Targeted deletion of mouse Rad1 leads to deficient cellular DNA damage responses

Abstract

The Rad1 gene is evolutionarily conserved from yeast to human. The fission yeast Schizosaccharomyces pombe Rad1 ortholog promotes cell survival against DNA damage and is required for G₂/M checkpoint activation. In this study, mouse embryonic stem (ES) cells with a targeted deletion of Mrad1, the mouse ortholog of this gene, were created to evaluate its function in mammalian cells. Mrad1^−/− ES cells were highly sensitive to ultraviolet-light (UV light), hydroxyurea (HU) and gamma rays, and were defective in G₂/M as well as S/M checkpoints. These data indicate that Mrad1 is required for repairing DNA lesions induced by UV-light, HU and gamma rays, and for mediating G₂/M and S/M checkpoint controls. We further demonstrated that Mrad1 plays an important role in homologous recombination repair (HRR) in ES cells, but a minor HRR role in differentiated mouse cells.

Graphical abstract

Keywords

Rad1 / DNA damage / checkpoint signaling / DNA repair / homologous recombination repair

INTRODUCTION

Cells face endogenous and exogenous assaults that damage genomic DNA. But eukaryotic cells have conserved surveillance mechanisms, which could detect the DNA lesions and send the signals to the DNA repair system and the cell cycle control machinery, to coordinate DNA repair and minimize negative effects of these lesions. The cell cycle delay induced via the checkpoint mechanism is thought to provide extra time for DNA damage repair, and to prevent cell cycle progression into critical phases that could lead to lethality (Hartwell and Weinert, 1989; Paulovich and Hartwell, 1995; Zhou et al., 2010).

Rad9, Rad1 and Hus1 are a group of genes conserved from yeast to human that play key roles in the cell cycle signaling networks. Their protein products form a ring-shaped heterotrimer, named the 9-1-1 complex (Doré et al., 2009; Sohn and Cho, 2009; Xu et al., 2009). It is believed that this complex is important for the functions of DNA repair as well as the activation of cell cycle checkpoints (Shiomi et al., 2002; Bermudez et al., 2003; Ellison and Stillman, 2003). Interestingly, human Rad1 (i.e., RAD1) also exists as monomer besides forming the 9-1-1 complex in cells, and the function of this form of the protein is unknown (Burtelow et al., 2001). In fission yeast Schizosaccharomyces pombe, disruption mutants of the three genes resulted in similar phenotypes, including viability, sensitivity to UV-light, the replication inhibitor hydroxyurea (HU), as well as gamma rays, and defective S/M and G₂/M checkpoint control (al-Khodairy and Carr, 1992; Enoch et al., 1992; Lieberman et al., 1992; Murray et al., 1991; Rowley et al., 1992). Disruption of the budding yeast Saccharomyces cerevisiae counterparts, Mec3 (schus1), Rad17 (scrad1) and Ddc1 (scrad9), also caused similar phenotypes in the corresponding mutants, including hypersensitivity to UV light, HU and gamma rays, and G₂/M checkpoint defect, but not a disruption of the S/M checkpoint defect (Longhese et al., 1997; Lydall and Weinert, 1997). Mouse cells with a disruption of Mrad9 or Mhus1, the mouse homologues of rad9 or hus1, were successfully created, and also exhibited significantly higher sensitivity to UV light, HU and gamma rays than the wild-type cells (Weiss et al., 2000; Hopkins et al., 2004). The cell cycle checkpoint functions of Mrad9 and Mhus1 were reported to be different but the comparison was based on the data using two different cell types (Weiss et al., 2000; Hopkins et al., 2004; Wang et al., 2004). The Mhus1^−/− cells are mouse embryonic fibroblasts (MEF), while the Mrad9^−/− cells are mouse embryonic stem cells (ES). Mhus1^−/− MEFs were defective in the UV light-induced intra-S phase checkpoint, but functioned normally with respect to the G₂/M checkpoint (Weiss et al., 2003). In contrast, Mrad9^−/− ES cells were not markedly defective in the UV light-induced intra-S phase checkpoint, but failed to maintain G₂/M checkpoint control following the exposure to gamma rays (Weiss et al., 2000; Hopkins et al., 2004).

