Rad18 guides polη to replication stalling sites through physical interaction and PCNA monoubiquitination
2004; Springer Nature; Volume: 23; Issue: 19 Linguagem: Inglês
10.1038/sj.emboj.7600383
ISSN1460-2075
AutoresKenji Watanabe, Satoshi Tateishi, Michio Kawasuji, Toshiki Tsurimoto, Hirokazu Inoue, Masaru Yamaizumi,
Tópico(s)Genomics and Chromatin Dynamics
ResumoArticle9 September 2004free access Rad18 guides polη to replication stalling sites through physical interaction and PCNA monoubiquitination Kenji Watanabe Kenji Watanabe Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto, Japan Department of Cardiovascular Surgery, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan Search for more papers by this author Satoshi Tateishi Satoshi Tateishi Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto, Japan Search for more papers by this author Michio Kawasuji Michio Kawasuji Department of Cardiovascular Surgery, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan Search for more papers by this author Toshiki Tsurimoto Toshiki Tsurimoto Department of Biology, School of Sciences, Kyushu University, Hakozaki, Higashi-ku, Fukuoka, Japan Search for more papers by this author Hirokazu Inoue Hirokazu Inoue Department of Regulation Biology, Faculty of Science, Saitama University, Urawa, Japan Search for more papers by this author Masaru Yamaizumi Corresponding Author Masaru Yamaizumi Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto, Japan Search for more papers by this author Kenji Watanabe Kenji Watanabe Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto, Japan Department of Cardiovascular Surgery, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan Search for more papers by this author Satoshi Tateishi Satoshi Tateishi Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto, Japan Search for more papers by this author Michio Kawasuji Michio Kawasuji Department of Cardiovascular Surgery, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan Search for more papers by this author Toshiki Tsurimoto Toshiki Tsurimoto Department of Biology, School of Sciences, Kyushu University, Hakozaki, Higashi-ku, Fukuoka, Japan Search for more papers by this author Hirokazu Inoue Hirokazu Inoue Department of Regulation Biology, Faculty of Science, Saitama University, Urawa, Japan Search for more papers by this author Masaru Yamaizumi Corresponding Author Masaru Yamaizumi Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto, Japan Search for more papers by this author Author Information Kenji Watanabe1,2,‡, Satoshi Tateishi1,‡, Michio Kawasuji2, Toshiki Tsurimoto3, Hirokazu Inoue4 and Masaru Yamaizumi 1 1Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto, Japan 2Department of Cardiovascular Surgery, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan 3Department of Biology, School of Sciences, Kyushu University, Hakozaki, Higashi-ku, Fukuoka, Japan 4Department of Regulation Biology, Faculty of Science, Saitama University, Urawa, Japan ‡These authors contributed equally to this work *Corresponding author. Institute of Molecular Embryology and Genetics, Kumamoto University, Kuhonji 4-24-1, Kumamoto 862-0976, Japan. Tel.: +81 96 373 6601; Fax: +81 96 373 6604; E-mail: [email protected] The EMBO Journal (2004)23:3886-3896https://doi.org/10.1038/sj.emboj.7600383 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The DNA replication machinery stalls at damaged sites on templates, but normally restarts by switching to a specialized DNA polymerase(s) that carries out translesion DNA synthesis (TLS). In human cells, DNA polymerase η (polη) accumulates at stalling sites as nuclear foci, and is involved in ultraviolet (UV)-induced TLS. Here we show that polη does not form nuclear foci in RAD18−/− cells after UV irradiation. Both Rad18 and Rad6 are required for polη focus formation. In wild-type cells, UV irradiation induces relocalization of Rad18 in the nucleus, thereby stimulating colocalization with proliferating cell nuclear antigen (PCNA), and Rad18/Rad6-dependent PCNA monoubiquitination. Purified Rad18 and Rad6B monoubiquitinate PCNA in vitro. Rad18 associates with polη constitutively through domains on their C-terminal regions, and this complex accumulates at