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CREST Research Project, Radiation Genetics, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan,1 Medical Research Council, Laboratory of Molecular Biology, Division of Protein and Nucleic Acid Chemistry, Hill Roads, Cambridge CB2 2QH, United Kingdom,2 Molecular Genetics, Institute of Development, Aging and Cancer, Tohoku University, Sendai 980-8575, Japan,3 Biological Chemistry Department, School of Medicine, University of California, Irvine, California 92697-1700,4 Department of Genetics, Microbiology and Toxicology, Arrhenius Laboratory, Stockholm University, S-106 91 Stockholm, Sweden,5 Department of Pathologic Biochemistry, Medical Research Institute, Tokyo Medical and Dental University, 2-3-10 Kandasurugadai, Chiyoda-ku, Tokyo 101-0062, Japan,6 Institute of Molecular Radiobiology, GSF, Neuherberg, Germany7
Received 8 July 2006/ Returned for modification 7 August 2006/ Accepted 17 November 2006
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ABSTRACT |
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INTRODUCTION |
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RAD6/RAD18 are central to PRR in budding yeast (reviewed in reference 6), and the amino acid sequences of both proteins are well conserved from yeast to human cells. Rad6 is a ubiquitin-conjugating enzyme (E2) (25), forming a tight complex with the Rad18 protein (4, 6). Rad18 has DNA-binding activity and may recruit the Rad6 protein to DNA lesions (3). Rad18 contains a RING finger motif, which is common to E3 ubiquitin ligases. The complex of Rad6 and Rad18 transfers ubiquitin onto PCNA, a clamp protein for DNA polymerases, in response to DNA damage (22, 55, 60). The ubiquitylation at Lys164 of PCNA appears to facilitate the recruitment of translesion DNA polymerases to DNA damage sites in mammalian cells, as well as in yeast (29, 60, 62). Although, like yeast, Rad18 deficiency in DT40 and murine embryonic stem cells results in hypersensitivity to a variety of DNA-damaging agents, it is unclear whether the function of Rad18 has been fully conserved during evolution (58, 63). In fact, PRR in vertebrates seems to be more complex than in yeast, not least because the number of TLS polymerases is larger (39) than in budding yeast (37). Accumulating evidence points to a degree of independence between Rad18 and at least three components of TLS, Pol kappa (
), Rev3 (Pol
), and Rev1, in DT40 cells (40, 48, 53). This observation is in agreement with the finding that Rev3 deficiency compromises cellular proliferation more significantly than does Rad18 deficiency in both mouse and DT40 cells (42, 53, 61). Likewise, the mutation of Lys164 of PCNA to arginine (pcnaK164R) in DT40 cells results in a more pronounced phenotype than does the disruption of the RAD18 gene (2, 52). Therefore, Rad18 apparently has lost some regulatory roles in PRR during evolution from yeast to mammals.
DSBs occur as a consequence of collapsed replication forks at sites of replication blockage or due to the action of environmental factors, such as IR. There are two major DSB repair pathways, HR and nonhomologous end joining (NHEJ), the functions of which are conserved from yeast to mammals (reference 18; reviewed in references 24 and 49). HR relies on the presence of a stretch of undamaged homologous DNA to act as a template for repair and is therefore largely accurate. NHEJ is able to directly rejoin the ends of a break and often results in errors in the form of sequence deletions (35). The initial steps of NHEJ involve the association of the DNA end-binding proteins Ku70 and Ku80 with the break, followed by the recruitment of the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs). The Ku-DNA-PKcs complex ultimately recruits ligase IV, which completes the repair of the break (12).
In vertebrates, HR and NHEJ differentially contribute to DSB repair, depending on the nature of the DSB and the phase of the cell cycle (57). IR induces DSBs in the genomic DNA of packed chromatin structure. The resulting DSBs are repaired primarily by NHEJ in the G1 phase and partly by HR between a damaged and an intact sister chromatid in the late S and G2 phases (reference 35; reviewed in reference 64). Cells deficient in both HR and NHEJ are extremely sensitive to IR compared to the respective single mutants, indicating that the two DSB repair pathways are complementary to each other in the repair of IR-induced DSBs (11, 57). Furthermore, several studies suggest that the Ku heterodimer interferes with HR-dependent DSB repair in various organisms, indicating competition between NHEJ and HR (14, 17, 44). In addition to IR, DSBs are generated as a consequence of replication blockage. This type of DSB is induced by exposing cells to camptothecin (CPT), which causes the stabilization of a complex of topoisomerase I covalently linked to nicked DNA. Such complexes interrupt replication and cause DSBs in one of the sister chromatids (reviewed by 45, 50). The resulting DSBs are repaired primarily by HR with the other, intact sister chromatids rather than by NHEJ (50). Accordingly, HR-deficient cells, but not ku70 or ligaseIV cells, are hypersensitive to killing by CPT (reviewed in reference 10). Moreover, ku70 DT40 cells tend to be more resistant to CPT than wild-type cells, suggesting that NHEJ may even be toxic to the cells (1).
