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Genes to Cells (2006) 11, 1305-1315. doi:10.1111/j.1365-2443.2006.01020.x
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Rad54 is dispensable for the ALT pathway

Koichi Akiyama1,2, Kosuke Yusa3, Hideharu Hashimoto1,2, Anuradha Poonepalli4, Manoor Prakash Hande4, Naoki Kakazu5, Junji Takeda3, Makoto Tachibana1,2 and Yoichi Shinkai1,2,*

1 Department of Life Science, Graduate School of Biostudies;
2 Experimental Research Center for Infectious Diseases, Institute for Virus Research, Kyoto University, 53 Kawara-cho, Shogoin, Sakyo-ku, Kyoto Kyoto 606-8507, Japan
3 Department of Social and Environmental Medicine, Graduate School of Medicine, Osaka University, Suita, Osaka 565-0871, Japan
4 Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Block MD9, 2 Medical Drive, Singapore 117597, Singapore
5 Department of Environmental and Preventive Medicine, Shimane University School of Medicine, 89-1 Enya-cho, Izumo Shimane 693-8501, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Some immortal cells use the alternative lengthening of telomeres (ALT) pathway to maintain their telomeres instead of telomerase. Previous studies revealed that homologous recombination (HR) contributes to the ALT pathway. To further elucidate molecular mechanisms, we inactivated Rad54 involved in HR, in mouse ALT embryonic stem (ES) cells. Although Rad54-deficient ALT ES cells showed radiosensitivity in line with expectation, cell growth and telomeres were maintained for more than 200 cell divisions. Furthermore, although MMC-stimulated sister chromatid exchange (SCE) was suppressed in the Rad54-deficient ALT ES cells, ALT-associated telomere SCE was not affected. This is the first genetic evidence that mouse Rad54 is dispensable for the ALT pathway.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
Telomeres are specialized structures at the ends of chromosomes, consisting of DNA and associated proteins that protect against degradation and fusion (Cech 2004; de Lange 2005). Mammalian telomeric DNA consists of tandem arrays of (5'-TTAGGG-3') that end with a 3' single-stranded overhang on the G-rich strand. Lack of telomerase causes progressive telomere shortening, and below a critical length telomeres can not maintain "functional telomere structure" so that DNA damage or activation of cell cycle check point pathways results, leading to senescence or apoptosis. Therefore, telomerase is normally one of requirement factors for "immortality." Germ-lineage cells and approximately 90% of human cancer cells possess telomerase activity.

The remaining approximately 10% of the cancer cells are telomerase negative, but still maintain their telomeres by the alternative lengthening of telomeres (ALT) pathway (Shay & Bacchetti 1997). Previous studies have identified ALT-associated PML bodies (APBs) in various human ALT cell lines (Yeager et al. 1999) that contain telomeric DNA, telomere-specific binding proteins TRF1, TRF2 and Rif2, DNA repair proteins including MRE11, RAD50, NBS1, RAD51, RAD51D, RAD52, ERCC1, XPF, RAD1, RAD9, RAD17, HUS1 and phospho-H2AX ({gamma}H2AX), the replication protein RP-A, and the RecQ helicases BLM and WRN (Yeager et al. 1999; Henson et al. 2002; Nabetani et al. 2004). Furthermore, many ALT cells exhibit a high rate of postreplicative exchange between telomeres of sister chromatids, T-SCE (Wu et al. 2000; Bailey et al. 2004; Bechter et al. 2004; Londono-Vallejo et al. 2004). These data suggest that the DNA double-strand break (DSB) repair machinery, especially for homologous recombination (HR), contributes to the ALT mechanism (Dunham et al. 2000; Reddel 2003). Reporter assays using a neo gene stably integrated in the telomeric regions of ALT cells also suggested that telomeres are lengthened by recombination (Dunham et al. 2000). Taking these observations into account, break-induced replication (BIR) and recombination-based models of ALT have been proposed in which polymerization-mediated extension of one telomere uses the DNA of a second telomere as a template (Reddel et al. 1997; Bosco & Haber 1998; Teng et al. 2000; Reddel 2003; McEachern & Haber 2006).