the foci after UV irradiation. Furthermore, polη interacts preferentially with monoubiquitinated PCNA, but polδ does not. These results suggest that Rad18 is crucial for recruitment of polη to the damaged site through protein–protein interaction and PCNA monoubiquitination. Introduction Exposure of cells to ultraviolet (UV) light causes several types of DNA damage. Among these, cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts are major DNA lesions. In normal vertebrate cells, 6-4 photoproducts are efficiently repaired by nucleotide-excision repair, but nearly 50% of CPDs remain unrepaired even at 24 h after UV irradiation (Mitchell and Nairn, 1989). In such a situation, the DNA replication machinery often encounters the lesion during the S-phase of the cell cycle, and stalls at the replication fork, resulting in a gap opposite the site of damage in the newly synthesized DNA strand. Cell death may be imminent unless the gap is filled. This gap-filling process is operationally defined as postreplication repair (PRR), which is characterized by restarting of DNA replication without removal of the lesion on a template strand. PRR is observed in diverse species from Escherichia coli to humans. It is hypothesized that PRR is mediated by either translesion DNA synthesis (TLS) or recombination to resolve the stalled replication fork (Broomfield et al, 2001). In the budding yeast Saccharomyces cerevisiae, genes belonging to the RAD6 epistasis group are involved in the PRR pathway, where Rad18 (a putative ubiquitin ligase) and Rad6 (a ubiquitin-conjugating enzyme, E2) play a pivotal role (Bailly et al, 1994, 1997a). rad6 and rad18 mutants are highly susceptible to various DNA-damaging agents including UV and methylmethanesulfonate (MMS) (Hynes and Kunz, 1981). rad6 and rad18 mutants, however, show reduced mutation frequency following treatments with UV and MMS, possibly because error-prone TLS does not work without Rad18/Rad6. Because Rad18 protein binds to single-stranded DNA and forms a tight complex with Rad6 protein (Bailly et al, 1994, 1997b), it is proposed that Rad18 recruits Rad6 protein at replication stalling sites through binding to gap regions, and that the Rad18 complex ubiquitinates some target molecules on the stalled replication forks. Recently, proliferating cell nuclear antigen (PCNA) was shown to be monoubiquitinated in a Rad18/Rad6-dependent manner, which is necessary for tolerance to DNA damage (Hoege et al, 2002; Stelter and Ulrich, 2003). Interaction with PCNA is essential for the function of Rad30 (Haracska et al, 2001a), a yeast homolog of polymerase η, which is a member of RAD6 epistasis group (McDonald et al, 1997). These results suggest that PCNA might be a major target of Rad18/Rad6 in the PRR process. In vertebrate cells, thus far only a single homolog of RAD18 has been identified (Tateishi et al, 2000). Human and mouse Rad18 interacts with two forms of the Rad6 homolog, Rad6A and Rad6B, both in vitro and in vivo (Tateishi et al, 2000, 2003; Xin et al, 2000). RAD18 knockout mouse embryonic stem (ES) cells and chicken DT40 cells manifest sensitivity to various DNA-damaging agents and enhanced genomic instability as determined by increased sister-chromatid exchange (SCE) and frequency of stable transformation (Yamashita et al, 2002; Tateishi et al, 2003). Vertebrate polymerase η (polη), a homolog of the RAD30 gene product of the yeast, is a member of a recently discovered Y-family of novel DNA polymerases including polι and polκ (Burgers et al, 2001; Ohmori et al, 2001). They are shown to be involved in TLS in vitro, and structurally related to each other, but unrelated to the replicative polymerases (polδ and polε). Polη has a highly distributive, rather than processive, mode of DNA synthesis on undamaged templates and a relatively low stringency (Johnson et al, 2000; Matsuda et al, 2000). However, polη can insert correct nucleotides opposite CPDs in TLS (Johnson et al, 1999; McCulloch et al, 2004). The gene encoding polη is mutated in a cancer-prone hereditary disorder, xeroderma pigmentosum variant (XPV) (Masutani et al, 1999). It is possible that without normal polη CPD becomes highly mutagenic probably due to TLS by some other error-prone