Telomeric DNA Ends Are Essential for the Localization of Ku at Telomeres in Fission Yeast*

Tomoichiro MiyoshiDagger , Mahito SadaieDagger , Junko Kanoh§, and Fuyuki IshikawaDagger §

From the Dagger  Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501 and § Graduate School of Biostudies, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan

Received for publication, August 28, 2002, and in revised form, November 4, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The Ku70-Ku80 heterodimer is a conserved protein complex essential for the non-homologous end-joining pathway. Ku proteins are also involved in telomere maintenance, although their precise roles remain to be elucidated. In fission yeast, pku70+, the gene encoding the Ku70 homologue, has been reported. Here we report the identification and characterization of pku80+, the gene encoding Ku80. Both pku70+ and pku80+ are essential for efficient non-homologous end-joining. We also found that the pku70 and pku80 mutants are sensitive to methyl methanesulfonate and hydroxyurea, suggesting their roles in the S phase. The pku80 mutant shows telomere shortening and tandem amplification of a subtelomeric sequence but no defects in the telomere position effect, as was previously reported for the pku70 mutant. By using the chromatin immunoprecipitation assay, we demonstrated that Pku70 and Pku80 physically interact with telomeric repeats and subtelomeric sequences. Interestingly, this telomere association of Pku proteins is independent of Taz1, a telomeric DNA-binding protein. We also showed that the Pku proteins do not associate with ectopically integrated telomeric repeats in the internal region of circular chromosomes. These results indicate that the physical end of DNA is necessary for the localization of Pku80 at telomeres.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

DNA double strand breaks (DSBs)1 cause serious damage to genetic materials in living cells. Indeed, a single chromosomal break is lethal if left unrepaired (1). DSBs are introduced by a variety of DNA-damaging agents, such as ionizing radiation and radiomimetic chemicals, or even spontaneously. DSB results in the loss of the continuity of DNA and the formation of two physical ends. These non-physiological ends of DNA are repaired in eukaryotes by two major pathways, the error-prone NHEJ pathway and the more accurate homologous recombination (HR) pathway (reviewed in Ref. 2). NHEJ ligates two broken DNA ends with little or no requirement for sequence homology between the two ends. In contrast, HR requires an intact DNA that is homologous with the DNA to be repaired and that serves as a template for the DNA synthesis to repair the break. The HR pathway requires products of the RAD52 epistasis group genes, including Rad51 and Rad54 in Saccharomyces cerevisiae. On the other hand, a different set of gene products is necessary for the NHEJ pathway, which includes the DNA-dependent protein kinase (DNA-PK), XRCC4, DNA ligase IV proteins, and the recently described Artemis protein in human cells (3). Artemis possesses nuclease activity that functions to process double-stranded DNA ends and is regulated by DNA-PKcs (4). The DNA-PK holoenzyme is a serine-threonine protein kinase consisting of DNA-PKcs and the Ku70-Ku80 heterodimer. The Ku70-Ku80 complex binds to the very end of broken DNA and then recruits and activates the DNA-PKcs (reviewed in Ref. 5).

The function of Ku proteins is not limited to the repair of non-physiological DNA ends. Ku is a bona fide component of telomeres, the physiological end of linear DNA in eukaryotes (6-8). Telomeres consist of telomeric DNA, tandem repeats of short G-rich sequences in most cases, and many associating proteins (reviewed in Refs. 9 and 10). By forming a large protein-DNA complex, telomeres protect the physiological end from DNA degradation and HR- or NHEJ-mediated DNA repair reactions. Accordingly, telomeres are essential for the maintenance of genome integrity. Besides this essential role, telomeres show several characteristic features. It is known that genes located close to telomeres become silenced. This is a phenomenon related to the position effect shown by a variety of heterochromatins, and it is called the telomere position effect (TPE). Indeed, telomeres are uniquely localized at the nuclear periphery in budding and fission yeasts (11, 12), a finding commonly observed with heterochromatin. These observations indicate that telomeres compose a class of heterochromatin. Indeed, many proteins are shared by telomeres and other classes of heterochromatin, such as the silent mating type loci in S. cerevisiae. In addition to these properties shown by the telomeres as heterochromatin, other functions performed by telomeres can be viewed as unique to the telomeres themselves. For example, by recruiting telomerase, a specialized reverse transcriptase synthesizing telomeric DNA, telomeres compensate for the continuous telomere length reduction caused by the end replication problem. Without this, telomeres become shorter and eventually lose telomeric DNA, leading to genetic instability.