Results from human RAD1 knockdown using siRNA suggested that the gene is an important element for cell growth and is required for the recovery of DNA synthesis following HU treatment (Bao et al., 2004). The same study showed that reduced RAD1 protein level caused a defect in the intra-S phase checkpoint but did not affect the G₂/M checkpoint. However, rad1-disrupted yeast cells failed to arrest in response to ionizing radiation exposure (al-Khodairy and Carr, 1992; Enoch et al., 1992; Rowley et al., 1992; Lydall and Weinert, 1997).

Although targeted deletion of Mrad9 and Mhus1 in mouse cells and mice have been reported (Weiss et al., 2000; Hopkins et al., 2004; Levitt et al., 2005; Levitt et al., 2007; Hu et al., 2008; Yazinski et al., 2009; An et al., 2010), equivalent studies for Mrad1 have not been published. Such investigation is important to reveal the gene functions that are not detectable when RAD1 protein is only partially expressed in siRNA knockdown cells (Bao et al., 2004) or heterozygous cells (Han et al., 2010). In the present study, we constructed mouse ES cells with a targeted deletion of Mrad1 gene and investigated Mrad1 function in these cells. Our results showed that Mrad1 homozygously deleted ES cells were viable, but were defective in G₂/M checkpoint maintenance as well as the HU-induced S/M checkpoint, and were highly sensitive to UV light, HU and gamma rays. Interestingly, the differentiation of Mrad1^−/− ES cells modulated the capability of double-strand breaks (DSB) repair.

RESULTS

Construction of mouse ES cells with homozygous disruptions of Mrad1

Mrad1^+/− ES cells were obtained as previously described (Han et al., 2010). The neo gene product can destroy antibiotic G418, and the Mrad1^+/− ES cells contained one allele of disrupted genomic Mrad1 bearing a copy of neo gene. We hypothesize that increasing G418 concentration in the medium might force the amplification of the copy number of neo and even replace the remaining wild type genomic Mrad1 with the neo-bearing disrupted genomic Mrad1. To obtain Mrad1^−/− clones, the Mrad1^+/− ES cells were incubated with 3.2–4.4 mg/mL G418 instead of the original 300 μg/mL G418 for 20 days, and from 96 survivors, six colonies bearing Mrad1 homozygous deletion were identified by Southern blotting (Fig. 1A). These results were confirmed using Northern blotting (Fig. 1B) and RT-PCR (Fig. 1C).

Mrad1 deletion retards cell proliferation and alters cell cycle phase distribution

RAD1 knockdown by siRNA reduced the proliferation rate of human cells (Bao et al., 2004). Consistent with this result, Mrad1^−/− ES cells grew significantly slower than the wild type control population (Fig. 2A), and formed much smaller colonies (Fig. 2B). We examined the cell cycle phase distributions of Mrad1^−/− and Mrad1^+/+ cells with flow cytometry, and found that significantly more Mrad1^−/− cells accumulated in the G₂/M phase than the wild-type cells (Fig. 2C), suggesting the mutant cells proceeded through G₂/M at a significantly slower pace. Bromodeoxyuridine (BrdU) incorporation analysis showed that S phase progression rate was reduced by homozygous deletion of Mrad1 (Fig. 2D). All the aforementioned changes in the cell cycle caused by Mrad1 deletion were reversed by ectopically expressing Mrad1 (Fig. 2C and 2D; data not shown), and thus these alterations were due to the lack of Mrad1 function.

