Interaction with DNA polymerase η is required for nuclear accumulation of REV1 and suppression of spontaneous mutations in human cells

DNA Repair 8 (2009) 585–599

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DNA Repair journal homepage: www.elsevier.com/locate/dnarepair

Interaction with DNA polymerase ␩ is required for nuclear accumulation of REV1 and suppression of spontaneous mutations in human cells Jun-ichi Akagi a,b,1 , Chikahide Masutani a,∗ , Yuki Kataoka a,b , Takashi Kan a,c , Eiji Ohashi d , Toshio Mori e , Haruo Ohmori d , Fumio Hanaoka a,2 a Graduate School of Frontier Biosciences and Solution Oriented Research for Science and Technology, Japan Science and Technology Agency, Osaka University, 1-3 Yamada-oka, Suita, Osaka 565-0871, Japan b Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka, Suita, Osaka 565-0871, Japan c Graduate School of Medicine, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan d Institute for Virus Research, Kyoto University, 53 Shogoin-Kawaracho, Sakyo-ku, Kyoto, Kyoto 606-8507, Japan e Radioisotope Research Center, Nara Medical University School of Medicine, Kashihara, Nara 634-8521, Japan

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Article history: Received 11 September 2008 Received in revised form 27 November 2008 Accepted 9 December 2008 Available online 21 January 2009 Keywords: Translesion synthesis (TLS) DNA polymerase ␩ REV1 Protein–protein interaction Cellular localization Mutation avoidance

a b s t r a c t Defects in the gene encoding human Pol␩ result in xeroderma pigmentosum variant (XP-V), an inherited cancer-prone syndrome. Pol␩ catalyzes efficient and accurate translesion DNA synthesis (TLS) past UV-induced lesions. In addition to Pol␩, human cells have multiple TLS polymerases such as Pol␫, Pol␬, Pol␨ and REV1. REV1 physically interacts with other TLS polymerases, but the physiological relevance of the interaction remains unclear. Here we developed an antibody that detects the endogenous REV1 protein and found that human cells contain about 60,000 of REV1 molecules per cell as well as Pol␩. In un-irradiated cells, formation of nuclear foci by ectopically expressed REV1 was enhanced by the coexpression of Pol␩. Importantly, the endogenous REV1 protein accumulated at the UV-irradiated areas of nuclei in Pol␩-expressing cells but not in Pol␩-deficient XP-V cells. UV-irradiation induced nuclear foci of REV1 and Pol␩ proteins in both S-phase and G1 cells, suggesting that these proteins may function both during and outside S phase. We reconstituted XP-V cells with wild-type Pol␩ or with Pol␩ mutants harboring substitutions in phenylalanine residues critical for interaction with REV1. The REV1interaction-deficient Pol␩ mutant failed to promote REV1 accumulation at sites of UV-irradiation, yet (similar to wild-type Pol␩) corrected the UV sensitivity of XP-V cells and suppressed UV-induced mutations. Interestingly however, spontaneous mutations of XP-V cells were only partially suppressed by the REV1-interaction deficient mutant of Pol␩. Thus, Pol␩–REV1 interactions prevent spontaneous mutations, probably by promoting accurate TLS past endogenous DNA lesions, while the interaction is dispensable for accurate Pol␩-mediated TLS of UV-induced lesions. © 2008 Elsevier B.V. All rights reserved.

1. Introduction DNA lesions generally block replicative DNA polymerases and cause replication fork arrest. Translesion DNA synthesis (TLS) is a major mechanism for overcoming replication fork arrest and involves the direct replication of damaged DNA templates by specialized TLS DNA polymerases.

∗ Corresponding author. Tel.: +81 6 6879 7978. E-mail address: [email protected] (C. Masutani). 1 Present address: Biosignal Research Center, Organization of Advanced Science and Technology, Kobe University, 1-1 Rokkodai, Nada-ku, Kobe 657-8501, Japan. 2 Present address: Department of Chemistry, Faculty of Science, Gakushuin University, 1-5-1 Mejiro, Toshima-ku, Tokyo 171-8588, Japan. 1568-7864/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.dnarep.2008.12.006

We have identified the defective gene product responsible for xeroderma pigmentosum variant (XP-V), an inherited cancer prone disease, as human DNA polymerase ␩ (Pol␩) [1,2]. Pol␩ catalyzes efficient and accurate TLS past cis–syn cyclobutane pyrimidine dimers (CPD, the most prominent lesions induced by UV irradiation), and thereby prevents UV-induced tumorigenesis [3,4]. In addition to CPD, Pol␩ has been shown to catalyze TLS past some other lesions induced by chemicals and oxidative stresses, although TLS past these lesions is often inefficient and inaccurate [5–10]. In human cells, 14 DNA polymerases (␣ to ␯ and REV1) have been identified and classified into four families: A, B, X and Y. Pol␩ belongs to the Y-family of DNA polymerases (also comprising Pol␫, Pol␬ and REV1) [11]. At present, 10 DNA polymerases including all Y-family members have been reported to exhibit TLS activity in vitro [12]. TLS polymerases are generally error-prone if they replicate undamaged DNA templates [13]. Therefore, appropriate mechanisms are

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necessary to ensure that TLS polymerases are recruited only to replication forks that encounter damaged DNA. Monoubiquitination of PCNA, which is catalyzed by the RAD6-RAD18 complex, is considered to be a crucial trigger for TLS in DNAdamaged cells [14]. All of the Y-family DNA polymerases possess ubiquitin-binding domains [15], which are hypothesized to mediate their recruitment to the stalled replication fork via direct interaction with ubiquitinated PCNA. However, it is unclear how these DNA polymerases, which have different lesion specificities [16], are appropriately recruited to stalled replication forks in a lesionspecific manner. Appropriate regulation of TLS polymerase activities is likely determined by protein–protein interactions. We and others have shown that Pol␩ interacts with REV1 [17–19] and Pol␫ [20]. Pol␫ is the paralogue of Pol␩ [21], and has the ability to incorporate nucleotides opposite 6–4 photoproducts [22,23], the second major UV-induced DNA lesions. Recent studies with mice defective for Pol␫ revealed that Pol␫ is involved in preventing UV-induced skin carcinogenesis in addition to Pol␩ [4,24]. Interestingly, formation of Pol␫ nuclear foci in UV-irradiated human cells has been shown to be largely dependent on Pol␩, suggesting that Pol␩ recruits Pol␫ to arrested replication forks [20]. REV1 has a deoxycytidyl transferase activity that incorporates dCMP opposite abasic sites [25]. Genetic analyses of S. cerevisiae and antisense RNA experiments in human cells revealed that REV1 participates in the majority of UV-induced mutagenesis by cooperating with Pol␨ [26–29]. Since the dCMP transferase activity of yeast Rev1 is not required for UV-induced mutagenesis, Rev1 is suggested to have non-catalytic roles in error-prone TLS [30]. Importantly, in addition to Pol␩, REV1 interacts with Pol␫, Pol␬ and REV7, a non-catalytic small subunit of Pol␨ via its C-terminal region [17,18,31–33]. Thus, REV1 is thought to act as a scaffold for recruiting TLS polymerases, although the physiological relevance of protein–protein interactions between REV1 and other TLS polymerases has not yet been elucidated. Recently, Ohashi and colleagues showed that two consecutive phenylalanines (483-4FF and 531-2FF) of human Pol␩ are crucial for the interaction with REV1 [34]. Here, we have investigated the biological significance of Pol␩–REV1 interactions. We have examined the Pol␩-dependency of REV1 localization to replication foci and sites of UV-induced DNA damage in human cells. Additionally, we have performed complementation analyses of XP-V cells using a REV1-interaction-deficient (FF-AA) mutant of Pol␩. Our results indicate that accurate TLS of UVinduced DNA lesions is predominantly dependent on Pol␩ and does not require interactions with REV1. In contrast, accurate TLS of spontaneously occurring DNA lesions requires Pol␩–REV1 interaction. 2. Materials and methods 2.1. Plasmids and siRNAs The full-length human XPV cDNA subcloned into pBS-KS(+), pBS-XPV, and constructs for untagged and hexahistidine tagged human Pol␩ at its C-terminus, pET-XPV and pET-XPV-His, respectively, were constructed as described previously [2,5]. To produce the FLAG-Pol␩-His expression construct, XPV-His fragment was obtained by using NdeI site at translation initiation site and Bpu1102I site, which was blunted, of the pET-XPV-His, and cloned into NdeI and SmaI sites of pBS-FLAG vector. Then the FLAG-XPVHis fragment was recovered by digesting the resulting plasmid with EcoRI and SacI, blunted, and cloned into the blunted XbaI site of the pMK10 vector in which the gene of interest is expressed under the ␤-actin promoter and CMV enhancer, resulting in the pMK10-FLAGPol␩-His.