polymerase(s), resulting in skin cancers of sun-exposed areas. When the replicative machinery encounters unrepaired CPD lesions, it is expected that the replicative polymerase is switched to polη to carry out TLS in normal cells. In UV-irradiated human cells, polη forms discrete nuclear foci in a UV-dose- and time-dependent manner (Kannouche et al, 2001). The sites of these foci are colocalized with PCNA, suggesting that these are sites of stalled replication. Because a polη deletion mutant, which has a polymerase activity but does not show focus formation following UV irradiation, cannot complement the sensitivity of XPV cells to UV irradiation, foci formation is an essential step of polη function. However, molecular mechanisms of how polη forms nuclear foci in UV-irradiated cells are largely unknown. In the study reported here, in order to understand the role of Rad18 in tolerance to UV-induced DNA damage, we used RAD18−/− mouse fibroblasts from RAD18 knockout mice to show that Rad18 functions as an essential coordinator of the formation of polη foci through PCNA monoubiquitination and physical interaction with polη. Results Requirement of Rad18 for PCNA monoubiquitination To investigate the role of Rad18 in UV-induced TLS, we established cell lines from RAD18 knockout mice (Tateishi et al, 2003). These cells did not express detectable levels of Rad18 protein, but showed normal levels of Rad6A/B (Figure 1A) and normal growth rates (Figure 1B). In wild-type (WT) cells, a band of PCNA corresponding to 44 kDa increased in a UV dose- and time-dependent manner, while in RAD18−/− cells, the band remained at the control level up to 8 h after UV irradiation even at 40 J/m2 (Figure 1C and D). Similar modification of PCNA in MMS-treated HeLa cells was reported (Hoege et al, 2002). We concluded that this band represented a monoubiquitinated form of PCNA for two reasons. (i) Unmodified PCNA was detected at 36 kDa in SDS–PAGE (Figure 1C–E) and, when lysates of UV-irradiated cells expressing transfected HA-tagged ubiquitin were immunoprecipitated, bands of 45 and 44 kDa were detected by immunoblotting with an anti-HA antibody and anti-PCNA antibody, respectively (Figure 1E, lanes 2 and 3). (ii) Unmodified PCNA was converted to a 44 kDa band of monoubiquitinated PCNA in vitro by purified Rad18 and Rad6B of human origin plus ubiquitin (Figure 1E, lanes 7, 11, and 12). When ubiquitin was replaced with FLAG-tagged ubiquitin in this system, a 45 kDa band appeared (Figure 1E, lane 13). These results indicate that Rad18 is a ubiquitin ligase for the monoubiquitination of PCNA. Figure 1.Rad18 dependent monoubiquitination of PCNA by Rad18 and Rad6A/B in vivo and in vitro. (A) Western blot of Rad18 and Rad6A/B in RAD18−/− cells. α-Tubulin was included as a control. An asterisk shows nonspecific bands. (B) Growth curves of RAD18−/− cells. (C, D) Monoubiquitination of PCNA as determined by Western blot. Cells were harvested 5 h later following various doses of UV irradiation (C). In (D), cells were irradiated at 30 J/m2 and harvested at the indicated times. (E) In vivo (left, lanes 1–6) and in vitro (right, lanes 7–13) monoubiquitination of PCNA. GM637 cells were transfected with HA-ubiquitin (lanes 1–4) and irradiated with UV (13 J/m2, 6 h). Lysates were immunoprecipitated and blotted as indicated. In lanes 5 and 6, GM637 cells without transfection were irradiated at 0 and 13 J/m2 (6 h), respectively. Lane 7 represents an in vitro ubiquitination product. In lane 12, two-fold amounts of E2 and E3 were included in the reaction. Download figure Download PowerPoint In budding yeast, Rad18 binds to Rad6 through its Rad6-binding domain (R6BD) (Bailly et al, 1997b). This domain is highly conserved among various species. To confirm that the putative R6BD in hRad18 (amino-acid residues 340–395; Figure 2A) was a binding site for Rad6A/B, we transfected a Rad18 plasmid lacking R6BD together with a Rad6A/B plasmid into COS-7 cells, and performed co-immunoprecipitation experiments. Rad18 protein lacking R6BD localized in the nuclei like WT Rad18 (data not shown), but did not interact with human Rad6A/B (Figure 2A, lanes 1, 2, 5, and 6). To confirm whether the failure of PCNA ubiquitination in