The increased accumulation of Mrad1^−/− cells in the G₂/M phase might result from activation of the G₂/M checkpoint by DNA lesions, and therefore, we monitored the DNA breaks in wild type and Mrad1^−/− ES cells using an alkaline comet assay for all types of DNA lesions and a histone γ-H2AX assay for DSBs. The comet tail moment in Mrad1^−/− ES cells was significantly higher than that in Mrad1^+/+ cells (Fig. 3A and 3B), indicating the presence of more DNA lesions in the mutant. These results were confirmed by the histone γ-H2AX assay, in which more foci (Fig. 3C and 3D) as well as a higher level of histone γ-H2AX were detected in the mutant population (Fig. 3E), reflecting enhanced DNA DSBs in Mrad1^−/− ES cells.

Failure of Mrad1^−/− ES cells to maintain ionizing radiation-induced G₂/M checkpoint control

DNA damage-induced arrest in G₂ phase is one of the most prominent cell cycle checkpoints in eukaryotic cells. Fission yeast S. pombe rad1 is required for this cell cycle arrest in response to ionizing radiation exposure (Freire et al., 1998; Udell et al., 1998). Therefore, we examined whether the role of Mrad1 in the G₂/M checkpoint is evolutionarily conserved. Mrad1^+/+ and Mrad1^−/− cells, as well as the Mrad1^−/− cells ectopically expressing Mrad1 were irradiated with 10 Gy of gamma rays, harvested at 4, 6, 8, 10 and 12 h after exposure, and then processed for flow cytometric analysis to assess cell cycle phase distribution. Only data from untreated, 6 and 10 h time points are presented here because the rest of data essentially indicated the same trends. The percentage of cell populations in each phase of the cell cycle is shown in graphic (Fig. 4) and tabular formats (Table 1). Subpopulations of both Mrad1^+/+ and Mrad1^−/− ES cells increased in the G₂/M phase and decreased in the G₁ and S phases post irradiation. This pattern, lacking G₁ arrest but exhibiting radiation-inducible G₂ arrest, is a typical response of wild-type ES cells to gamma rays (Aladjem et al., 1998). This result indicated that Mrad1 is not indispensable to activate the G₂/M checkpoint. However, in contrast to the wild-type cells, Mrad1-deficient cells accumulated in the G₁ phase (arrows in Fig. 4). To assess whether the small G₁ subpopulation of cells came from the G₂/M phase post irradiation, colcemid, which disrupts the mitotic spindle and traps cells in mitosis, was added to the cells. The results showed that incubation of the cells with colcemid eliminated the small G₁ subpopulation accumulation, and therefore the cells progressed from the G₂/M phase (Fig. 4), suggesting the important role of Mrad1 in maintaining the DNA damage-induced G₂/M checkpoint control. This conclusion was confirmed by the fact that the G₂/M checkpoint defect was rescued by the ectopic expression of Mrad1.

Mrad1 disruption in ES cells alters S/M checkpoint control

S. pombe rad1 is required to block cells with incomplete DNA replication from moving into the M phase of cell cycle (S/M checkpoint), while the S. cerevisiae ortholog, Rad17 (scRad1), is dispensable for the checkpoint. To determine whether the S/M checkpoint in mouse ES cells is Mrad1-dependent, we examined the level of phospho-histone-H3 (γ-H3) throughout the cell cycle. Histone-H3 is specifically phosphorylated during mitosis. After treatment with HU for different times, ES cells were labeled with anti-γ-H3 antibody, stained with propidium iodide (PI) for DNA content, and then analyzed by flow cytometry. Incubation with HU reduced the number of γ-H3 positive Mrad1^+/+ cells with 2N DNA content because most cells were blocked in S phase and thus fewer moved into the M phase. Few Mrad1^+/+ cells with less than 2N DNA content were γ-H3 positive after HU treatment, indicating that the wild type cells have a normal S/M checkpoint (Fig. 5 and Table 2). In contrast, the number of γ-H3 positive Mrad1^−/− cells with less than 2N DNA content dramatically increased after HU treatment. Mrad1-deficient ES cells ectopically expressing Mrad1 showed the same pattern as wild-type cells. Therefore, Mrad1 is essential for the S/M checkpoint control.