To produce GFP-tagged human Pol␩, XPV cDNA fragment was obtained by the digestion of pET-XPV by NdeI and XhoI, blunted, and cloned into blunted XhoI and BamHI sites of the pEGFP-C3 vector (BD Bioscience), resulting in the pEGFP-Pol␩. To produce untagged human Pol␩, an EcoRI site was added to the upstream of XPV cDNA by PCR. The PCR product was digested by EcoRI and then cloned into EcoRI and blunted NotI sites of the pIRESneo2 (BD Bioscience) vector, resulting in the pIRESneo2-Pol␩. FF-AA mutant Pol␩ cDNAs were generated by using a site-directed mutagenesis system as described elsewhere [34]. The REV1 cDNA coding for the full-length REV1protein (REV1(11251)) was obtained by PCR from human cDNA libraries. The translation initiation site of REV1 was converted to an NdeI site. The plasmid coding for C-terminally truncated REV1(1–1098) were generated by introducing the stop codon with a site-directed mutagenesis system, Mutan-K (TaKaRa). To add the Myc-tag to the full-length or truncated REV1 at its N-terminus, the oligonucleotide for c-Myc epitope was inserted between EcoRI and BamHI sites of the pBluescript II-KS(+) vector, and then the REV1 fragment was cloned into the NdeI site of the vector. The Myc-tagged REV1 fragment from the resulting plasmid was cloned into pIREShyg2 and pTRE2hyg vectors (BD Bioscience). To produce human REV1 in insect cells, full-length REV1 tagged with hexahistidine at its Nterminus was cloned into the pFASTBAC DUAL vector (GIBCO BRL). Recombinant virus was prepared by using BAC to BAC Bacurovirus Expression System (GIBCO BRL) according to the manufacturer’s protocol. pGEX6P-2-REV1(810-1251) was generated from pPC86REV1(810-1251) which obtained from yeast two-hybrid screenings of pPC86-Human Spleen cDNA library (GIBCO BRL) using pDBLNXPV as a bait [18]. The REV1(810-1251) fragment was recovered by digesting the plasmid with SalI and NotI, and cloned into the same restriction sites of pGEX6P-2 vector. Sequences of siRNA oligonucleotides (Dharmacon) used are as follows. REV1(A): 5 -CCG CUG AGG AAU UGA GAA AdTdT-3 . REV1(B): 5 -GAA CAG UGA CGC AGG AAU AdTdT-3 . Non-silencing control: 5 -AUU GUA UGC GAU CGC AGA CdTdT-3 . 2.2. Proteins Recombinant human Pol␩-His protein was prepared as described previously [5]. Human REV1 protein tagged with hexahistidine at its Nterminus was expressed in Sf9 cells infected for 48 h with the recombinant virus. Following procedures were done at 4 ◦ C or on ice. Cells from 25 dishes (150 mm) were collected, washed twice with PBS, and then suspended with six packed cell volumes (PCV) of buffer 1 (20 mM sodium phosphate, pH 7.2, 10% glycerol, 10 mM 2-mercaptoethanol, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 1.0 ␮g/ml antipain, 1.0 ␮g/ml aprotinin, 0.5 ␮g/ml leupeptin, 0.4 ␮g/ml pepstain A) containing 100 mM NaCl and 1% Triton X-100. The sample was stood on ice for 15 min with occasional mixing, and then centrifuged at 40,000 × g for 30 min to remove Triton X-100 extractable materials. The pellet was suspended with 4× PCV of buffer 1 containing 300 mM NaCl. The suspension was centrifuged at 40,000 × g for 30 min to obtain 0.3 M NaCl extracts. The extracts were adjusted to 15 mM imidazole and passed through a HiTrap DEAE column (5 ml, GE Healthcare) equilibrated with buffer A (40 mM Hepes-KOH, pH 7.4, 300 mM NaCl, 10% glycerol, 10 mM 2-mercaptoethanol, 0.01% Triton X-100). The flowthrough materials were directory loaded onto a Ni-NTA Superflow (1 ml, Qiagen) column. After washing the column with the same buffer, bound materials were eluted with a linear gradient of imidazole from 30 to 150 mM in 10 ml of buffer A. The fractions containing His-REV1 protein were dialyzed against buffer B (25 mM HepesKOH, pH 7.4, 10% glycerol, 0.1 mM EDTA, 10 mM 2-mercaptoethanol,

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0.01% Triton X-100) containing 0.3 M NaCl and then loaded onto a POROS/HE column (1 ml, Applied Biosystems) equilibrated with the same buffer. Bound proteins were eluted stepwise by 0.3, 0.4, 0.6, 0.8 and 1 M NaCl in buffer B. The peak fractions, which were eluted by 0.6 M NaCl, were collected and dialyzed against buffer B containing 0.3 M NaCl and then loaded onto a MonoS PC1.6/5 column (GE Healthcare). After washing the column with the same buffer, bound materials were eluted with two-step linear gradients from 0.3 to 0.4 M NaCl in 0.5 ml of buffer B followed by 0.4 to 1 M NaCl in 0.5 ml of buffer B. His-REV1 protein was detected on SDS-PAGE followed by silver staining and Western blotting, and also by its dCMP transferase activity (data not shown). REV1-rich fractions were pooled and stored at −80 ◦ C until use. 2.3. Cell lines and cultures The SV40-transformed human fibroblasts, WI-38VA13, XP2SASV3, XP4PASV and XP2OSSV, were cultured at 37 ◦ C with 5% CO2 in Dulbecco’s-modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS). HeLa TetOn/Myc-REV1 cells in which exogenous production of Myc-tagged REV1 is induced in the presence of doxycycline were constructed by stable transfection of HeLa Tet-On cells (BD Bioscience) with pTREhyg2-Myc-REV1 construct. For transfection experiments with plasmid DNA, FuGENE6 reagent (Roche) was used according to the manufacturer’s protocol. Stable transformants of XP2SASV3/FLAG-Pol␩-His and XP2SASV3/Pol␩ (wild type or FF-AA mutants) were maintained in DMEM/10% FBS supplemented with 0.2 mg/ml G418. For siRNA experiments, transfection was performed with Dharmafect I reagent (Dharmacon) according to the manufacturer’s protocol.

