thawed and resuspended in 20 mL of A buffer (20 mM sodium phosphate pH 7.4, 0.5 M NaCl, 10% glycerol). Lysozyme and phenylmethanesulphonyl fluoride (PMSF) were added to a final concentration of 0.1 mg/mL and 0.1 mM, respectively. After incubation on ice for 30 min, the cells were lysed by sonication, and the cell lysates centrifuged at 32 000 r.p.m. For 30 min. The supernatant was applied to a Hi Trap Chelating column (Amersham Biosciences) connected to an FPLC system. The column was washed with a buffer containing 50 mM imidazole and then developed with a linear gradient of imidazole up to 500 mM. The truncated Pol proteins were eluted at approximately 140 mM imidazole. Fractions containing the truncated Pol proteins were combined and dialysed overnight against B buffer (20 mM sodium phosphate pH 7.4, 50 mM NaCl, 10% glycerol) and loaded on to Hi Trap SP Sepharose FF (Amersham Biosciences). The column was washed with B buffer and subsequently developed with a linear gradient of NaCl up to 1M. The truncated Pol proteins were eluted at an approximately 300 mM NaCl. About 4 mg of purified truncated Pol proteins were obtained from the original 1.5 L culture.
Antibodies
Rabbit polyclonal anti-hREV7 antibody was produced by immunization with a keyhole limpet hemocyanin-conjugated peptide containing the C-terminal 19 amino acids of hREV7 and affinity-purified as previously described (Murakumo et al. 2001). Mouse monoclonal anti-FLAG M2, anti-His and anti-GFP antibodies were purchased from Sigma, Qiagen, and Nacalai Tesque, respectively. Rhodamine-conjugated anti-mouse IgG antibody was purchased from ICN Biomed. Inc.
In vitro protein–protein interaction assay
GST-fusion proteins were expressed in E. coli cells transformed with pGEX4T-2 plasmids after induction with 0.5 mm IPTG. After incubation for 2 h at 30 °C, the E. coli cells were harvested and lysed in bacterial lysis buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, 1 mM PMSF, 5 µg/mL leupeptin) by sonication. The lysates were centrifuged at 15 000 xg for 10 min and the GST-fusion proteins were immobilized on Glutathione-Sepharose beads (Amersham Biosciences). His-tagged Pol proteins were purified as described above. 25 µL of Glutathione-Sepharose beads containing 5 µg of the GST-fusion proteins were incubated with the His-tagged purified Pol proteins at 4 °C for 2 h in 250 µL of binding buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM EDTA, 10% glycerol, 0.1% Tween-20, 0.75 mg/mL bovine serum albumin (BSA), 1 mM PMSF, 5 µg/mL leupeptin). The beads were washed 3 times with 500 µL of wash-1 buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 5 mM EDTA, 10% glycerol, 0.1% Tween-20, 0.75 mg/ml BSA, 1 mM PMSF, 5 µg/mL leupeptin, 5 mM glutathione), then 3 times with 500 µL of wash-2 buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM EDTA, 10% glycerol, 0.1% Tween-20, 1 mM PMSF, 5 µg/mL leupeptin), and bound proteins were eluted by boiling in 1x sample buffer and analysed by SDS-PAGE followed by immunoblotting assay with anti-His antibody (Qiagen).
Immunoblotting assay
After SDS-PAGE, separated proteins were transferred to an Immobilon P membrane (Millipore) and probed with anti-FLAG, anti-His, anti-REV7, or anti-GFP antibody. The target proteins were visualized using the ECL Western blotting analysis system (Amersham Biosciences).
In vivo protein–protein interaction assay
Human 293T cells were transfected with pEGFP-hREV1 and pcDNA3.1(+)-FLAG-Pol or with pEGFP-hREV1 and pMK10 carrying FLAG-Pol in the downstream of the ß-actin promoter. The cells were harvested 48 h after transfection and disrupted in cell lysis buffer (20 mM HEPES, pH 7.6, 150 mM NaCl, 1 mM EDTA, 10% glycerol, 1 mM dithiothreitol, 0.1% Tween-20, 1 mM PMSF, 10 µg/mL leupeptin, 5 µg/mL pepstatin A) by three freeze-and-thaw cycles. The cell lysates were centrifuged at 15 000 xg for 10 min and incubated with protein G-Sepharose beads (Sigma) for 30 min at 4 °C to eliminate the nonspecific binding of proteins to the beads. After a brief centrifugation (1000 r.p.m. for 30 s), the supernatants were incubated with 4 µg of anti-FLAG or anti-GFP antibody for 10 h. The antigen-antibody complex was immobilized on protein G-Sepharose beads and the beads were washed five times in lysis buffer. The bound proteins were eluted by boiling in 1x sample buffer and subjected to SDS-PAGE and Western blotting with anti-GFP, anti-FLAG or anti-hREV7 antibody.
Microscopic study
MRC5 cells were transfected with plasmids encoding GFP- and FLAG-tagged fusion proteins. 24 h after transfection, the cells were grown on sterile cover slips and incubated for another 24 h. The cells were rinsed in PBS and fixed with 4% paraformaldehyde for 30 min, and then permealized with 0.25% Triton X-100 for 10 min. Cells were subsequently washed twice with PBS and incubated for 30 min at room temperature with 1% BSA to avoid nonspecific binding of proteins, and then incubated for 1 h with the primary antibody. Cells were then washed three times with PBS and incubated for 30 min with Rhodamine-conjugated anti-mouse IgG secondary antibody. Cells were subsequently washed twice with PBS and mounted with Permafluor Aqueous Mountant (Immunone) and photographed with an OLYMPUS FV500 system.
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: hohmori{at}virus.kyoto-u.ac.jp
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References |
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Received: 25 December 2003
Accepted: 29 March 2004
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