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1 Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto 862-0976, Japan
2 Department of Surgery II, Kumamoto University School of Medicine, Kumamoto 860-0811, Japan
3 Department of Science for Laboratory Animal Experimentation, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita City, Osaka 565-0871, Japan
4 Department of Regulation Biology, Faculty of Science, Saitama University, Urawa 338-8570, Japan
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Abstract |
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Introduction |
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Like rad6 mutants, rad18 mutants are severely sensitive to DNA damaging agents such as UV and alkylating agents (Haynes & Kunz 1981), and show an elevated spontaneous mutation frequency (Kunz et al. 1991). However, unlike rad6 mutants, rad18 mutants do not manifest UV-induced mutagenesis (Lawrence & Christensen 1976; Jones et al. 1988). Recently, Rad18 was shown to be involved in the ubiquitination of PCNA following DNA damage together with Rad6 (Hoege et al. 2002). Mono-ubiquitinated PCNA is further poly ubiquitinated at lysine 63 of the attached ubiquitin by the Rad5/Mms2/Ubc13 complex (Hoege et al. 2002). Higher eukaryotes contain one homolog of RAD18 and two homologs of RAD6 (HHR6A, HHR6B). Similar to lower eukaryotes, human and mouse homologs of Rad18 (hRad18 and mRad18, respectively) bind to Rad6 homologs (Tateishi et al. 2000; Xin et al. 2000). Human cell lines over-expressing mutant Rad18 at the RING finger motif show enhanced sensitivity to multiple DNA damaging agents and defective postreplication repair (Tateishi et al. 2000). More recently, RAD18 knockout cells were prepared from mouse ES cells (Tateishi et al. 2003) and chicken DT40 cells (Yamashita et al. 2002). These knockout cells are moderately sensitive to various DNA damaging agents and show genomic instability as shown by increased rates of sister chromatid exchange (SCE) (Yamashita et al. 2002; Tateishi et al. 2003), and integration of exogenous DNA (Tateishi et al. 2003).
In the budding yeast, transcription levels of RAD18 and RAD6 are up-regulated following genotoxic treatments and during meiosis (Jones & Prakash 1991). However, they remain constant during the mitotic cell cycle (Jones & Prakash 1991). Although some data have been presented about the expression of Rad18 in higher eukaryotes (van der Laan et al. 2000; Xin et al. 2000), thus far no information is available on regulation of the expression and localization of Rad18 under various culture conditions. In the present study, we showed that Rad18 protein levels remained constant even while RAD18 mRNA levels ranged from 25% to 175% of the control level. Furthermore, Rad18 dynamically translocated in the nucleus following various genotoxic assaults.
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Results |
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The expression of RAD18 mRNA in various tissues of mammals was examined by Northern blotting. RAD18 mRNA was expressed ubiquitously as multiple bands in humans, hamsters and mice. The major two bands corresponded to 2.9 kb and 3.6 kb. Expression levels varied among tissues (Fig. 1A), being moderate in spleen, ovary and prostate, and exceptionally high in testis. Estimation by densitometry revealed that expression levels were more than five-fold higher in the testis than other tissues. Inside the seminiferous tubules, spermatogonia, early spermatocytes, late spermatocytes, round spermatids and elongated spermatids are included. Among these cells, early spermatocytes showed a very high level of expression of mRAD18 mRNA (Fig. 1B). To examine the expression of Rad18 protein in individual cells in testis, we performed immunostaining using a mouse-specific antibody against Rad18. In the early meiotic stage, whole nuclei of spermatocytes were stained (Fig. 1C). These results are consistent with the results published elsewhere (van der Laan et al. 2000).