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To synchronize cells in S phase, cells were cultured with growth medium containing 2.5 mM thymidine (Sigma) for 24 h, cultured with normal growth medium for 12 h, again cultured with 2.5 mM thymidine-containing medium for 24 h, and then released for 3 h in the growth medium. To synchronize in G1 phase, cells were cultured with medium containing 2.5 mM thymidine for 24 h, released into the normal medium for 3 h, and cultured in the medium containing 30 ng/ml nocodazole (Sigma) for 12 h. Then, mitotic cells were collected by shaking-off and cultured in normal medium for 3 h. 2.4. Cell extracts For preparing cell-number determined lysates, cells were trypsinized, washed three times with ice-cold PBS, resuspended with 3× PCV of PBS, and then an aliquot of the suspension was sampled for cell counting. The remaining cells were lysed by adding final 1% SDS. The lysates were boiled at 95 ◦ C for 5 min, sonicated, and used for the analysis. Triton X-100 soluble fractions and solubilized chromatin fractions used for immunoprecipitation experiments and TLS assays were prepared as follows. Cells were harvested by gentle scraping and washed twice with PBS. The cell pellets were suspended with 0.5% Triton X-100 buffer (20 mM Hepes-KOH, pH 7.4, 155 mM KCl, 1.5 mM MgCl2 , 0.5% Triton X-100, 0.5 mM PMSF, 2 mM DTT, 1.0 ␮g/ml antipain, 1.0 ␮g/ml aprotinin, 0.5 ␮g/ml leupeptin, 0.4 ␮g/ml pepstain A) and allowed to stand for 5 min on ice and then centrifuged at 2,000 × g for 10 min at 4 ◦ C. The supernatants were collected as Triton X-100 soluble fractions. The pellets were suspended with 3× volumes of micrococcal nuclease buffer (20 mM Hepes-KOH, pH 7.4, 5 mM KCl, 1.5 mM MgCl2 , 2 mM CaCl2 , 0.5 mM PMSF, 2 mM DTT, 1.0 ␮g/ml antipain, 1.0 ␮g/ml aprotinin, 0.5 ␮g/ml

Fig. 1. Detection and estimation of endogenous REV1 and Pol␩ proteins in human cells. (A) Lysates (10 ␮g protein) from untreated WI-38VA13 cells (lane 1) and from cells transfected with non-silencing (lane 2) or REV1-specific siRNA oligonucleotides (lanes 3 and 4) were separated by 6% SDS-PAGE and subjected to Western blot with antiREV1C (upper) and anti-Pol␩ (5H10) (lower) antibodies. (B and C) Indicated amounts of purified recombinant His-REV1 (B) or Pol␩-His (C) proteins (lanes 1–4) and whole cell lysates from indicated numbers of WI-38VA13 (lanes 5-7) and XP2SASV3 (lanes 8-10) cells were separated by 6% SDS-PAGE and subjected to Western blot with anti-REV1C or anti-Pol␩ (5H10) antibodies. Numbers of REV1 and Pol␩ molecules in WI-38VA13 cells were calculated from the intensities of Western blots and are indicated in boxes.

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leupeptin, 0.4 ␮g/ml pepstain A). Micrococcal nuclease (Roche) was added to make final 200 U/ml. The mixtures were incubated for 10 min at 27 ◦ C and then centrifuged at 17,600 × g for 15 min at 4 ◦ C. The supernatants were collected as solubilized chromatin fractions. 2.5. Antibodies To generate guinea pig polyclonal REV1C antibodies against human REV1 protein, the C-terminal portion of human REV1(8101251) was expressed in E. coli Rosetta (DE3) cells as a GST-fused protein at its N-terminus. Logarithmically growing cells carrying pGEX6P-2-REV1(810-1251) were cultured in 4 litters of Luria broth containing 0.5 mM IPTG for 24 h at 16 ◦ C. Then, cells were harvested by centrifugation and resuspended with 0.3 M NaCl buffer (25 mM Tris–HCl, pH 7.5, 300 mM NaCl, 10% glycerol, 0.01% Triton X-100, 1 mM EDTA, 5 mM DTT, 0.25 mM PMSF, 0.4 ␮g/ml antipain, 0.4 ␮g/ml aprotinin, 0.2 ␮g/ml leupeptin, 0.16 ␮g/ml pepstain A) and lysed by three cycles of freezing and thawing followed by sonication. Clarified supernatant was obtained by centrifugation at 40,000 × g for 30 min. The supernatant was mixed with 4 ml of Glutathione Sepharose 4B resin (GE Healthcare) equilibrated with 0.3 M NaCl buffer, incubated at 4 ◦ C for 1 h with gentle mixing. The resin was washed with 10 ml of cleavage buffer (50 mM Tris-HCl, pH 7.0, 150 mM NaCl, 1 mM EDTA, 1 mM DTT) and then incubated with 4 ml of cleavage buffer containing 320 units of PreScission Protease

(GE Healthcare) for 16 h at 4 ◦ C to cleave REV1(810-1251) from the GST-tag. The eluted REV1(810-1251) rich fractions were combined and dialyzed against 20 mM sodium phosphate, pH 7.2, 20 mM NaCl, and then centrifuged at 40,000 × g for 30 min. The supernatant was then loaded onto a MonoQ HR5/5 column equilibrated with the dialysis buffer. The column was washed with 20 ml of the same buffer and the protein was eluted with a linear gradient of NaCl from 20 to 500 mM in 20 mM sodium phosphate, pH 7.2. The REV1(8101251) eluted in the wash and the beginning of the gradient fractions was precipitated with final 35% and 75% saturations of ammonium sulfate, and then pellets were resuspended with 800 ␮l of 20 mM sodium phosphate, pH7.2. The REV1(810-1251) was recovered in both 35% and 75% precipitated fractions, and so these fractions were combined and subjected to SDS-PAGE. The REV1(810-1251) protein was cut out from the CBB-stained gels and served as an antigen source. Mouse monoclonal TDM-2 antibody against CPD was generated as previously described [35]. Mouse monoclonal antibodies 5H10 and 5C6 against human Pol␩ were generated using N-terminal 511 amino acids of human Pol␩ as an antigen source. Rabbit polyclonal antibody against human Pol␩ (H-300) was purchased from Santa Cruz Biotechnology. Mouse monoclonal anti-Myc-tag antibody (PL14) was purchased from Medical & Biological Laboratories. Mouse monoclonal anti-BrdU antibody with nucleases was purchased as a component of BrdU Labeling and Detection Kit I (Roche).

Fig. 2. Co-localization of ectopically expressed GFP-Pol␩ and Myc-REV1. (A) Full-length Myc-REV1, (B) full-length Myc-REV1 and GFP-Pol␩, (C) Myc-REV1(1-1098) or (D) Myc-REV1(1-1098) and GFP-Pol␩ were transiently expressed in XP2SASV3 cells. Either Myc-REV1 or Myc-REV1(1-1098) was stained with anti-REV1C antibody and visualized with Alexa Fluor 568-conjugated secondary antibody. Intensity profiles were generated with MetaMorph. Bar = 10 ␮m.

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Fig. 3. Localization of endogenous REV1 to UV-damaged sites in Pol␩-proficient but not in Pol␩-deficient cells. (A) WI-38VA13, (B) XP2SASV3 or (C) XP2SASV3/Flag-Pol␩-His cells were locally irradiated with UV through polycarbonate isopore membrane filters and incubated for 3 h. After extraction and fixation, REV1 was stained with anti-REV1C antibody and visualized with Alexa Fluor 594-conjugated secondary antibody (red). FLAG-Pol␩-His was stained with anti-Pol␩ (H-300) antibody followed by Alexa Fluor 488-conjugated secondary antibody (green). UV-damaged DNA was stained with anti-CPD antibody (TDM2) followed by Alexa Fluor 350-conjugated secondary antibody (blue). Bar = 10 ␮m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

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Fig. 5. Localization of REV1 to UV-damaged sites in G1- and S-phase synchronized cells. (A) Outlines of experimental protocols. (B) WI-38VA13 cells of asynchronous or synchronized in G1 or S phase were locally UV-irradiated. Cells were extracted and fixed at 1 h after irradiation, and then REV1 were visualized as described in Fig. 4. (C) Populations of REV1-accumulated cells were counted. More than 200 cells were scored in three independent experiments, and averages were plotted with standard deviations. When CPD were examined under similar conditions, about 50% of irradiated cells gave positive signals. Bar = 10 ␮m.