For the molecular basis of ALT, the MRE11/RAD50/NBS1 complex has been shown to be important (Jiang et al. 2005). Furthermore, WRN helicase represses T-SCEs in ALT type cells (Laud et al. 2005). Other proteins involved in DNA recombination and repair including the Ku/DNA-PKcs complex components (Bailey et al. 1999; Samper et al. 2000; Goytisolo et al. 2001), BLM (Stavropoulos et al. 2002), RAD51D (Tarsounas et al. 2004), and Rad54 (Jaco et al. 2003), are also reported to be important for telomere maintenance, but their contributions to the ALT pathway have not been fully examined.

To further elucidate molecular mechanisms underlying ALT, we here inactivated Rad54 in the mouse ALT embryonic stem (ES) cells. Rad54 is a double-stranded DNA-dependent ATPase and an important accessory factor for Rad51, a key molecule in HR (Tan et al. 2003). Although Rad54-deficient mice are viable and fertile, they are sensitive to ionizing radiation and some DNA-damaging agents, and show reduced HR efficiency as measured by gene targeting and aberrant DSB repair (Essers et al. 1997; Dronkert et al. 2000). Rad54-deficiency also induces telomere shortening and increased frequency of end-to-end telomere fusions in mice, suggesting important roles of Rad54 in telomere length regulation and telomere capping (Jaco et al. 2003). Furthermore, genetic studies of the yeast survivors from the telomerase inactivation demonstrated that RAD54 is involved in the RAD51-dependent telomere lengthening pathway (Chen et al. 2001). Therefore, we decided to inactivate Rad54 in mouse ALT ES cells and examine the impact of Rad54-deficiency on telomere homeostasis and ALT-associated phenotypes.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Establishment of mouse RAD54-deficient ALT ES cells

Two independent mTERC-deficient survivor ES cell lines were established as previously reported (Niida et al. 1998; Niida et al. 2000). Both survivor lines contain only fused chromosomes (Niida et al. 2000 and Fig. S1), but maintain telomeres in the telomerase-independent fashion. Furthermore, one of the survivors named DKO741ALT showed amplification of the same tandem arrays of telomeric and non-telomeric sequences at most chromosome ends, suggesting cis/trans recombination/amplification as one of the mechanisms for ALT (Niida et al. 2000). To inactivate Rad54, we deleted exon IV of the Rad54 gene, resulting in elimination of approximately 90% of the protein, as used earlier for Rad54 disruption (Essers et al. 1997). However, if inactivation of Rad54 were to immediately induce a growth defect of DKO741ALT ES cells due to severe telomere dysfunction, we would not expect to obtain Rad54-deficient ALT ES cells by simple Rad54 targeting. To avoid this possibility, we introduced a conditional KO allele by inserting loxP sites flanking exon IV of the Rad54 gene. As illustrated in Fig. 1A, we sequentially replaced the endogenous Rad54 allele with the conditional KO vector and finally established conditional Rad54 KO (Rad54flox/floxneo) DKO741ALT cell. Western blot analysis revealed that Rad54 expression was detected in this conditional Rad54 KO ALT ES cell line, D107A9 (Fig. 1D). After infection with adenovirus expressing cre recombinase (Adeno-Cre), exon 4 could be deleted from both Rad54 conditional KO alleles. We treated the Rad54+/flox (D107) and Rad54flox/floxneo (D107A9) ALT ES cell lines with Adeno-Cre and analyzed the Rad54 expression 3 days postinfection. As clearly shown in Fig. 1D, Rad54 was mostly absent in the Adeno-Cre-treated D107A9 ALT ES cells (–/–) but not in the Rad54+/flox ALT ES cells (+/–). Southern blot analysis further confirmed deletion of Rad54 exon IV by the Adeno-Cre treatment (Fig. 1C). Genotypes of the cells used in this study are summarized in Fig. 1B.