In this study, we evaluated DSB repair in rad18-deficient DT40 cells (63) (Table 1) , as well as Rad18-depleted SW480sn3 human cells (36). We present genetic evidence that, in addition to a role for Rad18 in DNA damage bypass and PRR, Rad18 has a direct role in DSB repair. The evidence includes hypersensitivity of rad18 DT40 cells, but not pcnaK164R mutant or rev3 DT40 cells, to CPT; reduced HR-dependent DSB repair in a recombination reporter substrate; and the complete suppression of these phenotypes by concomitant inactivation of Ku70. A strikingly similar phenotype is also observed in DT40 cells deficient in poly(ADP-ribose) polymerase 1 (PARP1) (20), which is activated at DSBs, as well as single-strand breaks, and mediates the covalent attachment of ADP-ribose moieties derived from NAD to target proteins (reviewed in reference 7). To explore the functional interactions between Rad18 and PARP1, we created rad18/parp1 double-mutant and rad18/parp1/ku70 triple-mutant clones and found that rad18/parp1 cells displayed a synergistic increase in CPT sensitivity and that this phenotype was also normalized by additional inactivation of the KU70 gene. In summary, like PARP1, Rad18 facilitates HR by inhibiting the toxic effect of NHEJ, particularly at the DSBs that occur as a consequence of replication blockage. Thus, Rad18 has acquired this new function, in parallel with its critical regulatory role in facilitating lesion bypass, during evolution from unicellular to multicellular organisms.
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MATERIALS AND METHODS |
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Chromosome analysis. Analysis of chromosome abnormalities was performed as described previously (13, 54). Briefly, cells were treated for 3 h with medium containing 0.1 µg/ml Colcemid. Harvested cells were incubated in 1 ml of 75 mM KCl for 30 min at room temperature and fixed in 5 ml of a freshly prepared 3:1 mixture of methanol-acetic acid. The cell suspension was dropped onto an ice-cold wet glass slide and immediately flame dried. Then, the slides were stained with 3% Giemsa solution at pH 6.4 for 30 min. Chromosomal aberrations were assessed for at least 60 mitotic cells by microscope. Data for DT40 cells are presented as macrochromosomal (1 to 5 and Z) aberrations per 60 metaphase spreads (54).
siRNA transfection. Short interfering RNA (siRNA), 19-mer RNA with a protruding 3'-TT sequence (total, 21-mer), was synthesized and purified by high-performance liquid chromatography (Hokkaido System Science). The sense sequence, 5'-ACTCAGTGTCCAACTTGCT-3', which includes nucleotides 172 to 190 relative to the start codon, was used for designing siRNA for human Rad18 (hRad18). One hundred nanomolar siRNA was used for transfection. Transfection was performed using oligofectamine (Invitrogen), and as a control, cells were mock transfected with oligofectamine alone. Immunoblot assays with anti-hRad18 antibody and exposure of cells to cisplatin for colony formation assays were all performed 72 h after transfection. Determination of the cisplatin sensitivity of RNA interference-treated SW480 cells by using a colony formation assay was performed as described by Chang et al. (8). Forty-eight hours after siRNA transfection, the cells were treated continuously for 48 h with CPT, and then a colony formation assay was done (8).
Measurement of HR frequencies induced by I-SceI-induced DSBs in an artificial substrate. Modified SCneo (17) was inserted into the previously described OVALBUMIN gene construct and then targeted into the OVALBUMIN locus in wild-type, rad18, ku70, and rad18/ku70 DT40 clones. In transient transfections, 5 x 106 cells were suspended in 100 µl of Nucleofector solution T; mixed with each of several circular-plasmid DNAs (5 µg) (chicken Ku70, chicken RAD18, and I-SceI expression vector [pCBASce]); and electroporated with an AMAXA machine (Koeln, Germany) using either the A30 or B23 program. At 24 h after electroporation, the cells were plated in methylcellolouse containing 2.0 mg/ml G418 or without G418. The cells were grown for 7 to 10 days, and surviving colonies were counted. To distinguish between short-tract gene conversion (STGC) and long-tract gene conversion (LTGC)/sister chromatid recombination, genomic DNA was digested with HindIII and probed with a fragment of neo DNA (see Fig. 4A) (59). For the nucleotide sequence analysis of the repair products, genomic DNA was PCR amplified using the primers P1 and P2 (see Fig. 4A). To measure HR-dependent DSB repair in human cells, experiments were done as previously reported (50).