2.6. Western blotting

2.7. Immunofluorescence

Proteins were subjected to SDS-PAGE and transferred to PVDF membranes (Millipore) using a Trans-Blot SD Semi-Dry Transfer Cell (Bio-Rad). After blocking with 5% skim-milk in TBS-T (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 0.1% Tween 20), the membranes were incubated with primary antibodies, washed extensively, and incubated with appropriate secondary antibodies conjugated with the horseradish peroxidase. Chemiluminescence was used for detection.

Cells growing in 35-mm glass bottom dishes (Matsunami) were washed twice with ice-cold PBS, fixed with 3.5% paraformaldehyde/PBS on ice for 20 min, and then permiabilized with 0.5% Triton X-100/PBS on ice for 5 min. The cells were subsequently washed three times with PBS and incubated with blocking buffer (3% skim milk, 0.02% NaN3 in PBS) at room temperature for 30 min, incubated with primary antibodies at room temperature for 1 h, washed with PBS, incubated with Alexa Fluor dye-conjugated secondary anti-

Fig. 4. Localization of Pol␩ and REV1 to UV-damaged sites in BrdU-negative cells. Locally UV-irradiated (A) WI-38VA13 or (B) XP2SASV3/Pol␩ cells were pulse labeled with 10 ␮M BrdU for 1 h. The cells were extracted, fixed with 70% ethanol/50 mM glycine, pH 2.0, and incubated with (A) anti-REV1C/anti-BrdU/nucleases or (B) anti-REV1C/antiPol␩ (H-300)/anti-BrdU/nucleases. In (A), DNA was visualized with Hoechst 33342. In (B), REV1, Pol␩ and BrdU were visualized with Alexa Fluor 594 (red), Alexa Fluor 488 (green), and Alexa Fluor 350-conjugated secondary antibodies, respectively. Note that some non-specific spots were observed in outside of nuclei due to ethanol fixation. Bar = 10 ␮m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

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bodies (Molecular Probes) at room temperature for 1 h. Then cells were washed with PBS, and nuclear DNA was stained with 1 ␮g/ml Hoechst 33342 (Molecular Probes) in PBS at room temperature for 5 min. The dishes were preserved with 0.02% NaN3 /PBS. Fluorescence microscopy was performed with OLYMPUS IX-81 microscope. Image modification for figures and generation of intensity profiles were performed with the MetaMorph software.

2.8. Micropore UV experiments Cells were cultured in 35-mm glass bottom dishes for 2 days. The cells were washed with PBS, covered with a polycarbonate isopore membrane filter (pore size, 5 ␮m; Millipore), irradiated with 100 J/m2 UV (predominantly 254 nm) at a dose rate of 3.33 J/(m2 s), and then incubated in the growth medium for indicated peri-

Fig. 6. Requirement of REV1 interaction domains of Pol␩ for the REV1 localization to UV-damaged sites. (A) Schematic representation of REV1-interaction domains of Pol␩. The interaction domains suggested by yeast two-hybrid assays by Tissier et al. and ours are depicted green and orange, respectively. Blue represents the Y-family conserved region. Amino acid sequences flanking the two FF-motifs are shown. (B) Interaction of REV1 and Pol␩ proteins in human cells. HeLa Tet-On/Myc-REV1 cells were transiently transfected with pMK10 vector (vec), pMK10-FLAG-Pol␩-His (wt) or pMK10-FLAG-Pol␩-His double FF-AA mutant (FF-AA). Four hours after the transfection, the culture medium was supplemented with 1 ␮g/ml doxycyclin, and cells were cultured further 20 h to induce expression of Myc-REV1. Then the cells were irradiated with 15 J/m2 UV-C (or were left unirradiated for controls), cultured for 1 h, harvested and fractionated into Triton X-100 soluble and solubilized chromatin fractions as described under ‘Materials and methods’. The fractions were immunoprecipitated with FLAG-M2 agarose beads, and precipitates were subjected to Western blot analyses with antiREV1C (upper) and anti-Pol␩ (5H10) (lower) antibodies. (C and D) Localization of endogenous REV1 to UV-irradiated areas. (C) XP2SASV3/Pol␩ or (D) XP2SASV3/Pol␩ (double FF-AA) cells were locally irradiated with UV and incubated for 3 h. REV1 was visualized with anti-REV1C and Alexa Fluor 594-conjugated secondary antibodies (red), and Pol␩ was visualized with anti-Pol␩ (H300) and Alexa Fluor 488-conjugated secondary antibodies (green). DNA was stained with Hoechst 33342 (blue). (E–H) Foci formation of REV1 and Pol␩ in unirradiated cells. (E) Wild-type Pol␩, (F) wild-type Pol␩ and Myc-REV1, (G) double FF-AA mutant Pol␩, or (H) double FF-AA mutant Pol␩ and Myc-REV1 were transiently expressed in XP2SASV3 cells. Pol␩ was stained with anti-pol␩ (H-300) antibody and visualized with Alexa Fluor 488-conjugated secondary antibody. REV1 was stained with anti-REV1C antibody and visualized with Alexa Fluor 568-conjugated secondary antibody. Bar = 10 ␮m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

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Fig. 6. (Continued ).

ods. After washings with ice-cold PBS, cells were treated with extraction buffer (0.5% Triton X-100, 0.5 mM PMSF, 0.4 ␮g/ml antipain, 0.4 ␮g/ml aprotinin, 0.2 ␮g/ml leupeptin, 0.16 ␮g/ml pepstain A in PBS) on ice for 5 min to extract soluble materials. Then remaining materials were washed with ice-cold PBS and fixed as described above. After blocking, the cells were labeled with primary antibodies followed by Alexa Fluor dye-conjugated secondary antibodies. For visualization of the UV-damaged DNA, indirect immunostaining for REV1 and Pol␩ was performed as described above, and then the cells were treated with 2 M HCl at room temperature for 30 min. After washings, the cells were sequentially treated with anti-CPD (TDM-2) antibody for 1 h and Alexa Fluor 350 goat antimouse IgG for 1 h. For simultaneous detections of the proteins and BrdU, cells were incubated with growth medium supplemented with 10 ␮M BrdU for 1 h after micropore UV irradiation. The cells were then extracted as described above, and subsequently fixed with ethanol fixative (70% ethanol, 50 mM glycine, pH 2.0) at −20 ◦ C for 20 min. Indirect immunofluorescence staining was performed as described above

except that cells were incubated with primary antibodies containing anti-BrdU antibody with nucleases (1:10 dilution in incubation buffer) at 37 ◦ C for 30 min.

2.9. Immunoprecipitation To detect Pol␩–REV1 interaction in human cells, Triton X100 soluble and solublilized chromatin fractions from about 6 × 106 cells were incubated with anti-FLAG M2 agarose beads at 4 ◦ C for 1 h. Then, the beads were washed three times with wash buffer B (20 mM Tris-HCl, pH 8.0, 100 mM KCl, 5 mM MgCl2 , 0.2 mM EDTA, 10% glycerol, 0.1% Tween 20, 10 mM 2mercaptoethanol, 0.25 mM PMSF), and FLAG-Pol␩ was eluted twice with FLAG peptide at room temperature for 30 min. The eluted fractions were concentrated by trichloroacetic acid (TCA) precipitation and resolved with SDS-PAGE sample buffer (60 mM Tris–HCl, pH 6.8, 1% SDS, 3% glycerol, 0.1% bromophenol blue) and subjected to SDS PAGE followed by Western blot analyses.