In S. cerevisiae, Yku70/Hdf1 and Yku80/Hdf2, the homologues of human Ku70 and Ku80, respectively, are components of telomeres and are involved in such telomere functions as TPE, telomere localization at the nuclear periphery, and telomere length control (13-19). However, the presence of the Ku proteins at telomeres is enigmatic, because Ku proteins promote NHEJ at the DSB ends, a reaction that evidently should be inhibited at telomeres. Therefore, the physiological significance of the Ku proteins at telomeres is not known precisely.

Here we describe the identification and characterization of pku80+, the gene coding for the Schizosaccharomyces pombe Ku80 homologue. We found that Pku80 is involved in both NHEJ and telomere metabolism, indicating the conserved roles of this protein in a diverse set of eukaryotes. Interestingly, we found that Pku80 is bound to the native telomere but not to the ectopically integrated internal telomeric repeats. This result suggests that the physical end of DNA is necessary for the recruitment of Pku80.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

Strain and General Techniques-- The fission yeast strains used in this study are listed in Table I. Cells were grown in the rich medium YES or the synthetic medium SD and supplemented with amino acids as required. Growth media as well as basic genetic and biochemical techniques for fission yeast have been described elsewhere (20).

                              
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Table I
Yeast strains used in this study

Gene Disruption and Integration-- For the disruption of the pku80+ gene, the pku80+ ORF was amplified by PCR with a primer set of pku80NdeI (5'-GGAATTCCATATGATGAGTGATAAGGAATGTACTG-3'; the NdeI site is underlined) and pku80BamHI (5'-CGCCGGATCCAATGTTATTAAAATTATCATGTCTAG-3'; the BamHI site is underlined) using wild-type genomic DNA as a template. The ORF was then cloned into pT7Blue (Novagen). The resultant plasmid (pMP16) was digested with EcoRV, and a ura4+ cassette was inserted (pMP19). pMP16 was digested with EcoRV, and a LEU2 cassette or a kanr cassette was inserted (pMP20 or pMP31, respectively). pMP19, pMP20, and pMP31 were digested with NdeI and BamHI, and the pku80::ura4+, pku80::LEU2, or pku80::kanr fragment was used for transformation. For the disruption of the pku70+ gene, the pku70+ ORF containing its own promoter regions was amplified by PCR with a primer set of pku70-1027s (5'-AAATATTGTATCGCATTTCGAGC-3') and pku70-PstI (5'-AAAACTGCAGTAATTTTTTGACATAGTTCGTTAC-3') using wild-type genomic DNA as a template. This pku70+ DNA was then cloned into pT7Blue. The resultant plasmid (pMP37) was digested with EcoRV and SnaBI, and a ura4+ cassette was inserted (pMP39). pMP39 was digested with NdeI, and the pku70::ura4+ fragment was used for transformation.