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RESULTS |
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rays compared with wild-type cells (Fig. 1B). Moreover, the level of unrepaired chromosomal breaks scored in mitotic spreads following irradiation in the G2 phase was on average 2.5-fold higher in rad18 than in wild-type cells (Fig. 1C). These observations suggest that Rad18 can affect DSB repair in the G2 phase even after completion of DNA replication.
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) or rev3 cells (30, 53, 65), exhibited no increase in sensitivity to CPT (Fig. 1D). Conceivably, if topoisomerase I is covalently linked to DNA, the resulting broken template strands could not be bypassed by any TLS polymerases. Furthermore, pcnaK164R cells were far more CPT resistant than were rad18 cells (Fig. 1E), indicating that Rad18 can contribute to CPT tolerance independently of the modification of PCNA. Taken together, these data suggest that Rad18 may contribute to the repair of DSB arising during, as well as after the completion of, replication.
The increased sensitivities of rad18-deficient cells to IR and camptothecin are reversed by disruption of Ku70.
To understand which major DSB repair pathway is regulated by Rad18, we abolished an essential early NHEJ factor, Ku70, in rad18 cells. As a control, we also deleted Ku70 in the rev3 background. The resulting double-mutant cells were viable and did not show any further growth retardation (data not shown). Surprisingly, the -ray sensitivity of rad18 cells, but not that of rev3 cells, was completely reversed by the additional disruption of the KU70 gene (Fig. 2A and B). Accordingly, the patterns of IR sensitivity in ku70 and rad18/ku70 cells were indistinguishable (Fig. 2A). Likewise, the deletion of KU70 in rad18 cells normalized induced chromosomal breaks at 3 hours post-IR exposure (Fig. 2C). These observations indicate that IR sensitivity of rad18 cells depends on the Ku protein. This is in marked contrast with the findings in budding yeast, where the IR sensitivity of rad18 is not dependent on ku70 (9). Alternatively, this phenotype could arise as a result of an increase in recombinogenic lesions due to a PRR defect in rad18 cells. Additionally, these lesions could be repaired more efficiently by HR, particularly in the absence of Ku70. If this scenario is true, deletion of Ku70 should also suppress other PRR mutants, such as rev3 mutants. However, deletion of KU70 in rev3 cells slightly increased their IR sensitivity (Fig. 2B), as opposed to the suppression of IR sensitivity in rad18/ku70 cells. These data suggest that Ku70-dependent NHEJ and rev3-dependent TLS contribute to cellular tolerance of IR independently of each other and that the predominant role of Rad18 in tolerance of IR is distinct from that of Rev3.
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The reversion of rad18 cell hypersensitivity to IR and CPT by additional inactivation of Ku70 led us to measure cellular sensitivity to UV, MMS, and cisplatin by comparing rad18 and rad18/ku70 cells. The deletion of KU70 reversed the hypersensitivity of rad18 cells to UV, MMS, and cisplatin by more than 50% (Fig. 3A, B, and C). This phenotype is in marked contrast to the additive effect of KU70 deletion on cells deficient in Rev3, which plays a critical role in TLS past a wide variety of DNA damage (Fig. 3D, E, and F). As discussed above, this observation suggests that Ku70 does not necessarily interfere with PRR. In conclusion, Rad18 contributes to cellular tolerance of a wide range of DNA damage in the following two distinct manners: avoidance of the toxic effect of NHEJ on HR and facilitation of PRR.