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2.10. TLS assay The 30-mer oligomers containing CPD or 6-4PP were chemically synthesized as described previously [36,37]. The 5 -[32 P] primertemplate DNA was prepared by mixing the 16-mer primer, which was labeled at its 5 end using T4 polynucleotide kinase and [␥32 P]ATP, with the 30-mer DNA containing the lesion at a molar ratio 5:4. Triton X-100 soluble fractions from XP2SASV3, XP2SASV3/Pol␩, and XP2SASV3/double FF-AA Pol␩ cells were immunoprecipitated with an anti-Pol␩ (5C6) antibody bound to Protein G Sepharose. The precipitates were washed each three times with wash buffer C (20 mM Tris–HCl, pH 8.0, 100 mM KCl, 5 mM MgCl2 , 0.2 mM EDTA, 10% glycerol, 0.1% Tween 20, 10 mM 2-mercaptoethanol, 0.25 mM PMSF) and reaction buffer (40 mM Tris–HCl, pH 8.0, 30 mM KCl, 5 mM MgCl2 , 0.25 mg/ml BSA, 5% glycerol, 5 mM DTT, 0.1 mM dNTPs). Then 5 -[32 P] primer-template was added to make final 32 nM (total 10 ␮l), and incubated at 37 ◦ C for 15 min. The reaction was terminated by the addition of 10 ␮l of 98% formamide/10 mM EDTA followed by boiling. Products were electrophoresed on a 20% polyacrylamide/7 M urea gel and autoradiographed. 2.11. UV sensitivity assay Cells were plated into six-well plates before 24 h of UV irradiation. Then, the cells were exposed to increasing doses of UV-C and allowed to grow for 4 days in growth medium containing 1 mM caffeine, which increased the UV sensitivity of XP-V cells [38]. The number of living cells was estimated by measuring OD495 after the 1 h incubation with CellTiter 96 Aqueous One Solution Cell Proliferation Assay reagent (Promega). UV sensitivity was expressed as the percentage of OD495 in treated versus untreated cells. 2.12. Mutagenesis HPRT mutants were selected and mutation frequencies were determined according to published protocols [39,40]. Cells were pre-selected for functional HPRT by expanding the cultures for 10–14 days in medium supplemented with 1× HAT (100 ␮M hypoxantine, 0.4 ␮M aminopterin, 16 ␮M thymidine), and several aliquots of selected cells were stored at −80 ◦ C or in liquid nitrogen. A new aliquot was thawed for every experiment to ensure consistency in the age of cultures. Logarithmically growing cells were plated at 1 × 106 cells/10 cm dish and cultured for 2 days in medium supplemented with 1× HT (100 ␮M hypoxantine, 16 ␮M thymidine) to remove aminopterin, and then treated with or without 8 J/m2 UV-C. In UV-induced mutagenesis experiments, the cells were maintained in logarithmic growth by replating the cells until they underwent 4–7 population doublings for mutation expression. In spontaneous mutagenesis experiments, unirradiated cells were maintained by replating occasionally and PDL (n) was determined from the number of cells seeded (Ni) and recovered (Nf) by the equation Nf/Ni = 2n . Mutant selection was done by replating cells at 0.25, 0.5, and 1 × 105 cells/10 cm dish (three dishes for each cell density) into medium containing 40 ␮M 6-thioguanine. Colony-forming efficiency at the time of selection was determined by plating 200 cells/6 cm dish without 6-thioguanine (three dishes for each conditions). 3. Results 3.1. Detection of endogenous human REV1 protein To analyze intracellular dynamics of human REV1 protein, we prepared an anti-REV1 polyclonal antibody, anti-REV1C, which recognizes the C-terminal portion of human REV1. In Western blots,

the anti-REV1C antibody recognized purified recombinant REV1 protein and a protein of approximately 140 kDa (the predicted Mw for REV1 is 138 kDa) in WI-38VA13 whole cell lysates. Expression of the 140 kDa protein was suppressed by siRNAs against REV1, but not by control scrambled siRNA oligonucleotides, further validating the anti-REV1C antibody (Fig. 1A and B). The anti-REV1C antibody was also applicable for immunocytochemical analyses (Fig. S1). Using quantitative immunoblot analyses with anti-REV1C, we estimated that WI-38VA13 and XP2SASV3 (XP-V) cells contain approximately 60,000 molecules of REV1 per cell. In similar experiments using anti-Pol␩ antibodies we estimated that WI-38VA13 cells also contain 60,000 molecules of Pol␩ per cell (Fig. 1C). These results indicate that human cells contain equivalent amounts of Pol␩ and REV1 proteins, and that Pol␩-deficiency does not affect cellular levels of the REV1 protein. 3.2. Pol enhances foci formation of ectopically expressed full-length REV1 We examined the intracellular localization of ectopically expressed REV1 and Pol␩ proteins. In transient expression experiments we observed very little punctuated distribution of Myc-tagged REV1(1-1251, the full-length form) in nuclei of XP-V cells (Fig. 2A). When GFP-Pol␩ was co-expressed with Myc-REV1(11251), GFP-Pol␩ foci were observed in about 10% of the transfected cells. Moreover, most GFP-Pol␩ foci co-localized with clear foci of Myc-REV1(1-1251). Therefore, Pol␩ expression confers formation of Myc-REV1 nuclear foci in XP-V cells. We performed similar experiments to determine the distribution of Myc-REV1(1-1098) which lacks the C-terminal domain required for interaction with Pol␩· The basal distribution pattern of C-terminal truncated Myc-REV1(1-1098) expressed in the absence of Pol␩ was similar to that of full-length Myc-REV1(11251) (Fig. 2C). However, in contrast with full-length Myc-REV1, Myc-REV1(1-1098) formed only dim foci whose intensities were not enhanced by co-expression of Pol␩, even though Pol␩ exhibited clear foci (Fig. 2D). The ectopic protein level was much higher than the endogenous REV1 (Fig. S1). Therefore, the C-terminal Pol␩interacting region of REV1 is necessary for efficient formation of Myc-REV1 foci in response to Pol␩ co-expression.

Fig. 7. UV sensitivity of XP-V cells expressing wild-type and mutant Pol␩. WI38VA13 (black circle), XP2SASV3 (dark blue diamond), and XP2SASV3 derivative cells stably transformed by pIRESneo2 vector (XP2SASV3/vector cl.6; light blue diamond), pIRESneo2-Pol␩ (XP2SASV3/Pol␩ cl.2 and cl.13; dark and light green circles, respectively), pIRESneo2-Pol␩ (double FF-AA) (XP2SASV3/Pol␩ double FF-AA cl.23, 27-3, and 41; orange, magenta, and brown triangles, respectively), pIRESneo2-Pol␩ (483-4 FF-AA) (XP2SASV3/Pol␩ 483-4 FF-AA; orange square), or pIRESneo2-Pol␩ (531-2 FF-AA) (XP2SASV3/Pol␩ 531-2 FF-AA; purple square) were irradiated with UV-C, cultured for 4 days in medium containing 1 mM caffeine, and measured their viabilities by MTS assays. Data are presented as mean survival rates ± standard errors of three independent experiments. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