Figure 1
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  		Figure 1  Establishment of conditional mouse Rad54 KO ALT ES cells. (A) Partial restriction maps of the Rad54 locus (exons III–VIII of mouse RAD54), the Rad54 conditional targeting plasmid and the targeted loci are shown. (B) Genotypes of cells analyzed in this study are shown. (C) Southern blot analysis of Rad54 KO ALT ES cells. Genomic DNA from ES cells was digested with HpaI and XhoI and probed with the exon VII–VIII probe. Bands corresponding to the wild-type (+ 9.2 kb), targeted (floxneo; 7.6 kb), FLP/FRT-deleted (flox; 5.8 kb) and Cre/loxP-deleted (–; 8.0 kb) fragments are indicated. (D) Expression of Rad54 protein in the established ES cells was analyzed by Western blotting with anti-Rad54 antibodies.

 
No growth defect was found in the Adeno-Cre treated D107A9 cells after up to 1 week of cell culture, indicating that Rad54 is not essential for cell growth of the DKO741ALT ES cells, at least up to approximately ten cell divisions. Then, Rad54-deficient DKO741ALT ES cell clones were isolated by subcloning.

Rad54-deficient DKO741ALT ES cells are sensitive to ionizing radiation, but do not show any growth defect

It has been reported that mouse Rad54-deficient cells are sensitive to ionizing irradiation and various other DNA-damaging agents (Essers et al. 1997). To examine whether inactivation of the Rad54 gene also induces DNA DSB repair defects in mouse ALT ES cells, we compared radiosensitivity between Rad54flox/floxneo ALT ES (D107A9) and Rad54–/– ALT ES (D107A9cre) cells. As shown in Fig. 2A, D107A9cre cells were found to be more sensitive to {gamma}-irradiation than D107A9 cells or their parental cell, DKO741ALT.


Figure 2
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  		Figure 2  X-ray sensitivity and growth characteristics of Rad54-deficient ALT ES cells. (A) The effect of ionizing radiation on the Rad54-deficient ALT ES cells. After plating, each indicated ES cell line was treated with different doses of X-rays and allowed to form individual colonies. All measurements were performed in triplicate. (B) Growth characteristics of DKO741ALT, D107A9, and D107A9cre. Population doublings (PDLs) of the ES cells are plotted.

 
To further examine whether Rad54 is critical for the long-term cellular proliferation potential, DKO741ALT, D107A9 and D107A9Cre were subjected to long-term culture and replated every 3–4 days for up to 230 days. At this time point, no growth defect could be detected in either D107A9 or D107A9Cre cells (up to > 230 PDL (population doubling level)) (Fig. 2B). In our previous studies, it was shown that DKO741ALT cells possess on average half of the telomeric DNA contents per chromosome end of the original mTERC+ ES cells with minimum cell division (Niida et al. 1998). If Rad54 is essential for the ALT pathway of DKO741ALT cells and same progressive telomere shortening is re-initiated in the Rad54-deficient situation as seen in the original mTERC-deficient ES cells, another approximately 200 PDL would be expected to be the critical division point for inducing telomere dysfunction and growth defect. Therefore, the lack of any growth defect of D107A9Cre cells after 200 cell divisions indicates that "functional telomeres" are maintained without Rad54.

Telomere dynamics in the Rad54-deficient DKO741ALT ES cells

To examine the telomere dynamics in Rad54-deficient DKO741ALT ES cells, we performed two different experiments. Because DKO741ALT cells amplify tandem arrays of telomeric and nontelomeric sequences at most chromosome ends and regular four- or five-base restriction enzymes digest their telomeric DNA arrays (nontelomeric sequences) (Niida et al. 2000), we could not apply standard telomeric DNA Southern blot analysis. Therefore, we performed slot-blot DNA hybridization to measure the telomeric DNA contents. As shown in Fig. 3A, the ALT-inactive parental mTERC-deficient ES cells demonstrated progressive reduction in telomeric DNA contents. On the other hand, we did not observe any progressive telomeric DNA loss in D107A9 ALT ES cells and their telomeric repeats were heterogeneous in nature at different PDLs, which is a typical phenotype for most human ALT cells. Furthermore, telomeres of the Rad54-deficient D107A9Cre also behaved as D107A9 (Fig. 3A, right panel). Therefore, these data strongly suggest that Rad54 is dispensable for the ALT pathway activated in mTERC-deficient survivor cells.