Mrad1 is not essential for the intra-S phase checkpoint induced by UV light

The intra-S phase cell cycle checkpoint monitors DNA replication and delays DNA synthesis in the presence of DNA damage. We demonstrated that Mrad1-null cells are highly sensitive to UV light (see below). Therefore, we determined whether the UV-induced intra-S phase checkpoint of the mutant cells was aberrant. Mrad1^+/+ and Mrad1^−/− cells were treated with UV light, and then pulse-labeled with 10 μmol/L BrdU at designated times post treatment to detect DNA replication by flow cytometry. The incorporation rates of BrdU into DNA in both cell populations dramatically reduced at 40, 90 and 180 min after irradiation, and the kinetics were similar (Fig. 6). Thus, these findings indicate that deletion of Mrad1 does not affect the intra-S phase checkpoint control after exposure to UV light.

Mrad1-deleted ES cells are hypersensitive to UV light, HU and gamma rays

Previous research showed that Rad1 associates with Hus1 and Rad9 in a 9-1-1 heterotrimer to respond to DNA damage (Hang and Lieberman, 2000; Rauen et al., 2000; Lindsey-Boltz et al., 2001; Roos-Mattjus et al., 2002; Parrilla-Castellar et al., 2004). Both Mhus1^−/− MEF cells and Mrad9^−/− mouse ES cells are highly sensitive to genotoxins, including UV light, HU and gamma rays (Weiss et al., 2000; Hopkins et al., 2004; Wang et al., 2004; Wang et al., 2006). S. pombe rad1::ura4⁺ cells are also extremely sensitive to these DNA damaging agents (Freire et al., 1998; Udell et al., 1998). Therefore, we examined whether Rad1 in mouse ES cells plays an important role in promoting resistance to these genotoxins. As shown in Fig. 7, Mrad1^−/− ES cells were extremely sensitive to UV light, HU and gamma rays compared to the wild type control population. To confirm the sensitivities are due to a defect in Mrad1, resistance was examined in the mutant cells ectopically expressing the wild-type gene. As indicated in Fig. 7, expression of wild-type Mrad1 compensated the resistance to UV light, HU and gamma rays in Mrad1^−/− ES cells, thus indicating that Mrad1 gene mediates the resistance to these agents.

Deletion of Mrad1 does not affect expression of other cell cycle checkpoint genes

p21, p53, Hus1 and Rad9 are important cell cycle checkpoint genes, and the expression levels of p21, p53, Hus1 and Rad9 were examined by northern blotting to gain a mechanistic insight into the potential influence of Mrad1 deletion on the regulation of these genes. The results indicated that homozygous deletion of Mrad1 did not affect expression of these cell cycle checkpoint genes (Fig. 8). Mrad1^−/− cells bearing the Mrad1 cDNA also displayed similar expression levels of these cell cycle checkpoint RNAs, except for the increased expression of Mhus1, and the deletion of Mrad1 did not affect Mhus1 RNA level. Therefore, Mrad1 deletion did not cause a dramatic shift in RNA levels corresponding to this group of cell cycle checkpoint genes, suggesting that the deletion caused defects of cell checkpoints and altered cell cycle distribution not through regulating the expression of p21, p53, Hus1 or Rad9.

Differentiated Mrad1-deleted ES cells have more efficient HR repair

It has been reported that ES cells have more efficient DNA repair than differentiated ES cells in response to various DNA-damage agents (Maynard et al., 2008; Tichy and Stambrook, 2008). However, in the cell survival assay, we found that Mrad1^−/− ES cells were hypersensitive to IR, but retinoic acid (RA)-induced differentiated Mrad1^−/− ES cells had nearly identical sensitivity as the wild type cells (Fig. 9A). Meanwhile, undifferentiated and differentiated Mrad1^+/+ ES cells displayed similar resistance to the same doses of irradiation (Fig. 9B). Leukemia inhibitory factor (LIF) is routinely added to ES cell medium to prevent ES cells from differentiation. Here we obtained similar results when Mrad1^−/− ES cells were cultured in RA-containing medium as well as LIF-free medium (Fig. 9C), confirming that mouse ES cell differentiation compensated for DNA repair defects caused by Mrad1 deletion. Treatment by gamma rays causes DSBs, which are repaired by two major pathways, non-homologous end joining (NHEJ) and homologous recombination (HR). Using an established in vivo HR assay (Pierce et al., 2001), we found that the loss of Mrad1 caused significant reduction in HR repair capacity, but differentiation could largely compensate it in Mrad1^−/− ES cells (Fig. 9D and 9E).