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3.3. Pol enhances localization of endogenous REV1 at UV-damaged sites Next we examined the distribution of endogenous REV1 protein using our anti-REV1C antibody. Due to weak signal intensities, we were unable to detect endogenous foci. Therefore, in an alternative approach to studying the subcellular distribution of endogenous REV1 we employed micropore UV irradiation to induce photoproducts in a localized area of the nucleus [41]. When WI-38VA13 cells were locally UV-irradiated and sequentially stained with antibodies against REV1 and CPD at 3 h post-irradiation, REV1 spots were detected which co-localized with sites of CPD (Fig. 3A). The endogenous REV1 spots detectable in micropore irradiation experiments were sensitive to siRNA against REV1 (Fig. S2). Therefore, endogenous REV1 protein accumulates at UV-damaged nuclear regions in Pol␩-proficient cells. The accumulation of endogenous REV1 evident in micropore-irradiation experiments was observed 10 min after UV-irradiation in WI-38VA13 cells. The number of cells containing endogenous REV1 spots gradually increased, reaching a maximal level of about 35% of total cells at 1 h and persisted for 3 h post-irradiation (Fig. S3). In contrast with micropore UV-irradiated WI-38VA13 cells, in Pol␩-deficient XP-V cells the population of nuclei which accumulated endogenous REV1 was very small (Fig. 3B and S3). Importantly, ectopic expression of Pol␩ in the XP-V cells restored the REV1 accumulation to UV-damaged areas (Fig. 3C). Although nuclear accumulation of REV1 was largely dependent on Pol␩, Pol␩ often accumulated without REV1, suggesting that Pol␩ is recruited to UV damaged sites preferentially and independently of REV1. However, we have no explanation for the differences between Pol␩ accumulations accompanied and not accompanied with REV1 at present. It is possible that the differential recruitment of Pol␩ and REV1 to UV-

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irradiated nuclei reflects ectopic overproduction of Pol␩ in these experiments (as we have not yet succeeded in detecting endogenous Pol␩). Indeed, when we transiently overproduced Myc-REV1 in XP-V cells, nuclear accumulation of REV1 signals was occasionally observed following UV-irradiation (Fig. S4).

3.4. REV1 and Pol accumulate at UV-damaged sites both in replicating and non-replicating cells It has been reported that the ectopically expressed REV1 accumulates to the UV-damaged sites even in non-replicating cells [42]. Here we examined behaviors of the endogenous protein. In replicating cells, incorporation of BrdU was decreased in UV-irradiated areas, probably reflecting DNA damage-induced inhibition of initiation and elongation phases of DNA synthesis. The nuclear accumulation of REV1 was observed in both replicating (BrdUpositive) and non-replicating (BrdU-negative) WI-38VA13 cells (Fig. 4A) but not observed in XP2SASV cells (data not shown). In XP2SASV3/Pol␩ cells, ectopically expressed Pol␩ re-localized to nuclear regions often with endogenous REV1 both in replicating and non-replicating cells (Fig. 4B). When we analyzed REV1 distribution in synchronized WI-38VA13 cultures in G1 and S phases as well as asynchronous culture, similar levels of UV-induced nuclear REV1 foci were observed in cells from S-phase-enriched and asynchronous populations (Fig. 5B and C). We also detected nuclear accumulation of REV1 during G1, although ∼30% fewer cells accumulated REV1 foci during G1 relative to S-phase. These results indicate that Pol␩ and REV1 are recruited to UV damaged sites both in replicating and non-replicating cells. Nuclear accumulation of REV1 was also evident in non-replicating XP-A and XP-C cells and is therefore NER-independent (Fig. S5).

Table 1 UV-induced mutation frequencies of XP-V cells expressing wild-type and double FF-AA mutant Pol␩. Cells

UV dose (J/m2 )

Assay no.

No. of clonable cells selected (×105 )

XP2SASV3

0

1 2 3

1.05 2.12 3.06

8

XP2SASV3/Pol␩ cl. 2

0

8

XP2SASV3/Pol␩ double FF-AA cl. 41

0

8

No.of 6-TG resistant colonies 7 4 9

Mutation frequency (×10−5 ) 6.68 1.89 2.95

Total

6.22

20

3.22

1 2

1.27 2.06

62 96

48.86 46.65

Total

3.33

158

47.5

1 2 3 4

1.98 1.96 1.76 2.66

0 0 0 3

0.00 0.00 0.00 1.13

Total

8.36

3

0.36

1 2 3 4

1.72 1.08 1.93 2.64

1 0 1 7

0.58 0.00 0.52 2.65

Total

7.37

9

1.22

1 2 3

2.52 2.74 1.89

7 3 1

2.78 1.09 0.53

Total

7.15

11

1.54

1 2 3

1.91 2.11 1.90

4 0 4

2.10 0.00 2.11

Total

5.92

8

1.35

Total mutation frequency was calculated from the sum of 6-thioguanine resistant colonies and the sum of total clonable cells of each experiment.

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3.5. FF-AA mutant Pol lost abilities to affect cellular localization of REV1

condition, some proportion of the REV1 protein was recovered in the chromatin fraction independently of Pol␩ or UV-irradiation. As shown in Fig. 6B, Myc-REV1 was co-precipitated more efficiently with wild-type FLAG-Pol␩-His than with the double FF-AA mutant of Pol␩ in both soluble and chromatin fractions. However, compared with our previous studies, enhanced Pol␩–REV1 interaction was not so evident after UV-irradiation in these experiments (Fig. 6B). This inability to demonstrate UV-induced interactions between epitopetagged forms of REV1 and Pol␩ most likely reflects the use of ectopic proteins in these experiments, and especially we have no evidence that the Myc-tagged REV1 works as well as the endogenous protein in cells. Although the extreme C-terminus of Pol␩ (containing two phenylalanines at 707–8) also contributes weakly to the REV1binding activity of Pol␩ [34], we did not alter these residues because this sequence is critical for the interaction with PCNA [44]. Next we examined nuclear accumulation of endogenous REV1 in UV-irradiated areas of XP-V cells expressing untagged form of the wild-type or the double FF-AA mutant Pol␩. As expected, endogenous REV1 co-localized with wild-type Pol␩ in nuclei from UV-irradiated cells (Fig. 6C). In contrast, nuclear accumulation of REV1 was rarely observed in XP-V cells expressing the double FFAA mutant form of Pol␩, although nuclear accumulation of mutant Pol␩ was readily observed (Fig. 6D).

To investigate the physiological relevance of the Pol␩–REV1 interaction, we examined phenotypes of XP-V cells complemented with an ectopic Pol␩ mutant which lacks REV1-binding activity due to minimum amino acid substitutions. Previously, we and Tissier et al. have reported that human Pol␩ interacts with REV1 via amino acid residues 509–557 and 369–491, respectively [18,19]. Recently, sequential phenylalanine residues (FF) at 483–4 and 531–2 of Pol␩ were found to be involved in the interaction with REV1 (Fig. 6A) [34]. So, we substituted all REV1-interacting phenylalanine residues of Pol␩ with alanine (designated ‘double FF-AA’ mutant). To examine the Pol␩–REV1 interaction in human cells, expression vectors encoding epitope (FLAG and His)-tagged WT or double FF-AA mutant Pol␩ were introduced into HeLa Tet-On/MycREV1 cells, in which Myc-REV1 was expressed in the presence of doxycyclin (Fig. S6). We have previously shown that the interaction of ectopic Pol␩ and endogenous REV1 is specifically enhanced in the chromatin fraction (rather than Triton X-100 soluble fraction) of UV-irradiated cells [43]. Therefore, we examined interactions between ectopic REV1 and Pol␩ in soluble and chromatin fractions from UV-irradiated and unirradiated cells. In our fractionation

Table 2 Spontaneous mutation frequencies of XP-V cells expressing wild-type and double FF-AA mutant Pol␩. Cells

Assay no.