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To gain an insight into the role of Rad18 in HR, we examined HR products recovered from 24 neomycin-resistant clones from each genotype, using Southern hybridization analysis with a probe shown in Fig. 4A. The majority of the repair products were associated with STGC (Fig. 4C), as previously reported (28, 50). To evaluate the accuracy of DSB repair, HR products were PCR amplified using the primers indicated in Fig. 4A (P1 and P2), and 24 fragments from each genotype were subjected to base sequence determination. Interestingly, three clones derived from rad18 cells, but not any from cells of other genotypes, contained 4- and 8-base pair deletions (see Fig. S1 in the supplemental material). In order to further evaluate the accuracy of DSB repair, we examined DSB repair products 24 h after transient expression of I-SceI without neomycin selection. To exclude the repair substrate in the PCR amplification products, genomic DNA was digested with I-SceI prior to the amplification. Accurate HR would replace the I-SceI site with an NcoI site, while inaccurate DSB repair, either HR or NHEJ, might result in loss of both I-SceI and NcoI sites. A higher percentage of the amplified fragments from rad18 cells were resistant to both NcoI and I-SceI digestion compared with those from wild-type and rad18/ku70 cells (Fig. 4D, bottom panel). Collectively, deletion of Rad18 resulted in an increase in the percentage of inaccurate DSB repair events. Conceivably, Rad18 may be directly associated with DSBs and protect them from inaccurate repair by NHEJ, thereby facilitating HR.
Human Rad18 facilitates DSB repair. We wished to know whether the findings from DT40 cells presented here are also relevant to human cells. To this end, we depleted Rad18 in human SW480 cells, a mismatch repair-proficient cell line from a colon cancer (36), using siRNA and evaluated DSB repair. SW480 cells were transfected with rad18 siRNA or mock transfected (control) (see Materials and Methods). The level of Rad18 protein was reduced by more than 80% at 72 h after siRNA transfection (Fig. 5A), while the growth rate of the cells was unaffected (data not shown). As seen in rad18 mutant DT40 cells, the SW480 Rad18-depleted cells exhibited a slight increase in sensitivity to CPT, as well as cisplatin (Fig. 5B and C). Increased sensitivities to both CPT and cisplatin were also observed in a RAD18–/– HCT116 human cell line (personal communication from T. Shiomi, National Institute of Radiological Sciences, Japan). The less prominent phenotype of the human cell line (Fig. 5) in comparison with DT40 cells may be attributed to the duration of CPT exposure, only 48 h for the siRNA-treated cells but continuous during clonal expansion for DT40 cells. To assess HR, we used an assay based on the SCneo substrate (26), a single copy of which is integrated into SW480sn3 cells (36). At 72 h after the addition of either control or Rad18 siRNA to the cells, they were transfected with an I-SceI expression plasmid and were subsequently subjected to selection with neomycin. Rad18-depleted cells produced Neor recombinants at a frequency fivefold lower than the control (mock-treated) cells (Fig. 5D). These observations indicate that human Rad18 is also required for optimal execution of HR.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Rad18 plays a role in HR independently of its contribution to PRR. In this study, we presented evidence that the CPT sensitivity of rad18 cells is mainly caused by a defect in HR-mediated DSB repair, but not by impaired PRR. We derived this conclusion from the following results. First, in contrast to yeast rad18 mutant cells (5, 51) or DT40 cells deficient in TLS polymerases, rad18 cells displayed a strong increase in CPT sensitivity (Fig. 1D). This phenotype cannot be explained by defective TLS, because the attachment of topoisomerase I to the backbone of template strands would prevent DNA synthesis by polymerases. Second, in contrast with rad18 cells, DT40 cells carrying the PCNA K164R mutation displayed only a modest increase in sensitivity to CPT (Fig. 1E). Thus, Rad18 may contribute to cellular tolerance of CPT independently of PCNA's posttranslational modification, which seems to play a key role in PRR in mammalian cells (2, 29, 52, 62). Third, the tolerance of CPT by rad18 cells was restored by deletion of KU70 and LigIV, which act only in DSB repair and not in PRR (Fig. 2D and E). Fourth, rad18 DT40 cells, as well as Rad18-depleted human cells, showed reductions in the rate of HR-mediated repair of I-SceI-induced DSBs (Fig. 4B and 5D). Furthermore, the accuracy of DSB repair was moderately compromised in rad18 cells (Fig. 4D; see Fig. S1 in the supplemental material). This result agrees with a recent report that in rad18 DT40 cells immunoglobulin gene conversion is frequently associated with deletions and disruptions (56). These observations provide compelling evidence for the dual roles of Rad18 in HR-dependent DSB repair and PRR in vertebrate cells.
In the cases of UV, MMS, and cisplatin treatments, deletion of Ku partly suppressed the sensitivity of rad18 cells (Fig. 3A to C). Clearly, this suppression does not depend on the PRR function of Rad18, because Ku does not interact in a similar manner with other PRR factors, such as Rev3 (Fig. 3D to F). A comparison between the rad18 and rad18/ku70 phenotypes as shown in Fig. 3 allows a clear dissection of the HR and PRR branches of Rad18.