DISCUSSION

In fission yeast S. pombe, rad1 is a key component that mediates multiple cellular responses to DNA damage, including a role in cell cycle checkpoint (Murray et al., 1991; al-Khodairy and Carr, 1992; Enoch et al., 1992; Lieberman et al., 1992; Rowley et al., 1992; Parker et al., 1998). However, the function of this gene in mammals is not clear. In this report, we examined the activities of Mrad1, the mouse ortholog of S. pombeRad1, by creating and characterizing the mouse ES cells with deletion of Mrad1. We demonstrated that Mrad1-deficient ES cells were highly sensitive to UV light, HU and gamma rays (Fig. 7), defective in S/M and G₂/M cell cycle checkpoint controls (Fig. 4–6), and prone to accumulate DNA lesions under normal growth conditions (Fig. 3). These data indicate that Mrad1 plays essential roles in the resistance to UV light, HU and gamma rays, as well as in the S/M and G₂/M checkpoints.

As shown by previous reports (Burtelow et al., 2001; Roos-Mattjus et al., 2002), as well as 9-1-1 complex crystal structure (Doré et al., 2009; Sohn and Cho, 2009; Xu et al., 2009), Rad1 along with Rad9 and Hus1 in a trimeric checkpoint complex were believed to have similar functions. Indeed as we showed above, many phenotypes such as the hypersensitivity to HU, UV light and gamma rays are similar among Mrad1-deletion, Mrad9-deletion and Mhus1-deletion mouse cells (Fig. 7) (Weiss et al., 2000, 2003; Hopkins et al., 2004). In addition, mouse ES cells with Mrad1-deletion and Mrad9-deletion are similarly deficient in G₂/M and S/M checkpoint maintenance, but intact in intra-S phase checkpoint, which is in contrast to Mhus1 embryonic fibroblasts (EF) (Weiss et al., 2000, 2003; Hopkins et al., 2004). Taken together, Rad1, Rad9 and Hus1 are likely to work in the 9-1-1 complex for the resistance to HU, UV light and gamma rays as well as for the maintenance of S/M and G₂/M checkpoints in mouse ES cells. The phenotype differences in intra-S phase, S/M and G₂/M checkpoints between Mrad1 or Mrad9-deleted mouse ES cells and Mhus1-deleted mouse EF cells are probably due to the various differentiation states, suggesting the different functions of these genes in cell cycle checkpoints in ES and EF cells.

Consistent with the above hypothesis, we found in this study that the differentiated Mrad1^−/− cells induced by RA and LIF-free media had similar resistance to gamma rays as undifferentiated or differentiated Mrad1^+/+ cells (Fig. 9). Interestingly, the resistance to HU or UV light was similar between undifferentiated and differentiated Mrad1^−/− cells (our unpublished data). These results together suggest that differentiation has various influence on different DNA repair pathways. It is still unknown whether differentiation of Mrad9^−/− cells can also enhance their resistance to gamma rays. This experiment is critical to clarify whether the differentiation-associated resistance change is 9-1-1 complex dependent or only Rad1-dependent. Indeed, there are significant amounts of individual Rad1 molecules in human cells (Burtelow et al., 2001; our unpublished data).

ES cells were reported to have higher DNA repair abilities than differentiated cells (Maynard et al., 2008; Tichy and Stambrook, 2008). Our results are inconsistent with these reports. It is possible that repair factors work differently at various stages of differentiation, and the comparison between ES and differentiated cells only at certain stages probably does not reflect all the DNA repair situations of mammalian cells during differentiation. In addition, various DNA repair pathways are probably differently influenced by cell differentiation as shown in this study while only the resistance to gamma rays, but not to HU or UV light, was altered by the differentiation of mouse ES cells. As already shown by many studies, differentiation is largely regulated and reflected by chromatin status and many chromatin remodeling factors play important roles in DNA repair pathways. DNA repair at different stages of differentiation attracts more researches and will generate further insights into DNA repair mechanisms.