RAD18−/− cells was really due to a defect in Rad18, we established multiple RAD18−/− cell clones stably expressing WT human Rad18 (hRad18) (Figure 2B). PCNA ubiquitination following UV irradiation was restored to the WT level, whereas control RAD18−/− cells transfected with an empty vector did not show such recovery (Figure 2B). To examine whether PCNA ubiquitination required Rad6A/B together with Rad18, we established RAD18−/− cells stably expressing Rad18 but lacking R6BD (hRad18DR6). In these cells, PCNA was not ubiquitinated after DNA damage (Figure 2B). Furthermore, to confirm the requirement of Rad6A/B for PCNA monoubiquitination directly, Rad6 siRNA corresponding to both Rad6A and Rad6B was transfected into human cells, and reduced levels of Rad6A/B protein levels were confirmed by Western blot. In these cells, PCNA monoubiquitination was substantially reduced (Figure 2C). These results clearly indicate that in UV-irradiated mammalian cells, PCNA is monoubiquitinated in a Rad18- and Rad6A/B-dependent manner. To evaluate the significance of the monoubiquitination activity of Rad18, we determined the UV sensitivity of RAD18−/− mouse cells stably expressing hRad18. These cells showed almost normal UV sensitivity, while stable transformants with hRad18 lacking R6BD, or with the vector alone, remained sensitive to UV at the parent cell levels (Figure 2D). These results suggest that the UV sensitivity of RAD18−/− cells is caused at least in part by defects in the monoubiquitination of PCNA and subsequent foci formation of polη. Figure 2.Requirement of Rad6A/B for monoubiquitination of PCNA in UV-irradiated cells. (A) Interaction of WT and mutant hRad18 with hRad6A/B. Full-length and mutant Rad18 proteins are schematically shown on the top panel. Plasmids were transfected into COS-7 cells with different combinations indicated on the left of the middle panels, and immunoprecipitation was performed. Similar levels of expression of hRad18 and hRad6A/B proteins in the transformed cells were confirmed in the lower panel. (B) Restoration of PCNA monoubiquitination in RAD18−/− cells by expression of WT hRad18 but not of mutant hRad18. Cells were incubated for 6 h following UV irradiation at 20 J/m2. Cell lysates were immunoprecipitated and blotted with an anti-PCNA antibody (upper panel). Expression of FLAG-hRad18 or FLAG-Rad18DR6 was confirmed in individual clones of stable transformants of RAD18−/− mouse fibroblasts by Western blot with an anti-Rad18 rabbit antibody (lower panel). α-Tubulin was indicated as a volume control. (C) Inhibition of PCNA monoubiquitination by siRNA for Rad6A/B. WI38VA13 cells were transfected with Rad6A and Rad6B siRNA, incubated for 4 days, and then irradiated with 10 J/m2 of UV light. At the indicated times, protein levels of monoubiquitinated PCNA were determined by Western blot. An asterisk shows a nonspecific band that remained constant following the siRNA treatment. (D) Restoration of UV sensitivity of RAD18−/− mouse cells by introduction of human Rad18 as determined by a colony-forming assay. Two independent clones of stable transformants (WT#1 and WT#2 in (B)) were tested. Download figure Download PowerPoint Relocalization of Rad18 at stalling sites with PCNA In mammalian cells fixed with formaldehyde, a substantial fraction of Rad18 was homogeneously localized in the nucleus, while the remaining fraction existed as dots of irregular shapes and sizes (Figure 3A, left). Notably, most of the nuclear dots of Rad18 dispersed throughout the nucleus within 15 min with UV doses as low as 5 J/m2 (Figure 3A, middle). Rad18 dispersion occurred in the presence of cycloheximide (data not shown), suggesting that direct or indirect post-translational modification of Rad18 is involved in this process. Within a few hours after UV irradiation, nuclear foci of Rad18 with uniform sizes appeared (Figure 3A, right). Such dynamic intranuclear translocation of Rad18 was much more clearly detected in cells fixed with methanol (Figure 3B). To investigate the relationship between Rad18 and PCNA, we performed double immunostaining on methanol-fixed cells. Under normal conditions, partial colocalization of Rad18 with PCNA was observed (Figure 3B, upper). Within 1 h after