It is postulated that HR does not require Rad18 in budding yeast, as the Rad18 and Rad52 epistasis groups constitute different repair pathways, PRR and HR, respectively (reviewed in reference 21). However, a recent study showed that although the yeast rad18 mutant is very sensitive to IR, the pol30 (pcnaK164R) mutant exhibits only slight sensitivity to IR (9), indicating that Rad18 can promote HR-mediated repair of IR-induced DSBs independently of the PCNA ubiquitylation. Moreover, hypersensitivity to both CPT and topoisomerase II inhibitors is observed in fission yeast defective in the ortholog gene of RAD18 (A. M. Carr and K. Furuya, Sussex, United Kingdom, personal communication). Taken together, these observations may uncover a role of Rad18 in DSB repair in lower- as well as higher-eukaryotic cells.
Functional interaction between Rad18 and Parp1 in preventing NHEJ. The present data indicate that the hypersensitivity of rad18 cells to CPT may require proficient NHEJ, including both the Ku proteins and LigIV (Fig. 2D and E). Thus, the entire NHEJ pathway is unfavorable under these circumstances. Presumably, when a large number of DSBs arise within a replication factory following CPT treatment, active NHEJ makes inappropriate joins between DSB ends, which could eventually result in chromosomal translocation and deletion. Our findings postulate a role for Rad18 in suppressing such inappropriate NHEJ at stalled replication forks. We have recently found that cells deficient in PARP1, an enzyme that poly(ADP-ribose)ylates proteins at DNA breaks, shares a strikingly similar phenotype with Rad18-deficient cells with regard to their interactions with NHEJ (20). Furthermore, in the case of PARP, biochemical studies have already shown that the attachment of PAR to the Ku proteins reduces affinity to DSBs in vitro (33). Thus, both biochemical data and our genetic results consistently support the idea that PARP1 suppresses the access of the Ku proteins to DSB ends. Finding substrates of Rad18-mediated ubiquitylation in this novel pathway will be crucial for understanding the molecular mechanism of Rad18 function in DSB repair.
In this study, we showed that the sensitivity of a rad18/parp1 double mutant to CPT was dramatically increased compared to those of the relevant single mutants and that cellular tolerance of CPT was restored by deletion of KU70 in rad18/parp1 cells (Fig. 6). Based on these genetic findings, a model emerges in which Rad18-mediated ubiquitylation and PARP-mediated poly(ADP-ribose)ylation separately limit the access of NHEJ factors to DNA ends at stalled replication forks. Conceivably, higher eukaryotes have evolved a novel regulatory mechanism to keep their highly active NHEJ pathways in check. This may be necessary in order to avoid the unwanted ligation of DSB breaks at replication forks and to allow access of HR factors when this reaction is more favorable for cellular survival.
Multiple functions of DNA repair factors in vertebrates. Genetic studies in yeast have clearly established defined functions for the genes in the RAD3, RAD52, and RAD6 epistasis groups (23, 47). Vertebrate cells are faced with a more complex task in safeguarding their DNA, due to the sheer size of their genomes. As the vertebrate genetics of DNA repair become more accessible, it appears that, besides evolving new genes, the functions of old players have shifted during metazoan evolution to help cope with the more complex tasks of ensuring chromosome stability. This seems to be particularly true for PRR genes, which overlap much more with HR (30, 41, 53) than appears to be the case in yeast. The present study indicated that another example of such a PRR gene is the gene encoding Rad18, which may inhibit access of NHEJ to DSBs while at the same time losing its dominant role in controlling every PRR subpathway in the more complex system in vertebrates. These crossovers of DNA repair pathways in vertebrates have to be kept in mind when specific factors are targeted for therapeutic purposes. Multifunctional effectors, such as Rad18, could thus be ideal candidates for inhibitor screens or gene therapy in order to maximize the effects of DNA-damaging agents, such as CPT, during chemotherapy.
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ACKNOWLEDGMENTS |
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Financial support was provided in part by CREST.JST (Saitama, Japan) and the center of excellence (COE) grant for Scientific Research from the Ministry of Education, Culture, Sports and Technology. A. Saberi has a scholarship from the Health Ministry of Medical Education of Iran. D.S. was supported by the Leukemia Research Fund.
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FOOTNOTES |
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Published ahead of print on 22 January 2007.
Supplemental material for this article may be found at http://mcb.asm.org/.
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