XP2SASV3

1* 2* 3* 4 5 6 7

XP2SASV3/Pol␩ double FF-AA cl. 41

Generations

Mutation rate (×10−6 )

6.68 1.89 2.95 0.75 1.28 0.66 0.00

4.85 4.20 5.10 13.60 14.00 39.30 57.20

13.77 4.50 5.78 0.55 0.91 0.17 0.00

17.42

27

1.55

22.87

0.68

1.98 1.96 1.76 2.66 1.86 1.62 2.57

0 0 0 3 0 1 2

0.00 0.00 0.00 1.13 0.00 0.62 0.78

4.30 5.30 6.09 6.13 15.10 15.73 44.52

0.00 0.00 0.00 1.84 0.00 0.39 0.17

14.41

6

0.42

14.84

0.28

1 2 3

3.24 1.59 2.90

0 1 0

0.00 0.63 0.00

4.60 5.23 5.84

0.00 1.21 0.00

Total

7.73

1

0.13

5.19

0.25

1* 2* 3* 4 5 6

2.52 2.74 1.89 1.60 1.35 2.44

7 3 1 6 1 0

2.78 1.09 0.53 3.75 0.74 0.00

4.20 4.20 5.90 13.50 15.32 43.40

6.61 2.61 0.90 2.78 0.48 0.00

12.54

18

1.44

14.47

1.00

2.93 1.82 1.43 2.55 1.30 2.12

7 0 0 10 13 3

2.39 0.00 0.00 3.93 9.98 1.42

5.23 6.05 6.32 30.60 50.16 68.26

6.93 0.00 0.00 1.28 1.99 0.21

12.15

33

2.72

26.61

1.02

1* 2* 3* 4* 5 6 7

Total XP2SASV3/Pol␩ double FF-AA cl. 27-3

Mutation frequency (×10−5 )

7 4 9 2 3 2 0

Total XP2SASV3/Pol␩ cl. 13

No.of 6-TG resistant colonies

1.05 2.12 3.06 2.65 2.35 3.03 3.16

Total XP2SASV3/Pol␩ cl. 2

No. of clonable cells selected (×105 )

1 2 3 4 5 6 Total

Total mutation frequency, generations and mutation rate were calculated from (the sum of 6-thioguanine resistant colonies)/(the sum of total clonable cells), (the sum of (generations) × (number of clonable cells) in individual experiment)/(the sum of total clonable cells) and (total mutation frequency)/(total generations), respectively. * Same data in Table 1 are represented.

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To examine the formation of REV1 foci in the absence of UVirradiation, Myc-REV1 was co-expressed with wild-type or mutant Pol␩ in XP-V cells. Although signal intensities were lower than those observed with GFP-Pol␩ (compare with Fig. 2), untagged Pol␩ formed constitutive foci (Fig. 6E) which co-localized with MycREV1 (Fig. 6F). Although the double FF-AA mutant of Pol␩ also formed foci constitutively in a fraction of the cells (Fig. 6G), Pol␩ FF-AA failed to confer formation of Myc-REV1 nuclear foci (Fig. 6H). These results indicate that in contrast with wild-type Pol␩ the double FF-AA Pol␩ mutant fails to interact with REV1 and does not facilitate the recruitment of REV1 to nuclear foci in human cells. The double FF-AA mutant retained TLS activity past CPD which was comparable to that of wild-type Pol␩ (Fig. S7), demonstrating that the FF-AA substitutions do not perturb general Pol␩ function. 3.6. FF-AA mutant Pol fails to suppress spontaneous mutagenesis in XP-V cells We established XP-V cell lines stably expressing wild type Pol␩, double FF-AA mutant Pol␩, and Pol␩ harboring FF-AA mutations in

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each REV1-interacting region FF483-4AA or FF531-2AA. The complemented XPV cells expressed levels of Pol␩ that were >5-fold higher than endogenous Pol␩ levels in WI-38VA13 cells (data not shown). Interestingly, wild-type Pol␩ as well as the Pol␩ mutants harboring individual or combinatorial mutations in the REV1interacting FF motifs fully corrected UV sensitivities of the parental XP-V cells to the level of WI-38VA13 cells (Fig. 7). Therefore, the interaction of Pol␩ and REV1 is not important for viability of human cells after UV-irradiation. To examine whether the double FF-AA mutant Pol␩ can suppress UV-induced mutagenesis in XP-V cells, mutation frequency of the HPRT gene was measured by counting 6-thioguanine resistant colonies. In contrast to parental cells, in which HPRT mutation was drastically induced by UV irradiation, both wild-type and double FF-AA mutant forms of Pol␩ suppressed UV-induced HPRT mutations almost completely (Table 1 and Fig. 8A). Thus, Pol␩–REV1 interaction is likely not required for suppressing UV-induced mutagenesis. Interestingly, we noticed that the mutation frequency of un-irradiated XP2SASV3 cells was fully suppressed by expressing wild-type Pol␩ but was only partially suppressed by the double FF-AA mutant. To confirm this small difference of mutation frequencies, we measured spontaneous mutation frequencies of two clones of wild-type or double FF-AA mutant Pol␩ expressing cells over several generations. Repeated experiments revealed that mutation rates calculated as (mutation frequency)/(average of generations) in XP2SASV3 and in two cell lines expressing double FF-AA mutant Pol␩ were higher than observed in cells expressing wild-type Pol␩ (Table 2 and Fig. 8B). These results suggest that the Pol␩–REV1 interaction contributes to suppressing spontaneous mutations in mammalian cells probably by conducting accurate TLS past spontaneous DNA lesions, although Pol␩ can catalyze accurate TLS past UV-induced lesions in the absence of REV1. 4. Discussion

Fig. 8. (A) UV-induced mutation frequencies of XP-V cells expressing wild-type and double FF-AA mutant Pol␩. Plots show mutation frequency of individual experiment, horizontal bars represent the mean values (depicted under plots). (B) Spontaneous mutation rates of XP-V cells expressing wild-type and double FF-AA mutant Pol␩. Plots show mutation rate of individual experiment, horizontal bars represent the mean values (depicted under plots). Dashed bars represent the mean values of the two clones of XP2SASV3/Pol␩ or XPSASV3/Pol␩ double FF-AA. Note that these mean values differ from the total mutation frequencies or rates listed in Tables 1 and 2, which were calculated by total number of 6-TG resistant colonies/total numbers of clonable cells selected.

In this paper, we detected endogenous REV1 protein in human cells by using a specific antibody. Western blot analyses revealed that both of WI-38VA13 and XP2SASV3 (XP-V) cells have approximately 60,000 REV1 molecules per cell, suggesting that the presence or absence of Pol␩ did not affect REV1 levels. Pol␩ is also present at ∼60,000 molecules per cell in WI-38VA13, indicating that normal cells have similar levels of Pol␩ and REV1 proteins. The Pol␩–REV1 interaction, however, may be a dynamic association rather than a stable one, as less than 1% of REV1 could be co-precipitated with Pol␩. Cellular protein levels of REV1 and Pol␩ were unaffected by UV irradiation or cell cycle progression (data not shown), suggesting that these TLS polymerases respond to DNA damage via changes in activity, cellular localization or protein–protein interactions. We showed that localization of endogenous REV1 to UVirradiated areas in the nucleus is largely dependent on Pol␩. Point mutations of Pol␩ in critical phenylalanines for the REV1 interaction attenuated its ability to re-localize endogenous REV1, strongly suggesting that REV1 is recruited to DNA lesions predominantly through direct protein–protein interactions with Pol␩. Foci formation of the ectopically expressed REV1 in un-irradiated cells was also enhanced by the co-expression of Pol␩ in the interactiondependent manner. Previously, Tissier et al. [19] reported that ectopic YFP-REV1 co-localized with PCNA nuclear foci independently of Pol␩. We also observed accumulation of ectopically expressed Myc-REV1 at UV-damaged areas in XP-V cells. We consider it likely that cells have additional Pol␩-independent mechanism(s) to recruit REV1 at sites of DNA lesions, which are most readily detected when REV1 is present at high levels following ectopic expression. It has