UV irradiation, almost all of Rad18 became colocalized with PCNA (Figure 3B, lower) and such colocalization was observed at least up to 4 h, suggesting that Rad18 translocates to the replication stalling sites. To confirm directly this assumption, UV-irradiated cells were labeled with BrdU and stained for Rad18 and incorporated BrdU. Before UV irradiation, BrdU sites were partially colocalized with Rad18 (Figure 3C, upper), but after UV irradiation most of the BrdU sites were colocalized with translocated Rad18 (Figure 3C, lower). Since colocalization of Rad18 with PCNA was observed in XPV cells with a similar time course, it was inferred that translocation of Rad18 does not require functional polη (data not shown). To determine the subnuclear localization of PCNA, chromatin fractions were separated from UV-irradiated cells. Almost equal amounts of unmodified PCNA were obtained in the soluble and chromatin fractions irrespective of UV irradiation. In contrast, monoubiquitinated PCNA was exclusively recovered in the chromatin fraction of UV-irradiated cells, and it moved to the solubilized nuclear fraction after treatment with micrococcal nuclease (Figure 3D), suggesting that monoubiquitinated PCNA is tightly associated with chromatin. We could not detect any apparent physical interaction between Rad18 and PCNA before or after UV irradiation by co-immunoprecipitation, suggesting that the interaction is weak or transient (data not shown). Figure 3.Colocalization of Rad18 with PCNA on chromatin following UV irradiation. (A) Dispersion and relocalization of Rad18. GM637 cells irradiated at 15 J/m2 were fixed with formaldehyde and stained for Rad18. Bar=20 μm. (B) UV-induced colocalization of Rad18 with PCNA. GM637 cells irradiated at 15 J/m2 were fixed with methanol 4 h after UV irradiation and processed for double staining for Rad18 (green) and PCNA (red). Bar=10 μm. (C) Accumulation of Rad18 at the replication stalling sites. UV-irradiated (15 J/m2) GM637 cells were labeled for 2 h with BrdU, fixed with methanol, and processed for double staining for Rad18 (red) and BrdU (green). Bar=10 μm. (D) Binding of monoubiquitinated PCNA to chromatin. Chromatin fractions were isolated from UV-irradiated (15 J/m2, 6 h) or nonirradiated HeLa cells, and then treated with micrococcal nuclease (MNase). The distributions of PCNA in the total cell lysate (TCL), soluble fraction (S2), solubilized nuclear fraction (S3), and chromatin-enriched fraction (P3) are shown. Orc2 is shown as a chromatin fraction marker. Download figure Download PowerPoint Requirement of Rad18 for polη focus formation Using polη fused to enhanced green fluorescent protein (eGFP-polη), Kannouche et al (2001) found that polη, which localizes uniformly in the nucleus under normal conditions, formed distinct nuclear foci at the replication stalling sites after treatment with DNA-damaging agents including UV and MMS. This polη focus formation is essential for UV survival, because mutant polη, which is defective in focus formation, could not complement UV survival of XPV cells (Kannouche et al, 2001). To investigate whether Rad18 is required for polη focus formation, we introduced eGFP-hpolη into either RAD18−/− or RAD18+/+ mouse cells. While polη focus formation was clearly observed in the UV-irradiated WT cells, polη remained uniformly dispersed in the nucleus of UV-irradiated RAD18−/− cells (Figure 4A). The formation of polη foci proceeded gradually and reached a plateau at 6 h after UV irradiation at least with dosages ranging 10–20 J/m2 (Figure 4B). Defective focus formation in RAD18−/− cells could be restored by concomitant introduction of WT hRad18 with or without a FLAG tag in its N-terminal region (Figure 4A and C, data not shown). However, hRad18 lacking R6BD did not restore the focus formation (Figure 4C). Furthermore, the formation of polη foci was significantly inhibited in cells treated with Rad6A/B siRNA (Figure 4D). These results indicate that UV-induced polη focus formation is dependent on both Rad18 and Rad6A/B. Figure 4.Rad18- and Rad6-dependent formation of polη foci. (A) Focus formation of eGFP-polη following UV irradiation in WT cells but not in RAD18−/− cells. Cells were irradiated at 15 