Chromosomal Integration of pku70-3HA, pku80-3HA, and taz1-3HA-- The strain with circular chromosomes was obtained by successive plating of PM26-1 trt1::LEU2 on YPD (TN254). Loss of telomeric repeats and chromosome circularization were confirmed by Southern hybridization and pulsed-field gel electrophoresis, respectively. This strain was used for constructing the strains expressing pku70-3HA, pku80-3HA, or taz1-3HA. To tag Pku70 with the HA epitope at the C terminus, we amplified the pku70+ ORF by PCR with a primer set of pku70NotEGFP (5'-AAGGAAAAAAGCGGCCGCATAATTTTTTGACATAGTTCGTTAG-3', the NotI site is underlined) and pku70NotHA (5'-AAGGAAAAAAGCGGCCGCAACTCTAAAACCCTGGCACTG-3', the NotI site is underlined) using wild-type genomic DNA as a template. The NotI fragment was then cloned into pTN149, which contained three copies of HA epitope and a ura4+ marker (pMP34). pMP34 was digested with HindIII, and a kanr cassette was inserted (pMP41). pMP34 or pMP41 was linearized at the HpaI site in pku70+ and used for transformation. To tag Pku80 with the HA epitope at the C terminus, we amplified the pku80+ ORF by PCR with a primer set of pku80NotEGFP (5'-AAGGAAAAAAGCGGCCGCAAATGTTATTAAAATTATCATGTCTAG-3', the NotI site is underlined) and pku80NotHA (5'-AAGGAAAAAAGGCGGCCGCAATCTGATGCAGTGTCTACTG-3', the NotI site is underlined) using wild-type genomic DNA as a template. The NotI fragment was then cloned into pTN149 (pMP35). pMP35 was digested with HindIII, and a kanr cassette was inserted (pMP43). pMP35 or pMP43 was linearized at the ClaI site and used for transformation. To tag Taz1 with the HA epitope at the C terminus, pJK295 (21) was digested with HpaI of LEU2, and then a kanr cassette was inserted (pMP51). pMP51 was partially linearized at the EcoRV site and used for transformation. Expression of Pku70-3HA, Pku80-3HA, or Taz1-3HA was confirmed by immunoblotting using anti-HA antibodies (16B12; Babco). For the integration of telomere-ura4+-TAS fragment into the ade6 locus, pNSU70 fragment (SacI and HindIII) was cloned into pBluescript II SK(-). The resultant plasmid was digested with EcoRI, and a ura4+ cassette was inserted. The resultant plasmid was digested with HindIII, and the pNSU56 HindIII fragment was inserted. The resultant plasmid was digested with PvuI and inserted into the pYB26 NdeI-AatII site (pYB41). pYB26 consists of the ade6 locus BamHI-SpeI fragment inserted into pBluescript II SK(-). pYB41 was digested with BamHI and SpeI and used for transformation.

NHEJ Assay-- Undigested pAL19 (22) was used as a control plasmid to monitor the transformation efficiency. The linear substrate for the NHEJ assay was prepared by excision of the PvuII fragment from pAL19. Both undigested and linear DNAs were purified using the Qiagen Gel Extraction Kit (Qiagen). Logarithmically growing cells were transformed with an equal amount (0.3 µg) of undigested or linear DNA using the lithium acetate method (20). The NHEJ frequency was expressed as the percentage of the number of Leu+ colonies arising from cells transformed with the linearized plasmid divided by that of colonies transformed with the undigested plasmid. This value was normalized by the value obtained from wild-type cells. Experiments were performed three times, and the average is shown on bar graphs.

Yeast Two-hybrid Assay-- The pku70 cDNA was amplified by PCR and cloned into pGBKT7 (Clontech). The pku80 cDNA was amplified by PCR and cloned into pGAD-GH (Clontech). The S. cerevisiae Y190 strain (MATa ura3-52 his3-D200 lys2-801 ade2-101 trp1-901 leu2-3, 112gal4Delta gal80Delta LYS2::GAL-HIS3 URA3::GAL-lacZ cyhr) was transformed with each plasmid and assayed for beta -galactosidase activity according to the manufacturer's instructions.

Southern Hybridization-- Chromosomal DNA was isolated using the glass bead-phenol chloroform method (23). Genomic DNA was digested with ApaI, EcoT22I, or EcoRI, separated on 2.0 or 1.0% agarose gel in 1× TBE buffer, and transferred onto a Hybond-N+ nylon membrane (Amersham Biosciences) according to the manufacturer's instructions. Probes specific for the telomeric and telomere-associated sequence (TAS) were generated by random-primed labeling of DNA fragments of pNSU70 (24). Membranes were washed with 1× SSC, 0.5% SDS, and signals were visualized using Hyperfilms (Amersham Biosciences).

Chromatin Immunoprecipitation-- The ChIP assay described by Saitoh et al. (25) was adopted with modifications. Taz1-HA-, Pku70-HA-, or Pku80-HA-expressing cells grown in 50 ml of YES culture (2 × 107/ml) at 30 °C were fixed with formaldehyde. For immunoprecipitation, anti-HA antibodies (16B12; Babco) and protein A-Sepharose CL-4B (Amersham Biosciences) were used. After washing the beads, coprecipitated DNA was extracted and then suspended in TE. Portions of immunoprecipitated DNAs were used as PCR template. The locations of the primer sets are shown in Fig. 3.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

S. pombe Homologue of Ku80 Is Involved in DNA Repair-- In S. pombe, the Ku70 homologue has been reported (22, 26), whereas the Ku80 homologue remains to be identified. To investigate whether Ku plays a role in telomere maintenance in fission yeast, we searched for the candidate Ku80-homologue gene in the Sanger Institute S. pombe genome data base. By using the BLASTP program, we identified ORF SPBC543.03C (hereafter referred to as pku80+) that encodes a protein showing 20 and 16% identities to human Ku80 and budding yeast Yku80, respectively. The pku80+ gene encodes a protein of 695 amino acids with a calculated molecular mass of 80 kDa.