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The postreplication repair system works during or possibly after DNA replication. To examine the expression levels of RAD18 mRNA and Rad18 protein during the cell cycle, HeLa cells were synchronized by the double thymidine block method. Synchronization was confirmed by flow-cytometry (FACS) (data not shown) and immunostaining for the BrdU incorporated during pulse-labeling. Both methods showed that more than 90% of the treated cells were arrested at the G1/S border at the end of the second thymidine block (Fig. 2A). Immediately after their release from the block, cells entered the S-phase. RAD18 mRNA levels increased with incubation up to the 6 h point, reaching a maximal 1.7-fold increase in the late S-phase (6 h), and then decreased rapidly during the G2/M (9 h) and the early G1 phase where the RAD18 mRNA level was about 50–60% of that in G1/S (Fig. 2B,C). This is in sharp contrast to RAD18 transcription in the yeast where RAD18 mRNA levels do not change during the cell cycle (Jones & Prakash 1991). Although in budding yeast, Rad18 protein is detected as a single band corresponding to 60 kDa (Bailly et al. 1997a), the protein in HeLa cells (hRad18) was detected as two major bands corresponding to 75 kDa and 85 kDa (Fig. 2D). These two bands were detected in all human cell lines tested in this laboratory. Since the molecular weight of hRad18 is estimated to be 63 kDa from its amino acid sequence, hRad18 is probably modified post-translationally. In contrast to the apparent fluctuation of RAD18 mRNA levels, Rad18 protein levels remained constant throughout the cell cycle (Fig. 2D,E). Similarly to a previous report (Bravo & Celis 1980), PCNA protein levels increased in the early S-phase (Fig. 2D, lanes 1, 2). These results suggest that Rad18 translation is down-regulated during the S-phase and up-regulated during the G2-phase.
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In the yeast Saccharomyces cerevisiae, RAD18 and RAD6 are transcriptionally up-regulated following genotoxic treatments (Jones & Prakash 1991). To examine whether the up-regulation of RAD18 and RAD6 is a general cellular response of lower eukaryotes, we measured mRNA levels of uvs-2 and mus-8, homologs of RAD18 and RAD6 of the filamentous fungus Neurospora crassa, respectively, by Northern blotting after treatment with either UV or MMS. Both mRNA amounts increased over 10-fold within an hour in a dose-dependent manner (Fig. 3A,B). In contrast, when HeLa cells were irradiated with UV, the mRNA expression of RAD18 decreased rapidly in a dose-dependent manner: with 20 J/m2, mRNA levels dropped to as low as 30% of the control value at 8 h after irradiation (Fig. 4A,B). Similarly, RAD6 mRNA levels decreased by more than 50% following UV-irradiation (Fig. 4A). In sharp contrast, Rad18 protein levels remained at the control level for up to 24 h following UV-irradiation (Fig. 4C,D). These results indicate that Rad18 protein expression is up-regulated by mechanisms that compensate for the decrease in transcription levels following DNA damage. To estimate the half-life of the Rad18 protein, cycloheximide was added to the culture medium of undamaged HeLa cells to inhibit protein synthesis. The half-life of 85 kDa Rad18 was nearly 4 h, and that of the 75 kDa form seemed to be a little longer (Fig. 5A, lanes 1, 2, 3, and Fig. 5B). Again both protein levels remained at nearly initial values following UV-irradiation (Fig. 5A, lanes 1, 4, 5, and Fig. 5B). However, when de novo protein synthesis was inhibited by treatment of the cells with cycloheximide, Rad18 protein levels decreased at similar rates irrespective of UV-irradiation (Fig. 5A, lanes 1, 6, 7 and Fig. 5B), suggesting that degradation rates were not affected by UV-irradiation. Since the maintenance of Rad18 protein levels following UV-irradiation was observed in other human cell lines such as MCF7 (a mammary tumor cell line), GM637 (normal human fibroblasts transformed with SV40) and Mori (primary cells) (data not shown), we concluded that this is a general phenomenon of human cells. Interestingly, Rad6 as well as Rad18 protein levels were not affected by UV in the presence of cycloheximide (Fig. 5A,C). From these results, we inferred that translation rates for RAD18 mRNA increased following DNA damage. To test this possibility, we labeled HeLa cells with a mixture of 35S-methionine and 35S-cysteine for short periods at 6 h after UV-irradiation. At this time point, RAD18 mRNA levels of UV-irradiated cells became at nearly 40% of those of control cells (Fig. 4B). As shown in Fig. 5D, almost the same amount of Rad18 protein was synthesized in the UV-irradiated cells as in the non-irradiated cells, indicating that the translation rate of Rad18 is up-regulated by UV-irradiation.