Figure 3
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  		Figure 3  Quantification of telomeric DNA sequences in the Rad54-deficient ALT ES cells. (A) Ratios of amounts of the telomeric DNA to the total genome DNA during cell growth. The amounts of telomeric DNA sequences in the parental mTERC-deficient ES cells declined progressively with increasing PDLs. Those of DKO741ALT cell lines were slightly diminished at some PDLs (50 PDL for D107A9 and 60–90 PDLs for D107A9Cre) but recovered at later PDLs (150–200 PDLs for D107A9 and 148–190 PDLs for D107A9Cre). (B) Q-FISH analysis. The amounts and frequencies of telomeric signals in the parental mTERC-deficient ES cells show decline at 150 PDLs, but in the Rad54-proficient (D107A9) and deficient (D107A9cre) ALT cell lines no significant differences are evident.

 
To further analyze the telomere dynamics in Rad54-deficient D107A9Cre, we performed cytogenetic analysis of metaphase spread chromosomes by quantitative fluorescence in situ hybridization (Q-FISH) with the (C3TA2)3 PNA probe (Zijlmans et al. 1997; Hande et al. 1999). As shown in Fig. 3B, the parental ALT-inactive mTERC-deficient ES cell, DKO741 exhibited clear telomere shortening after 200 cell divisions (mean telomere fluorescence intensities of DKO741 were reduced from 3298.6 ± 60 at PDL 30– 1854.4 ± 39.6 at PDL 240). In contrast, no clear telomere shortening was observed for D107A9 and D107A9Cre cells after the same cell culture period. Therefore, from these two independent experimental observations, we conclude that Rad54 is dispensable for the ALT pathway, at least in DKO741 survivor cells.

Lack of changes of APB-associated {gamma}H2AX in Rad54-deficient DKO741ALT ES cells

Although Rad54 could be shown to be dispensable for the ALT pathway of the mTERC-deficient survivor ES cells, we further determined the impact of Rad54 deficiency on other ALT-associated phenotypes. Because {gamma}H2AX is known to accumulate in the APBs of various human ALT cells, we analyzed telomere accumulation of {gamma}H2AX in DKO741ALT ES cells. As seen in Fig. 4 and summarized in Table 1, {gamma}H2AX positive cells were increased in DKO741ALT (64.8%) and D107A9 (67.0%) as compared with the wild-type ES cells (25.0%). Furthermore, most {gamma}H2AX positive survivor ES cells possessed telomere (TRF1)-associated {gamma}H2AX foci (53.6 and 64.5% for DKO741ALT and D107A9 comparing to 1.9% for E14). Therefore, telomere-associating {gamma}H2AX was also shown to be linked with the mouse ALT cells. Further analysis of telomere-associated {gamma}H2AX foci in D107A9Cre cells demonstrated the percentage of positive cells to remain high with no significant decrease (51.8%).


Figure 4
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  		Figure 4  Colocalization of {gamma}H2AX on telomeres in mouse ALT ES cell lines. Indirect immunofluorescence studies were performed with wild-type and ALT mouse ES cells. TRF1 (green) and {gamma}H2AX (red) were stained with specific primary antibodies and detected with Alexa568- or Alexa488-labeled secondary antibodies. Small boxes with merged images highlight co-localization signals. Representative co-localizing foci of {gamma}H2AX and TRF1 are indicated by arrows.

 

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  		Table 1  Percentages of the cells with {gamma}H2AX nuclear foci on telomeres
 
Defect of MMC-induced SCE but intact T-SCE in Rad54-deficient DKO741ALT ES cells

As described earlier, many ALT cells feature a high rate of postreplicative exchange between telomeres of sister chromatids, T-SCE (Bailey et al. 2004; Bechter et al. 2004; Londono-Vallejo et al. 2004). Therefore, we lastly tested whether mTERC-deficient survivor ES cells had an activated T-SCE pathway. As shown in Fig. 5 (arrows) and summarized in Table 2, T-SCE was greatly increased in DKO741ALT ES cells (1.40 ± 0.11 events per chromosome (epc), mean ± SE) but not in wild-type ES cells (0.0015 ± 0.001 epc). This high rate of T-SCE was not affected by the Rad54 inactivation (1.48 ± 0.10 epc for D107A9Cre). General SCE was also examined for the parental E14, DKO741ALT and D107A9Cre cells (Table 3, top). The general SCE in DKO741ALT was increased about twice (11.6 ± 1.6 events per metaphase (epm)) as compared to E14 (6.56 ± 1.9 epm), and inactivation of Rad54 did not affect the general SCE rate (11.0 ± 2.1 epm for D107A9Cre). However, as earlier reported for mouse Rad54 deficient ES cells (Dronkert et al. 2000), MMC-stimulated general SCE was still suppressed by the Rad54 inactivation in the DKO741 survivor ES cells (4.57 ± 0.60 epc for DKO741ALT and 1.74 ± 0.30 epc for D107A9Cre) (Table 3, bottom).