HR repair was a major component that was altered in DSB repair from mouse ES Mrad1^−/− cells to RA-induced differentiated mouse Mrad1^−/− cells (Fig. 9D and 9E), but it only accounted for half of the altered DSB. It is likely that NHEJ also changed during the differentiation. If this is true, the differentiation would modulate the common part(s) of both repair pathways, and the chromatin status during DSB repairing process might be modulated

A study of human HCT116 cells with RAD1 siRNA demonstrated no effect of the corresponding reduced protein levels on the G₂/M checkpoint, but impaired intra-S phase checkpoint control was observed (Bao et al., 2004). Our study using Mrad1-deficient ES cells revealed the opposite results: a defective G₂/M and an intact intra-S phase checkpoint (Fig. 4 and 6). The difference in cell types might contribute to the different checkpoint responses. As for the lack of a role of human RAD1 in G₂/M checkpoint as shown by knockdown strategy, a possibility also exists that a low level of RAD1 is sufficient to support G₂/M checkpoint function.

MATERIALS AND METHODS

Growth of ES cells, gene targeting, and generation of Mrad1-deficient cells

Mrad1^+/− ES cells were prepared as previously described (Han et al., 2010). To generate Mrad1^−/− ES cells, Mrad1^+/− ES cells were grown in a medium containing 3.2–4.4 mg/mL G418. For the construction of Mrad1^−/− ES cells that ectopically express wild-type gene, the cells were transfected with pZeoSV2-Mrad1, grown in the presence of zeocin (30 μg/mL), and resistant clones were examined by RT-PCR to identify Mrad1 transcription.

The Mrad1 expression vector was made by PCR from mouse cDNA with the primers: 5′-ATTCGGCCGACTCGAGTCAAGACTCAGGAACTTCTTCATCAG-3′ and 5′-GTCCATAAGCTTGCCGCCACCATGCCTCTCCTAACCCAGTACAATG-3′. The product was cut with XhoI/HindIII and subcloned into pZeoSV2 (Invitrogen).

Retinoic acid (RA)-induced differentiated ES cells were prepared using normal Mrad1^+/+ and Mrad1^−/− ES cells cultured in 8 μmol/L RA for 5 days.

Southern blotting and PCR assays to assess genotypes

Genomic DNA was isolated from ES cells and mouse tails using published methods (Weiss et al., 2000). For southern blotting, DNA was digested with HindIII, separated on a 0.7% agarose gel, then transferred to a nylon membrane, and hybridized to a ³²P-labeled probe, which was generated by PCR using primers: 5'-GTGGCCTAGGTGGTTGCGTATCTGAAC-3' and 5'-GTCGGCTCCGAGAAGAAGGATGCTCC-3' with mouse genomic DNA as template.

To genotype ES cells and mice by PCR, the reaction was performed using genomic DNA templates and the following primer pairs: 5'-GTCTCAGGTTTTCACACATCTTCC-3' and 5'-GCTTATATTCTAGAAACCTTCCTGTATG-3'. After denaturation at 94°C for 5 min, 35 cycles of amplification (94°C for 13 s, 59°C for 30 s, at 72°C for 3 min 10 s) were followed, with a final extension at 72°C for 10 min.