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Subfractions of Rad18 protein localized to the nucleus as 50–100 discrete nuclear dots. Sizes of each dots differed in individual cell lines, and even in the same line. When cells were irradiated with UV, these nuclear dots dispersed within 20 min throughout the nucleus excluding the nucleolus in a dose-dependent manner, resulting in a rapid increase in the intranuclear concentration of Rad18 of the diffused type (Fig. 6A). The dispersion was observed even at a low dose of UV-irradiation (5 J/m2) (Fig. 6B). A similar dose- and time-dependent dispersion of Rad18 was observed after treatments not only with DNA-damaging agents such as an alkylating agent (MMS), cross-linkers (MMS, cisplatin), an oxidation agent (H2O2), and ionizing radiation, but also with inhibitors of DNA replication such as hydroxyurea and aphidicolin which inhibit ribonucleotide reductase and DNA polymerase 
, 
and 
, respectively (Fig. 6C). These results suggest that Rad18 dispersion is triggered not by DNA damage but by DNA replication stalling as a result of DNA damage. Furthermore, the dispersion of Rad18 was observed even in the presence of cycloheximide (data not shown), indicating that de novo protein synthesis is not required for the dispersion process. The re-appearance of nuclear dots in UV-irradiated cells occurred slowly, and it took nearly 24 h to restore the initial staining pattern (Fig. 6D).
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Discussion |
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Rad18 is very highly expressed in the testis. A more detailed examination revealed that the periods with the highest expression correspond to the zygothene–pachythene stages of spermatogenesis in mice, when genetic recombination takes place. Similarly, Rad18 levels in the budding yeast increase during meiosis. Since both SCE and illegitimate recombination frequencies increased several fold in RAD18–/– ES cells compared with wild-type cells (Tateishi et al. 2003), we speculate that Rad18 modulates recombination activity to suppress deleterious recombination reactions.
We determined the RAD18 mRNA and protein levels during the cell cycle. Although RAD18 mRNA levels in the budding yeast remained constant during the cell cycle (Jones & Prakash 1991), the mRNA expression of RAD18 in human cells was up-regulated in the late S-phase and down-regulated in the early G1-phase. Rad18 protein levels, however, remained constant during the cell cycle. Similarly, although transcription levels of RAD18 in S. cerevisiae and N. crassa increased rapidly more than several fold after DNA damage, those of mammalian cells decreased sharply. But again, Rad18 protein levels in mammalian cells changed little after the treatments, suggesting that some mechanism exists to maintain the level of Rad18 protein during the cell cycle or after genotoxic treatments. Since the maintenance of Rad18 protein levels was inhibited in the presence of cycloheximide, it required protein synthesis. There may be several explanations for this phenomenon of Rad18 maintenance. The simplest model would be autoregulation of the translation of RAD18 mRNA by the Rad18 protein itself: when Rad18 protein levels increase, more Rad18 protein molecules bind to RAD18 mRNA, thereby suppressing translation of the mRNA. Reciprocally, when Rad18 protein levels decrease, free RAD18 mRNA levels increase, resulting in an up-regulation of translation. We do not know why Rad18 protein levels are kept constant in mammalian cells under various conditions, but one plausible reason may be as follows. Rad6 is a ubiquitin-conjugating enzyme, and it binds to not only Rad18 but also Ubr1 (Watkins et al. 1993) and Bre1 (Wood et al. 2003), both of which are involved in the ubiquitination of histone H2B under normal conditions. Fluctuation of Rad18 protein levels might disturb the equilibrium of these complexes sharing Rad6, which might have detrimental effects on cell survival. Indeed, over-expression of Rad6 in mammalian cells results in chromosomal instability (Shekhar et al. 2002). Furthermore, in our preliminary experiment, we could not isolate stable transformants over-expressing Rad18 protein (data not shown). The balancing of these protein concentrations might be important for normal cell survival.