In mammals and S. cerevisiae, Ku80 forms a complex with Ku70 and is involved in the NHEJ DSB repair pathway (5, 14, 27). We first examined whether Pku80 associates with Pku70 in the yeast two-hybrid system. As shown in Fig. 1A, we found that Pku70 and Pku80 physically interact with each other in this system. We next investigated whether pku80+ is involved in NHEJ, as reported in other organisms, by using the in vivo plasmid DSB repair assay (22, 28). pAL19 plasmid was linearized and transformed into strains containing the leu1-32 mutation (Fig. 1B). In this assay, the DNA sequence around the linearized site bears no homology with any S. pombe genomic sequence. Therefore, the linearized DNA is not integrated into the S. pombe genome by HR and is repaired solely by NHEJ. The deletion of pku80+ caused an ~20-fold decrease in NHEJ efficiency compared with the wild-type strain, and this reduction in the pku80 strain was similar to that found in the pku70 strain (22, 26). Taken together, we found that Pku80 associates with Pku70 and is involved in NHEJ in fission yeast. We therefore conclude that Pku80 is the orthologue of Ku80.


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Fig. 1.   Pku70 and Pku80 physically interact and are required for DSB repair. A, binding between Pku70 and Pku80. Protein interaction between Pku70 and Pku80 was detected by yeast two-hybrid assay. beta -Galactosidase activities observed in yeast cells expressing the following proteins in different combinations are shown. GBD, GAL4 DNA binding domain; GAD, GAL4 DNA activation domain; Pku70, full-length Pku70; and Pku80, full-length Pku80. B, Pku70 and Pku80 are individually essential for efficient NHEJ. Wild-type, Delta pku70, or Delta pku80 strain was transformed with an equal amount of circular or linear PvuII-cleaved pAL19. The relative number of transformants obtained with PvuII-linearized pAL19 compared with that with circular pAL19 is shown for each host strain. C, Delta pku70 and Delta pku80 strains show moderate sensitivities to MMS and HU. Cells serially diluted at 1:10 of the indicated strains were plated and spotted on YES plates at 30 °C containing the indicated drugs or treated with UV. Temperature sensitivity was tested at 37 °C.

We next analyzed the role of Pku70 and Pku80 in the repair of chromosomal DNA damage. The pku70, pku80, and wild-type cells were exposed to HU, MMS, ultraviolet, or high temperature stress, and the number of surviving colonies was measured (Fig. 1C). The mutant strain of rad3+, a checkpoint gene essential for repairing a variety of DNA damages (29), was also examined as control. We found that both the pku70 and pku80 cells are sensitive to MMS or HU but not to UV or high temperature. We noticed that the sensitivities of the pku70 and pku80 cells were moderate; minimal numbers of pku70 or pku80 colonies were formed, whereas almost no rad3+ colony was formed in this assay. These data suggest that Pku70 and Pku80 are involved in repairing the DNA damage induced by MMS or HU.

Pku80 Is Involved in Telomere Length Control and in Protection of Telomeres from Subtelomeric Rearrangement-- It has been shown that Ku proteins are involved in telomere metabolism in diverse organisms. In fission yeast, it was reported that Pku70 is involved in telomere length control and suppression of subtelomeric recombination but not in TPE (22, 26). We next examined the role of Pku80 in telomere maintenance. We first analyzed TPE using the strain that has the ura4+ gene inserted within the telomeric region (30). The mutant strain of taz1+ that encodes the fission yeast telomeric DNA-binding protein is known to be defective in TPE (30, 31) and is therefore used as control. When TPE is proficient, the cells do not grow on a plate lacking uracil, because the ura4+ gene is silenced. As shown in Fig. 2A, both the wild-type and pku80 cells, but not the taz1 cells, did not grown on plates lacking uracil (SD-uracil). It was therefore concluded that pku80+ is not required for efficient TPE. Together with the previous study on pku70+ (22), both Pku70 and Pku80 are not necessary for TPE in fission yeast. This result is in contrast to what is known in budding yeast, where both Yku70 and Yku80 play crucial roles in TPE (15, 16, 18).