Northern blotting and RT-PCR

Total RNA was isolated from ES cells using RNeasy Mini kit (QIAGEN) as described by the manufacturer. For Northern blotting, 10 μg RNA was fractionated in a 1.2% (w/v) formaldehyde-agarose gel and then transferred to a Hybond-N membrane. Templates for probes were made by PCR using the following primers: Mhus1, 5'-ATGAAGTTTCGCGCCAAGAT-3' and 5'-AGTCTGGGATGGAGGGTTCT-3'; Mrad9, 5'-ACTATTGAGGATTCCTTGCTGGATG-3' and 5'-ACAGTGAACGAAACTTCTTGGGTG-3'; Mrad1, 5'-GGAGTTTCCTGCATTTCCAAAAG-3' and 5'-GTCCATAAGCTTCCTCTCCTAACCCAGTACAATGAAGAG-3'; neo, 5'-CTACGCGTCGACATTGAACAAGATGGATTGCACGC-3' and 5'-AGGAATTCAGACATGATAAGATACATTGATGAG-3'; p21, 5'-ATGTCCAATCCTGGTGATGTCCG-3' and 5'-CAGGCTGGTCTGCCTCCGTTTTC-3'. Then, the membrane was hybridized with the probes, which were made using [α-32P]-dCTP and the Prime-a-gene labeling system (Amersham). The labeled membrane was washed and used to expose X-ray film.

For RT-PCR, 2 μg total RNA was reverse-transcribed to cDNA using the SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen). PCR amplification was carried out using the following primer pairs: Mrad1 ORF, 5'-TCCATAAGCTTCCTCTCCTAACCCAGTACAATGAAGAG-3' and 5'-ACTGCCATAACTCGAGTCAAGACTCAGGAACTTCTTCATCAGG-3'; Mrad1 upstream, 5'-ATGCCTCTCCTAACCCAGTACAATG-3' and 5'-TTCTTCCTGAATGACAAATTCCTG-3'; Gapdh, 5'- GCAAAGTGGAGATTGTTGCC-3' and 5'-CCGTATTCATTGTCATACCA-3'.

Western blotting

Cell lysate for western blotting was prepared in 1 × SDS-sample buffer, with the final concentration of 10⁴ cells/μL. 3 μL lysates were resolved on a 10% SDS-PAGE gel, and proteins were transferred to a polyvinylidene difluoride membrane. The membrane was probed consecutively with primary and peroxidase-conjugated secondary antibodies, and the signal was detected using the SuperSignal West Pico Chemiluminescence Substrate system (Prod #34077, Pierce). Primary and secondary antibodies used in this study were mouse anti-phospho-H2AX (Upstate), mouse anti-tubulin (Sigma), mouse anti-p21 (Santa Cruz), rabbit anti-p53 (Santa Cruz), chicken anti-RAD9, anti-HUS1, peroxidase-conjugated anti-chicken IgY (A9046, Sigma), peroxidase-conjugated anti-mouse IgG (A9044, Sigma), and peroxidase-conjugated anti-rabbit IgG (A9169, Sigma). The anti-RAD9 and anti-HUS1 antibodies were isolated from the eggs of chickens immunized with full-length human RAD9 and HUS1 proteins, respectively.

Cell survival assays

ES cells were plated in duplicate or triplicate and grown for 16 h before treatment. To test hydroxyurea (HU) sensitivity, the drug was added to the medium to achieve the designated final concentrations. After 24 h incubation, cells were washed twice with phosphate-buffered saline (PBS), a fresh medium without HU was added back, and the cells were incubated for 10 more days before Giemsa stain and colony counting. To assess the ionizing radiation sensitivity, cells were exposed to graded doses of gamma rays using a ⁶⁰Co-based irradiator, and incubated for another 10 days to allow colony formation. To determine the sensitivity to 254-nm UV light, the medium was removed, and the cells were exposed to graded doses of the UV light, and then the fresh medium was added to the cells, which were incubated for 10 more days before colony number was assessed. Survival percentage was calculated as 100 × [(number of colonies in treated dishes/number of cells seeded in treated dishes)/(number of colonies in mock-treated control dishes/number of cells seeded in mock-treated control dishes)]. Mean values were derived from three independent replicates, and the standard deviations were calculated.