Rad18 exists in the nucleus in two forms, a diffused form and a condensed form. The condensed form localizes as discrete nuclear dots of different sizes and shapes. Whether these two forms correspond to the two major molecular species of Rad18 (75 kDa and 85 kDa) remains to be resolved. Following various types of DNA damage induced by UV, alkylating agents or DNA cross-linkers, the nuclear dots dispersed within 15 min even with mild treatments. As a result, the intranuclear concentration of the diffused type increased rapidly. Since such a dispersion of Rad18 also occurs on treatment with DNA replication inhibitors, it is assumed that the dispersion is induced not by the damage itself but by the stalling of DNA replication. This type of dynamic intranuclear translocation following DNA replication block has been observed with Brca 1 (Scully et al. 1997). In this case, phosphorylation of Brca 1 by checkpoint kinase 2 (chk2) is involved in the initial step of the event (Lee et al. 2000). It is possible that the same pathway is involved in the dispersion of Rad18. However, since Rad18 is phosphorylated at multiple sites immediately after translation (our unpublished data), it is difficult to test this hypothesis. Furthermore, we could not detect any changes in the mobility of Rad18 in cells treated with DNA damaging agents by SDS-PAGE under the present conditions (Fig. 5). Following its dispersion, Rad18 co-localized with PCNA for several hours, suggesting that it is involved in DNA damage tolerance at the stalled sites (Nikiforov et al. 2004; Watanabe et al. 2004). Because PCNA is monoubiquitinated in RAD18-dependent manner, and because polymerase 
showes a high affinity to the modified PCNA, it is assumed that the modification of PCNA would be the motive force for the polymerase switching from pol to pol (Kannouche et al. 2004; Watanabe et al. 2004). In the budding yeast, it is reported that following DNA damage, the Mms2/Ubc13 complex migrates from the cytoplasm to the nucleus (Ulrich & Jentsch 2000), but there is no report of a dynamic translocation of Rad18 in lower eukaryotes.
Compared with the dynamic movement of Rad18 following DNA damage, the intracellular localization of Rad6 changed little: it was mainly expressed in the nucleus diffusely. Some Rad6 was detected in the perinuclear region of the cytoplasm. No nuclear dots were observed with any of the polyclonal anti-Rad6 antibodies that we used. Since Rad6 is highly conserved from lower eukaryotes to higher eukaryotes, there is a possibility that all of our polyclonal antibodies recognize a unique epitope of Rad6 which is located at the binding site for Rad18. Thus, binding of these antibodies to Rad6 might be inhibited by the bounded Rad18. However, even if we expressed Rad6 with a tag, we could not observe any nuclear dots of Rad6 with an antibody against the tag (data not shown), suggesting that this possibility is low. Taken together, the present results strongly suggest that regulation of the postreplication pathway including Rad18 and Rad6 in higher eukaryotes differs significantly from that in lower eukaryotes.
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Experimental procedures |
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HeLa, MCF7, GM637 and Mori (primary human skin fibroblasts established in this laboratory) cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, penicillin G (100 units/mL), and streptomycin (100 µg/mL) in a humidified 5% CO2 incubator. For cell synchronization, the double thymidine block method was used (Thilly 1976). In brief, about 30% confluent HeLa cells were cultured in dishes with a diameter of 60 mm for 2 days in normal medium. After thymidine was added at a concentration of 1.5 mM, cells were cultured for 24 h, and then cultured for 8 h in normal medium. After a second addition of thymidine at 1.5 mM, cells were cultured for 14 h. These cells were then cultured again in normal medium, thereby releasing them from cell cycle arrest at G1/S. To determine the levels of cell synchronization, coverslips were included in the dishes. At the specific time points after the release, the coverslips were transferred to new dishes (30 mm in diameter), and labeled with BrdU.
Immunocytochemistry
To assess synchronization by the double thymidine block method, HeLa cells were labeled for 30 min with 10 µM BrdU at the indicated time points after release from the second thymidine block. Cells were fixed with 100% methanol for 10 min/at 0 °C. After washing with PBS, cells were treated with 2 N HCl for 30 min, neutralized with 0.1 M sodium tetrahydroborate (pH 8.5) for 1 min, and stained for BrdU with an anti-BrdU monoclonal antibody (Pharmingen) for 30 min. The percentage of BrdU-labeled cells was determined by counting at least 300 cells.
To examine the localization of Rad18 or Rad6, GM637 cells were prefixed with 3.7% formaldehyde for 2 min at room temperature, and then fixed with 80% methanol for 10 min at 0 °C. After washing with PBS, the cells were stained for either Rad18 or Rad6 with rabbit polyclonal antibodies for 30 min at room temperature by an indirect immunofluorescent staining method. Anti-human Rad18 antibody was raised by immunizing rabbits with a human Rad18 fragment (aa 383–495) as described (Tateishi et al. 2000). Anti-mouse Rad18 antibody was raised by immunizing rabbits with a mouse Rad18 fragment (aa 373–509) as described (Tateishi et al. 2003). Anti-human Rad6 antibodies were raised by immunizing rabbits with either full-length recombinant HHR6A or HHR6B protein. In some cases, anti-Mus8 (the Rad6 homolog of N. crassa) rabbit antibody, which cross-reacts well with human Rad6, was used (Tateishi et al. 2003). To examine dispersion of Rad18, GM637 cells treated with various DNA damaging agents were cultured for given periods and fixed as described above. For evaluation of Rad18 dispersion, the percentage of cells showing typical nuclear dots was determined by counting at least 300 cells.