Figure 5
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  		Figure 5  CO-FISH analysis of T-SCE. Left, E14 shows a normal CO-FISH pattern with two typical telomeric DNA signals. Middle, DKO741ALT has many T-SCE signals. Right, the T-SCE rate for D107A9cre is the same as that for DKO741ALT (summarized in Table 2). Small boxes highlight representative T-SCE signals.

 

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  		Table 2  The rate of telomere SCEs in wild-type and ALT+ ES cells
 

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  		Table 3  Induction of SCEs by MMC* in wild-type and ALT + ES cells
 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
The present study provided the first genetic evidence that mRAD54 is dispensable for the ALT pathway and ALT-associated characteristics, at least in the mTERC-deficient survivor ES cell line, DKO741ALT.

Generalization of our current findings to the mammalian ALT pathway(s)

Because DKO741ALT features amplification of tandem arrays of telomeric and non-telomeric sequences, it tends to be classified type of cell line distinct from the majority of mammalian ALT cell lines that amplify and maintain only telomeric DNA sequences on telomeres. Survivor cells of Saccharomyces cerevisiae from deletion of telomerase genes are also known to maintain their telomere lengths, using a RAD52-dependent HR pathway (Lundblad & Blackburn 1993; McEachern & Blackburn 1995; Teng et al. 2000; Chen et al. 2001). Survivors utilize two types of ALT pathways, I and II. Type I survivors have very short C1–3A/TG1–3 tracts but exhibit multiple tandem copies of the subtelomeric Y' element, depending on RAD51, RAD54, RAD57, and Sgs1 genes, whereas type II survivors only amplify C1–3A/TG1–3 tracts and have significant telomere lengthening and heterogeneity, depending on RAD50, RAD59 (Chen et al. 2001; Tsai et al. 2006). Based on such a different yeast telomeric DNA amplification phenotype, DKO741ALT seems to be a mammalian type I counterpart. However, these yeast survivors have been functionally characterized and classified so that type I survivors utilize > 100 base pairs of homology sequences of multiple Y' elements for maintaining telomeres by the RAD51-dependent HR pathway and type II survivors use only shorter homology TG1–3 tracts to elongate telomeres by the RAD50-dependent recombination pathway (Chen et al. 2001; McEachern & Haber 2006). Unlike yeast telomeres, mammalian telomeric DNA consists of highly homologous TTAGGG tracts (> kb). Therefore, based on the nature of the amplified telomeric DNA sequences, not only DKO741ALT but also the majority of human ALT cell lines can be classified as mammalian type I survivors. We also hypothesize that the nontelomeric sequences amplified in DKO741ALT originated from subtelomeric (mixture of telomeric and nontelomeric arrays) regions (Niida et al. 2000). Although the nontelomeric sequences amplified in DKO741ALT have still not been mapped to any mouse chromosomes by a mouse full-genome database search, this result suggests that they are likely to be located in highly repetitive sequence regions like telomeres. If so, intra- and interchromosomal BIR/recombination between telomeres and subtelomeres could easily amplify nontelomeric sequences and an additional ALT mechanism for DKO741ALT would not be expected. Furthermore, except for the telomeric DNA phenotype, DKO741ALT shows the typically observed phenotypes of human ALT cell lines, including telomere length heterogeneity (Fig. 3B), APB-associated {gamma}H2AX, and T-SCE. These phenotypes are also shared by another mTERC-deficient ALT ES cell line, DKO301ALT (Niida et al. 2000 and not shown). Even if such type I/II distinction is not well established in mammals, assuming the previously mentioned hypothesis to be correct, we can apply our conclusions obtained with DKO741ALT to the general case of mammalian ALT cells.