J/m2. After 6 h, the distribution of eGFP-polη was examined after fixation. Defective focus formation of polη was recovered by concomitant expression of Rad18. Bar=10 μm. (B) Time course of eGFP-polη focus formation in UV-irradiated cells. RAD18−/− mouse cells and WT cells were transfected with eGFP-polη. After 20 h, cells were irradiated with UV at the indicated doses. (C) Restoration of eGFP-polη focus formation in UV-irradiated (20 J/m2) RAD18−/− cells by expression of WT hRad18 but not of mutant hRad18 lacking the Rad6-binding domain. (D) Inhibition of polη focus formation by siRNA for Rad6. WI38VA13 cells were transfected with Rad6A and Rad6B siRNA, cultured for 3 days, and then transfected again with an eGFP-polη plasmid. After 20 h, cells were irradiated with UV (10 J/m2), and 6 h later cells containing eGFP-polη foci were counted. Download figure Download PowerPoint Association of Rad18 with polη Immunostaining for Rad18 clearly demonstrated that Rad18 colocalized with eGFP-polη at the foci following UV irradiation (Figure 5A, lower). To investigate the interaction between Rad18 and polη, HA-tagged polη was transfected into GM637 cells, and co-immunoprecipitation experiments were performed. Rad18 was consistently associated with polη, irrespective of UV irradiation (Figure 5B). Furthermore, purified polη bound to purified Rad18 in an immunoprecipitation assay, but polδ did not (Figure 5C, lanes 2 and 4), indicating that at least a part of Rad18 is directly associated with polη in a UV-independent manner. To determine the binding site of polη to Rad18, we overexpressed a series of deletion mutants of polη fused with GST at their N-terminal regions (Figure 6A) in insect cells, and purified them with glutathione beads (Figure 6B). GM637 cell lysates were pulled down with these beads. Rad18 interacted with full-length polη and a C-terminal fragment of polη (GST-polη158c) spanning amino-acid residues 556–713 (Figure 6C). We also determined the binding site of Rad18 to polη in a similar way. In this assay, Myc-tagged WT and deleted Rad18 proteins were overexpressed in COS-7 cells (Figure 6D and E), and cell lysates were pulled down with glutathione beads associated with GST-polη158c. Among the deletion mutants, only Rad18 lacking a region spanning amino-acid residues 402–444 could not interact with polη (Figure 6F). To evaluate the biological significance of the interaction between Rad18 and polη, hRad18 lacking the polη-binding domain (hRad18DC2) was transiently expressed in RAD18−/− mouse cells together with eGFP-polη. Formation of eGFP-polη foci was not restored following UV irradiation (Supplementary Figure S1). Furthermore, RAD18−/− cells stably expressing Rad18 lacking the polη-binding domain showed high UV sensitivity like cells transformed with an empty vector (Supplementary Figures S2 and S3), while they had normal levels of monoubiquitination of PCNA after UV irradiation. These results suggest that Rad18 recruits polη to replication stalling sites through direct interaction. Since eGFP-polη localized uniformly in the nucleus with Rad18 under normal conditions (Figure 5A), the nuclear dots of Rad18 in nonirradiated cells might be reservoirs of free Rad18. Figure 5.Direct interaction of polη with Rad18. (A) UV-induced colocalization of Rad18 with eGFP-polη in GM637 cells. Cells transfected with eGFP-polη and FLAG-Rad18 plasmids were irradiated at 15 J/m2 and incubated for 6 h. After fixation, cells were stained for Rad18 with an antibody against FLAG. Bar=10 μm. (B) Interaction of Rad18 with polη. HA-polη was transiently expressed in GM637 cells. Immunoprecipitation was performed at various times after UV irradiation (12.5 J/m2). As a control, UV-irradiated cell lysates (6 h) were immunoprecipitated with control IgG. (C) Direct binding of Rad18 with polη. Recombinant Rad18 and polη were purified from insect cells. After incubation of the mixture, Rad18 was immunoprecipitated and polη bound to Rad18 was detected by Western blot. Polδ was used as a control. Download figure Download PowerPoint Figure 6.Determination of binding sites. (A) Structural domains of GST-polη fusion proteins. P: putative PCNA-binding domain; Z: zinc-finger domain. (B) Purification of