Testes were fixed in 4% paraformaldehyde in PBS for 20 h at 4 °C and embedded in methyl methacrylate (MMA) resin (Blythe et al. 1997), and sections (4 µ) were collected on a microslide glass. After removal of the MMA resin by incubation in xylen for 5 min at 37 °C. They were blocked in blocking solution in TBS (pH 7.4) (Nacalai Tesque, Kyoto, Japan) for 30 min at room temperature before incubation with the Rad 18 poly clonal antibody diluted at 1 : 200 for 1 h at room temperature. The sections were incubated with FITC-conjugated anti-rabbit antibody (Jackson) for 30 min at room temperature and observed with a fluorescence microscope.
Western blotting
Cells treated with various DNA-damaging agents were washed once with PBS and lyzed in a buffer containing 1.7% sodium dodecyl sulfate, 17% glycerol, 0.1 M dithiothreitol, and 0.083 M Tris (pH 6.8). In some cases, cycloheximide was added to the culture medium to determine the stability of Rad18 protein. These cell lysates were boiled for 10 min and stored at –20 °C before use. SDS-PAGE and immunoblotting were performed as described elsewhere (Tateishi et al. 2000). Rad18 protein and Rad6 protein were detected with the rabbit polyclonal antibodies described above. In all cases, -tubulin was detected with an anti--tubulin monoclonal antibody (OPO 6, Calbiochem) to confirm loading of equal amounts. Immunoblots were developed with an ECL detection system (Amersham Pharmingen Biotech).
Experiments with N. crassa
Conidia of the Neurospora crassa wild-type strain were cultured for 6 h at 30 °C in liquid minimal medium containing 1.5% sucrose. Germinating conidia were UV-irradiated at doses of 0, 100, 200, 400, 800 J/m2 or treated with MMS at the concentration of 0, 0.0015, 0.005, 0.015, 0.05%, and cultured for 1 h at the same condition. Total RNAs were extracted as described elsewhere (Sokolovsky et al. 1990). Northern blot analysis was carried out as described elsewhere (Sambrook et al. 1989). The probe DNA was labeled with 32P using the Multiprime DNA labeling kit (Amersham). cDNA of uvs-2 and mus-8 were used as probes.
Northern blotting
Human total RNA from various organs was hybridized with 32P-labeled human RAD18 cDNA using Multiple Tissue Northern Blot (Clontech) and Total RNA Northern Blot (Biochain). Chinese hamster total RNA from various organs was hybridized with 32P-labeled mouse RAD18 cDNA. Northern blot analysis was carried out as described elsewhere (Sambrook et al. 1989). To obtain germ cell fractions at different stages of development, we separated spermatogenic cells from testes of wild-type adult mice into enriched subpopulations by centrifugal elutriation as described elsewhere (Bellve et al. 1977). These subpopulations were early pachytene spermatocyte, late pachytene spermatocyte, round spermatid, and elongating spermatid fractions. We extracted RAN from each fraction and used them for Northern blot analysis.
Labeling of Rad18 protein
Sub-confluent monolayers of HeLa cells in 6 cm dishes were incubated for 3 days in normal DMEM. Cells were irradiated with UV at 20 J/m2, and incubated for 4.5 h in normal DMEM. After incubation for another 1.5 h in DMEM containing 10% dialyzed FCS without methionine and cysteine, Promix (Amersham) containing 0.2 mCi 35S-methionine and 35S-cysteine was added to the medium. Cells were incubated for the periods indicated. These cells were washed twice with PBS, scraped, and suspended in RIPA buffer. Labeled hRad18 protein was immunoprecipitated with a polyclonal rabbit antibody against hRad18 and then protein G-Sepharose. After the Sepharose beads had been washed with RIPA buffer, bound proteins were separated by SDS-PAGE. The proteins were transferred to a PVDF membrane (Millipore). The membrane was dried for 10 min and placed in contact with a X-ray film or an imaging plate (Fuji film).
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: yamaizm{at}gpo.kumamoto-u.ac.jp
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References |
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Received: 14 April 2005
Accepted: 19 April 2005
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