Why is Rad54 dispensable for telomere lengthening in DKO741ALT?

In yeast, Rad54 plays crucial roles in the RAD51-dependent HR pathway and disruption of RAD54 inactivates RAD51-dependent telomere lengthening (Chen et al. 2001). If DKO741ALT is a mammalian type I survivor and the Rad51-pathway is critical for the telomere lengthening, why did Rad54-deficiency not affect telomere maintenance in our DKO741ALT? A number of explanations are possible. (1) As with yeast type I survivors, the Rad51-dependent pathway is crucial for the telomere maintenance of DKO741ALT, but Rad54 is not indispensable. Rad54-deficient cells grow normally (Essers et al. 1997), but Rad51 is essential for cell growth and survival (Lim & Hasty 1996; Tsuzuki et al. 1996; Sonoda et al. 1998). Therefore, even if Rad54 plays some roles in DSB repair and HR, the Rad51-dependent pathway may still function on BIR and recombination and maintain telomere length in its absence. (2) Rad54 paralogs could play important roles in telomere maintenance of DKO741ALT and one example, Rad54B, exists in mammals and is known to partly compensate for Rad54-defective phenotypes in mouse ES cells (Wesoly et al. 2006). However, Rad54B-inactivation phenotypes are much milder than those with Rad54-deficiency and Rad54/Rad54B double deficient mice are still viable and fertile, and double KO ES cells could be shown to grow normally. Therefore, even if Rad54 paralogs play some roles in ALT, Rad54B's contribution may be minimal (so far, only Rad54 and Rad54B are classified as the Rad54 paralogs in mouse). In this context it should be noted that we examined Rad54B expression before and after Rad54 inactivation, but no up-regulation of Rad54B was observed in D107A9Cre (data not shown). (3) The Rad51/Rad54-dependent pathway is dispensable for the telomere maintenance of DKO741ALT but (an)other DSB repair pathway(s) is crucial. Alternatively, multiple pathways including the RAD51-dependent pathway cooperatively elongate telomere length and disruption of a single pathway may not have a clear impact on telomere lengthening. Mammalian telomeres form T-loop structures (Griffith et al. 1999) that may contribute to the reduction of their dependence on the Rad51-dependent pathway for telomere elongation because TRF2 could potentially bypass Rad51-mediated strand-invasion. MMC-stimulated SCE was here found to be sensitive to Rad54 inactivation in DKO741ALT, but T-SCE was not. If TRF2 is also involved in strand-invasion between sister chromatid telomeres, this might explain why T-SCE was resistant to Rad54 deficiency because it is thought to be generated from the resolution of BIR/HR between sister chromatid telomeres.

It is still important to identify molecules that are directly involved in telomeric DNA elongation/amplification in ALT cells. However, studies of the WRN-deficient mouse embryonic fibroblasts clearly showed that inactivation of WRN alone is not sufficient for the activation of ALT (Laud et al. 2005). Thus, it is also important to elucidate how the ALT pathway(s) is normally suppressed or how such suppression might be canceled in ALT cells.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Targeting vector construction

A BAC (bacterial artificial chromosome) clone containing a region of the Rad54 locus of Mus musculus, RP23–319H15 was purchased from the BACPAC Resource Center (BPRC) at Children's Hospital Oakland Research Institute in Oakland, California, in the United States. The clone was modified as previously described (Liu et al. 2003).

Briefly, RP23–319H15 was transferred into the bacterial strain, EL350, which expresses cre recombinase on arabinose induction. A loxP site was inserted approximately 200 bp upstream of exon IV, and a FRT (Flp recognition target)-pgk neo-FRT-loxP cassette approximately 200 bp downstream of exon IV. The same region as previously reported for a Rad54 targeting strategy (Essers et al. 1997) was retrieved from the modified BAC, using a linearized plasmid, arms homologous to the approximately 7 kb upstream of exon IV and approximately 2 kb downstream of the FRT-pgk neo-FRT-loxP cassette, and a diphtheria toxin A (DTA) gene. The targeting vector was digested with PmeI for linearization, and electroporated.