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been reported that REV1 interacts directly with monoubiquitinated PCNA through two ubiquitin-binding motifs (UBM) near its C-terminus and the BRCT domain near its N-terminus [46,47]. Since the C-terminus of REV1 is also important for interactions with Pol␫, Pol␬ and Pol␨ in addition to Pol␩ [17,18,31–33], we could not exclude possibilities that interactions with these proteins also contribute to the recruitment of REV1. Actually, ectopic co-expression of Pol␬ increased the frequency of the foci formation of ectopically expressed REV1 [18], suggesting that multiple Y family polymerases can contribute to the REV1 recruitment. These protein–protein interactions with alternative Y-family polymerases or with other proteins may help recruit REV1 to DNA lesions independently of Pol␩. However, it should be noted that these observations were of ectopically over-expressed proteins. Our results suggest that Pol␩ guides REV1 to sites of DNA damage under the physiological protein concentrations. A previous report indicates that Pol␫ localizes in nuclear foci in a Pol␩-dependent manner [20]. Thus, Pol␩ is likely to be preferentially recruited to sites of DNA damage, and may subsequently facilitate the recruitment of additional TLS polymerases. Pol␩ is also known to interact with the RAD6–RAD18 complex [43,45], which ubiquitinates PCNA in cells that acquire DNA damage [14]. It has been proposed that RAD18 helps ‘guide’ Pol␩ to stalled replication forks [45]. Once guided to sites of replication fork stalling, Pol␩ is well poised to associate with ubiquitinated PCNA via its ubiquitin-binding motif immediately after the RAD6-RAD18 complex has ubiquitinated PCNA. Such preferential recruitment of Pol␩ to DNA lesions may be advantageous because Pol␩ has a broader range of lesion specificity than Pol␫ or Pol␬. It is generally considered that genotoxin-induced TLS polymerase nuclear foci represent stalled replication sites in replicating cells [48]. However, accumulation of REV1 and Pol␩ at UVirradiated areas in nuclei were observed not only in S-phase cells but also in BrdU-negative and G1 synchronized cells. Similar results have been reported by using GFP-REV1 protein [42]. The nuclear accumulation of REV1 was also observed in non-replicating XP-C and XP-A cells (which are deficient in the initiation steps of NER) and therefore is unlikely to contribute to DNA synthesis during NER. It has been proposed that yeast Rev1 has largely a G2 function as it is highly expressed during G2/M phase rather than S-phase [49]. It has been also reported that Rev1 and Rev3 are involved in the production of DNA replication-independent frameshift mutations in cell cycle arrested yeast cells [50] and that monoubiquitination of PCNA occurs in both proliferating and arrested cells independently of NER [51,52]. These observations suggest that TLS polymerases are recruited not only to the arrested replication machinery, but also to DNA lesions acquired outside S-phase via interaction with monoubiquitinated PCNA, although we have no data to address physiological relevance of these observations at present. Mutant Pol␩ containing substitutions of four phenylalanines at residues 483, 484, 531, and 532 showed decreased interaction with REV1 in human cells, yet corrected UV-sensitivities and suppressed UV-induced mutagenesis in XP-V cells almost completely. These results suggest that efficient and accurate TLS past UV-damaged lesions is predominantly performed by Pol␩ itself and does not require direct interaction with REV1. This is in sharp contrast to the observation that a FF-AA mutant Pol␬ devoid of REV1 binding failed to correct BPDE- and UV-sensitivities of the Polk−/− mouse embryonic fibroblast cells [34]. Although Pol␩–REV1 interaction is unlikely to be involved in UV-damage tolerance in human cells, we found that the interaction is required for formation of REV1 foci in un-irradiated cells and may contribute to suppression of spontaneous mutations. It has been reported that ectopic overexpression of Pol␩ does not induce spontaneous mutations [40] despite its error-prone nature when copying undamaged DNA [53]. Instead, our experiments have identified a novel role of Pol␩ in suppressing

spontaneous mutagenesis through interaction with REV1. We have not yet elucidated the molecular basis for REV1-dependent suppression of spontaneous mutations by Pol␩. Potentially, Pol␩–REV1 interactions might play a role in polymerase switching events that are required when Pol␩ is inappropriately recruited to lesions that it cannot bypass [54]. Pol␩-dependent recruitment of REV1 most likely represents a general response to many types of DNA lesions. In the case of UV, REV1 was dispensable for bypass of CPD lesions, which are bypassed efficiently by Pol␩ alone. However, REV1 is likely to be required for accurate bypass of endogenous lesions which cannot be bypassed by Pol␩ alone. DNA lesions induced by oxidative stress are the most likely sources of spontaneous damage and mutagenesis. Therefore, future experiments will be performed to investigate the significance of Pol␩–REV1 interaction in the response to oxidative DNA damage. In conclusion, our findings provide new insights into the physiological relevance of interactions between TLS polymerases, which contribute to genome maintenance in mammalian cells. Conflict of interest None. Acknowledgements We thank all the members of the Hanaoka laboratory at Osaka University and Dr. Cyrus Vaziri at Boston University School of Medicine for critical discussions and helpful comments. We also appreciate Dr. Kiyoji Tanaka and Dr. Shigenori Iwai at Osaka University, for kindly providing XP2SASV3 cells and lesion-containing DNA oligomers, respectively. This work was supported by KAKENHI (Grant-in Aid for Scientific Research) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (17013053 to F.H.) and by Solution Oriented Research for Science and Technology from the Japan Science and Technology Agency. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.dnarep.2008.12.006. References [1] C. Masutani, M. Araki, A. Yamada, R. Kusumoto, T. Nogimori, T. Maekawa, S. Iwai, F. Hanaoka, Xeroderma pigmentosum variant (XP-V) correcting protein from HeLa cells has a thymine dimer bypass DNA polymerase activity, EMBO J. 18 (1999) 3491–3501. [2] C. Masutani, R. Kusumoto, A. Yamada, N. Dohmae, M. Yokoi, M. Yuasa, M. Araki, S. Iwai, K. Takio, F. Hanaoka, The XPV (xeroderma pigmentosum variant) gene encodes human DNA polymerase ␩, Nature 399 (1999) 700–704. [3] Q. Lin, A.B. Clark, S.D. McCulloch, T. Yuan, R.T. Bronson, T.A. Kunkel, R. Kucherlapati, Increased susceptibility to UV-induced skin carcinogenesis in polymerase ␩-deficient mice, Cancer Res. 66 (2006) 87–94. [4] T. Ohkumo, Y. Kondo, M. Yokoi, T. Tsukamoto, A. Yamada, T. Sugimoto, R. Kanao, Y. Higashi, H. Kondoh, M. Tatematsu, C. Masutani, F. Hanaoka, UV-B radiation induces epithelial tumors in mice lacking DNA polymerase ␩ and mesenchymal tumors in mice deficient for DNA polymerase ␫, Mol. Cell. Biol. 26 (2006) 7696–7706. [5] C. Masutani, R. Kusumoto, S. Iwai, F. Hanaoka, Mechanisms of accurate translesion synthesis by human DNA polymerase ␩, EMBO J. 19 (2000) 3100–3109. [6] A. Vaisman, C. Masutani, F. Hanaoka, S.G. Chaney, Efficient translesion replication past oxaliplatin and cisplatin CpG adducts by human DNA polymerase ␩, Biochemistry 39 (2000) 4575–4580. [7] Y. Zhang, F. Yuan, X. Wu, O. Rechkoblit, J.S. Taylor, N.E. Geacintov, Z. Wang, Error-prone bypass by human DNA polymerase ␩, Nucleic Acids Res. 23 (2000) 4717–4724. [8] R. Kusumoto, C. Masutani, S. Iwai, F. Hanaoka, Translesion synthesis by human DNA polymerase ␩ across thymine glycol lesions, Biochemistry 41 (2002) 6090–6099. [9] Y. Zhang, X. Wu, D. Guo, O. Rechkobalt, N.E. Geacintov, Z. Wang, Two-step errorprone bypass of the (+)- and (−)-trans-anti-BPDE-N2-dG adducts by human DNA polymerase ␩ and ␬, Mutat. Res. 510 (2002) 23–35.

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