GST- polη fusion proteins by glutathione beads. Proteins bound to the beads were stained with Coomassie brilliant blue (CBB, arrowheads). (C) Pull-down assay. GM637 cell lysates were pulled down with GST-polη fusion proteins bound to glutathione beads. Interaction with Rad18 was analyzed by Western blot. (D) Structural domains of Myc-tagged Rad18 proteins. R: RING finger domain; Z: zinc-finger domain. (E) Deletion mutant proteins were overexpressed in COS-7 cells and their expression was confirmed by Western blot. (F) COS-7 cell lysates containing Myc-tagged mutant Rad18 proteins were pulled down with GST-polη158c bound to glutathione beads. Association of WT and mutant Rad18 proteins with polη was analyzed by Western blot using an anti-Myc antibody. Download figure Download PowerPoint Preferential binding of polη to monoubiquitinated PCNA To investigate the molecular mechanism of how UV-induced monoubiquitination of PCNA functions in polymerase switching to polη, the physical interaction between PCNA and polη was determined by a pull-down assay. GST-polη bound to glutathione beads was mixed with lysates prepared from UV-irradiated HeLa cells, and PCNA associated with the GST-polη beads was revealed by Western blot. While monoubiquitinated PCNA was a minor fraction of the total PCNA in the lysates, it was recovered predominantly from the precipitated beads in a time-dependent manner (Figure 7A, right). In contrast, monoubiquitinated PCNA was not associated with GST-polδ in the same assay (Figure 7A, middle). The affinity of monoubiquitinated PCNA for polη was much higher than that of unmodified PCNA, because even at higher salt concentrations, monoubiquitinated PCNA remained bound to polη (Figure 7B, left). Monoubiquitinated PCNA bound to polη was much more refractory to elution by high salt concentrations than unmodified PCNA (Figure 7B, right). To investigate whether polη interacted with monoubiquitinated PCNA in UV-irradiated cells, HA-polη was transiently expressed in GM637 cells, and co-immunoprecipitation assay was performed. In this experiment, cells were treated with 0.1% NP-40 before preparation of cell lysates. This treatment allowed specific crosslinking between chromatin-bound polη and monoubiquitinated PCNA probably by excluding unmodified PCNA and a diffused form of polη from nuclei. Monoubiquitinated PCNA was preferentially immunoprecipitated with HA-polη in the UV-irradiated cells (Figure 7C, lanes 5 and 6). In contrast, monoubiquitinated PCNA was not immunoprecipitated in nonirradiated cells (Figure 7C, lanes 2 and 3). Taken together, these results indicate that polη preferentially binds to monoubiquitinated PCNA both in vitro and in vivo. To prove that the interaction between polη and monoubiquitinated PCNA is direct, PCNA was monoubiquitinated in the in vitro PCNA ubiquitination reaction (Figure 1E). Rad18 and Rad6B were then removed from the in vitro PCNA ubiquitination reaction mixture (Figure 1E) by multiple cycles of immunodepletion with an anti-Rad18 antibody (Figure 7D, upper). Immunodepletion of Rad18 and Rad6B was confirmed by Western blot. Monoubiquitinated PCNA still bound to polη in a pull-down assay (Figure 7D, lane 3). In contrast, polη lacking the three putative PCNA-binding sites on the C-terminus (Kannouche et al, 2001) showed no interaction with PCNA (Figure 7D, lane 2). Immunostaining demonstrated that more than 50% of the transfected eGFP-polη colocalized with endogenous polδ 5 h after UV irradiation (Figure 7E). Furthermore, endogenous polδ colocalized with PCNA in UV-irradiated cells (Figure 7F), suggesting that both polymerases and Rad18 localize at the same stalling sites. Figure 7.Preferential binding of polη to monoubiquitinated PCNA. (A) Binding of polη to ubiquitinated PCNA. HeLa cells were irradiated with UV at 20 J/m2. PCNA in the cell lysates was pulled down with either GST-polη beads or polδ beads, and analyzed by Western blot using an anti-PCNA antibody. (B) Effects of different salt concentrations on the binding of PCNA to GST-polη (left) and on PCNA elution from GST-polη (right). PCNA pulled down was washed with buffer containing various concentrations of NaCl. PCNA in bound or eluted fractions was analy
Referência(s)