Generation of knockout ES cells

mTERC (mouse Telomerase RNA component) knockout ES cells and the survivor cell line, DKO741ALT were as previously described (Niida et al. 1998; Niida et al. 2000).

Ten micrograms of linearized Rad54 conditional targeting vector were electroporated into 107 mouse ALT cells at 300 V, 75uF with a Gene Pulsar II (BIORAD). The neomycin-resistance gene (neo) was used as a positive selection marker and DTA as a negative selection marker. G418-positive clones were screened and those that were PCR positive were checked by Southern blotting. Of 385 G418-resistant clones screened, four had undergone homologous recombination correctly. pCAGGS-FLPe-IRES-puro was a gift from Dr H. Hamada, Osaka University. To generate the conditional allele, the neo gene was deleted by transfecting the targeted cell with the Flp expression vector and selection with 1.5 µg/mL puromycin. Heterozygous targeted cells were retargeted in the same way as described previously. After infection with Adeno-cre, expression of the Rad54 protein in the established ES cells was monitored by Western blotting using an anti-hRad54 antibody generated against the N-terminus of human Rad54 (Sc-5849, Santacruz), which cross-reacts with mouse Rad54.

Immunofluorescence

Cytospun cells were fixed onto slides with 4% paraformaldehyde for 20 min, permeabilized with 0.1% Nonidet P-40 (NP-40) for 20 min, and incubated with blocking solution (4% bovine serum albumin, 0.1% NP-40 in phosphate buffered saline (PBS)) at 37 °C for 45 min. Incubation was with rabbit anti-mTRF1 Ab (Iwano et al. 2004) and mouse anti-{gamma}H2AX (UPSTATE) diluted to 1 : 200 at 37 °C for 60 min. An anti-mouse IgG conjugated with Zenon Alexa 568 Fluor (Molecular Probes) was used for detection of {gamma}H2AX, and an anti-rabbit IgG conjugated with Zenon Alexa 488 Fluor (Molecular Probes) for mTRF1. The slides were examined under a fluorescence microscope and analyzed with AxioVision software (Zeiss).

Cell survival assays

The sensitivity of the cells to increasing doses of ionizing radiation was determined by measuring their colony-forming ability. The cells were trypsinized and counted. Various cell dilutions were aliquoted onto gelatinized six-well plates, and after 12–24 h, the cells were irradiated. Cells were grown for 7–10 days and the number of colonies formed were scored. The measurements were performed in triplicate.

Sister chromatid exchange (SCE) assay

SCE assays were performed as previously described (Sonoda et al. 1999). Briefly, cells grown to approximately 50% confluence were incubated for 48 h (two cell cycles) with 20 mM 5-bromo-2'-deoxyuridine (BrdU) (Sigma) and then treated with 0.1 µg/mL colcemid for 2 h. After harvesting with trypsin, they were swollen for 20 min in 75 mM KCl and fixed for 30 min in methanol: acetic acid (3 : 1). The cells were then dropped onto moist prewashed (with 50% EtOH) glass slides, allowed to dry on a 42 °C hot plate and stained with 10 µg/mL Hoechst 33258 in Phosphate buffer (pH 6.8) for 20 min at 25 °C. The slides were then exposed to UV light for 1 h, using Stratalinker (1800) (Stratagene), washed with 2 x SSC (0.3 M NaCl, 0.03 M sodium citrate) at 62 °C, stained with 3% Giemsa for 50 min and finally air-dried and mounted with glass cover slips in Enteran neu (Merck, Darmstadt, Germany).

For mitomycin C (MMC)-induced SCE, MMC was added to the culture media at a final concentration of 0.1 µg/mL for 16 h prior to harvesting. Slides were analyzed with a bright-field microscope equipped with a 100 x objective.