Assays for cell cycle checkpoint functions

To evaluate G₂/M checkpoint control, 10⁶ cells were plated on 10-cm dishes and incubated at 37°C in 5% CO₂ overnight. Two sets of cells were exposed to 10 Gy of gamma rays, with one set mock treated as a control. Immediately after irradiation, colcemid (final concentration of 50 ng/mL) was added to one irradiated set of cells, which were subsequently incubated for various times at 37°C. Cells were processed, stained with propidium iodide (PI) and analyzed by an FACSCalibur flow cytometer (Becton Dickinson) using an established method (Hang and Fox, 2004).

S/M checkpoint function was examined using published procedures (Hu et al., 2008). Briefly, ES cells were grown to 70% confluence, and 1 mmol/L HU was added to the medium to achieve a drug concentration of 1 mmol/L. Cells were incubated at 37°C in 5% CO₂ for various times, processed and suspended in PBS. The cells were probed with rabbit anti-phospho-histone H3 (Upstate), then FITC-conjugated anti-rabbit antibodies (Jackson ImmunoResearch Laboratories, INC), and stained with PI before flow cytometric analysis.

Intra-S phase checkpoint function was also evaluated by radio-resistant DNA synthesis (RDS) assay in the BrdU labeling experiment (Hang and Fox, 2004). Briefly, cells were grown to 70% confluence. The medium was removed and cells were exposed to 20 J/m² UV light. Afterwards, pre-warmed medium was added back to dishes, and cells were re-incubated at 37°C. At various times after UV treatment, 10 μmol/L BrdU was added to the medium and cells were pulse-labeled for 10 min. After processed, probed with FITC-conjugated anti-BrdU antibody, and stained with PI, cells were subjected to flow cytometric analysis.

Comet assay

An alkaline comet assay for detecting DNA damage was carried out with the CometAssay kit as described by the manufacturer (TREVIGEN). Briefly, comet assay slides were loaded with a mixture of 10 μL of ES cell suspension (5 × 10⁵ cells/mL) and 90 μL of low-temperature melt agarose at a final concentration of 0.75%. After solidification, slides were lysed at 4°C in darkness for 1 h in lysis solution. The slides were soaked and subjected to electrophoresis in alkaline solution, washed and stained with SYBR Green (0.1 μg/mL). The comet images were captured using a fluorescence microscope (Nikon). The tail moment was analyzed using Euclid comet analysis software (Euclid Analysis, St. Louis, MO).

Immunofluorescence assay

Cells grown on coverslips were fixed with 4% paraformaldehyde in PBS for 15 min at room temperature. The coverslips were washed in PBS twice, incubated in PBS containing 0.5% Triton-X100 for 15 min, then in PBS containing 5% BSA and 0.1% Triton-X100 for 1 h, washed in PBS again, and incubated with anti-phospho-H2AX (Upstate) primary antibody (1:100 dilution) in PBS containing 5% BSA and 0.1% Triton-X100 for 1 h at 37°C. Afterwards, coverslips were washed twice for 5 min each in PBS and incubated with FITC-conjugated anti-mouse antibody (1:100 dilution in PBS containing 5% BSA and 0.1% Triton-X100) for 1 h at 37°C. Finally, the coverslips were counterstained with DAPI (10 ng/mL). The images were captured using a fluorescence microscope (Nikon).

Homology-directed recombination assay

ES cell clone with the integrated homologous recombination reporter DR-GFP was generated as described previously (Pierce et al., 2001). 70 μg of the hprtDRGFP plasmid digested with KpnI/SacI was transfected into 2 × 10⁷ cells in 0.8 mL of PBS using an electroporator at 800 V and 10 μF. Then cells were plated onto 5 plates, selected by puromycin (1.2 μg/mL) for 7 days and then by 2 μmol/L 6-thioguanine for another 7 days, and the remaining colonies were isolated. The I-SceI expression vector pCBASce was transfected using a Lipofectamine plus protocol. 10⁵ ES cells were plated onto a 6-well dish, and transfected with 1 μg I-SceI plasmid using the Lipofectamine plus mixture on the next day. Cells were incubated for 48 h, and then analyzed by FACSCalibur cytometer (Becton Dickinson).

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