Chromosome orientation fluorescence in situ hybridization (CO-FISH)

CO-FISH has been previously described in detail (Bailey et al. 2001). Briefly, confluent cultures were subcultured in a medium containing 20 µM BrdU and incubated at 37 °C for 24 h (one cell cycle). Colcemid (0.2 µg/mL) was added during the last 2 h. Cells were trypsinized and suspended in 0.9% citrate buffer at 37 °C for 25 min before fixing in 3 : 1 methanol : acetic acid. Fixed cells were dropped onto wet glass slides, washed with PBS once, treated with 0.5 µg/mL RNase A for 10 min at 37 °C, stained with 0.5 µg/mL Hoechst 33258 (Sigma) in 2 x SSC for 15 min at room temperature and then exposed to 365 nm UV light (Stratalinker 1800) for 30 min. Enzymatic digestion of the BrdU-substituted DNA strands with 3 units/mL of Exonuclease III (Takara) in buffer supplied by the manufacturer (50 mM Tris-Cl, 5 mM MgCl2, and 5 mM dithiothreitol, pH 8.0) was allowed to proceed for 10 min at room temperature. The probe for telomeric DNA was Cy3-labeled (CCCTAA)3 peptide nucleic acid (PNA). Following dehydration through an ethanol series (70, 85, 100%), a hybridization mixture containing 0.5 µg/mL probe PNA in 70% formamide was applied. Following 2 h hybridization in a moist chamber at room temperature, the slides were washed once for 15 min each in wash solution I (70% formamide, 0.1% BSA, 10 mM Tris-Cl-pH 7.5) and then washed 3 times for 5 min with 0.05% Tween-20 in Tris buffered saline. After a second dehydration through an ethanol series (70, 85, 100%), slides were mounted in a Vectashield containing 0.2 µg/mL 40,6-diamidino-2-phenylindole (DAPI) to counterstain chromosomal DNA, examined under a fluorescence microscope, and analyzed with AxioVision software (Zeiss).

Quantification of amounts of telomere repeat sequences (slot-blot DNA hybridization)

Genomic DNA (0.1, 0.3 and 0.5 µg) was loaded onto a 0.8% agarose gel in 1 x TAE and electrophoresed at 100 V for 15 min. The gel was stained with 0.01 µg/mL ethidium bromide and values for fluorescent intensity of each band were acquired by Fluor-S (Bio-Rad). Gels were dried at 70 °C for 40 min, denatured in a 1.5 M NaCl, 0.5 M NaOH solution for 20 min, neutralized in a 1.5 M NaCl, 0.5 M Tris-HCl pH 8.0 buffer for 15 min, and probed with 32P-labeled (C3TA2)4 telomeric DNA oligonucleotides in 5 x SSC, 5 x Denhardt's solution at 37 °C overnight. Following high stringency washes in 0.1 x SSC at room temperature for 1 h, autoradiography was performed and the radioactivity of each band was measured using BAS 5000 (Fuji Film).

Quantative fluorescence in situ hybridization

Cells were harvested after colcemid treatment (0.1 µg/mL) for 2 h, washed with PBS, swollen for 25 min in 0.03 M sodium citrate buffer and fixed in methanol: acetic acid (3 : 1). Cell suspensions were then placed on wet, clean slides and dried overnight. FISH with Cy3-labeled (C3TA2)3 peptide nucleic acid was performed as described earlier (Zijlmans et al. 1997; Hande et al. 1999) and metaphase spreads were visualized using a Zeiss Axioplan 2 imaging fluorescence microscope equipped with a CCD camera. Images were captured using ISIS imaging software (Metasystems, Germany) and telomere fluorescence intensities were measured using ISIS telomere software (Metasystems, Germany).


   Acknowledgements
 
We acknowledge the generosity of Dr H. Hamada (Osaka University) in providing the FLP expressing plasmid. We also thank Dr E. Sonoda (Kyoto University) for his useful comments and Dr T. Iwano (RIKEN) for helpful advice and the initial Rad54 targeting experiments. This work was supported by a grant-in-aid from the Ministry of Education, Science, Technology, and Culture of Japan (to Y.S. and M.T.), ARF, National University of Singapore and National Medical Research Council Ministry of Health, Singapore (to M.P.H.) and by the 21st Century COE Program of the Ministry of Education, Culture, Sports, Science, and Technology to the Graduate School of Biostudies and Institute for Virus Research, Kyoto University (to K.A. and H.H.).


   Footnotes
 
Communicated by: Fuyuki Ishikawa

* Correspondence: E-mail: yshinkai{at}virus.kyoto-u.ac.jp


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
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Received: 6 June 2006
Accepted: 19 August 2006





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