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Fig. 2.   Pku70 and Pku80 maintain telomere structure. A, proficient TPE in the Delta pku80 strain. Indicated strains carrying the ura4+ marker gene between the telomeric repeats and TAS at the left arm of chromosome II (30) were grown on YES plates and SD plates lacking uracil. Serial dilutions of cells were spotted. B, restriction map at the telomere and TAS of pNSU70 (24), and probes used for Southern analyses. C, telomere shortenings in Delta pku strains. ApaI-digested genomic DNAs from indicated strains were hybridized with telomeric repeats as a probe. Ethidium bromide staining confirmed that the same amount of DNA was loaded for each strain (data not shown). D, TAS rearrangements in Delta pku strains. EcoT22I-digested genomic DNAs from indicated strains were hybridized with the TAS1 probe (left panel). The position of the aberrant band ladder is shown by the bracket. After removal of the TAS1 probe, the same membrane was re-hybridized with the TAS2 probe (right panel). E, tandem amplification of TAS in Delta pku80 strains. EcoRI-digested genomic DNAs from indicated strains were hybridized with the TAS1 or telomere probe. The positions of additional amplicons (see text) are shown by the asterisk. As a loading control, the ade6 probe was also hybridized with the same blot.

We next investigated whether Pku80 is involved in telomere length control. Genomic DNAs were digested with ApaI to produce ~300-bp fragments derived from telomeric repeats in wild-type S. pombe (Fig. 2B). We found that the length of ApaI-digested telomeric fragments in the pku80 strain is ~100 bp less than that in the wild-type strain (Fig. 2C). A similar degree of telomere shortening was observed in the pku70 strain, as reported previously (22, 26) and shown in Fig. 2C. It is known that the rad3 strain shows telomere shortening (32, 33). Telomere lengths in the pku70 or pku80 strain were slightly longer than those in the rad3 strain. This result indicates that both Pku70 and Pku80 play a role in telomere length control.

In S. pombe, telomeric repeat sequences are flanked by a conserved sequence called TAS (24). Depending on the strain background, TAS is found in four, five, or all six chromosome ends (24, 26, 34). It was reported that TAS very frequently undergoes recombination in the pku70 strain (26). We examined whether a similar instability of TAS is observed in the pku80 strain. Two probes, TAS1 and TAS2, located within TAS were used as probes in Southern hybridization experiments (26, 34) (Fig. 2B). TAS1 is positioned immediately centromeric to the telomeric repeats, whereas TAS2 is positioned more internally. In the wild-type strain, digestion of total DNA with EcoT22I generated several TAS1-containing fragments, and the restriction pattern remained constant through 160 generations in liquid growth (Fig. 2D, left panel). In contrast, aberrant TAS1-positive bands were frequently observed in the pku70 and pku80 strains. During extended growth, the frequency and length of the aberrant bands were increased, as shown by the appearance of slower migrating bands with higher intensities (Fig. 2D, left panel). Several hypotheses can be proposed for these results. Among them, it is possible that the absence of Pku gave rise to unusual DNA, such as single-stranded DNA, in the subtelomeric region, which was resistant to restriction digestion. The failure of restriction digestion in the subtelomeric region would render the telomere-derived fragments apparently longer than the actual size. However, when TAS2 was used as a probe, no aberrant bands were detected, indicating that the two EcoT22I sites that demarcate TAS2 were efficiently digested by EcoT22I (Fig. 2D, right panel). This result makes the hypothesis unlikely, and we concluded that the aberrant telomeric bands in the pku80 strain were produced most probably by subtelomeric rearrangements, as reported in the pku70 strain (26). These rearrangements appear to occur in a relatively small region close to telomeres, because it was detected by the TAS1 probe, but not by the TAS2 probe.

The TAS1-hybridizing aberrant bands in the pku70 and pku80 strains showed regular spacing, forming a ladder (Fig. 2D, left panel). The simplest interpretation of this observation was that the aberrant bands were produced by tandem amplification of a specific TAS1-containing sequence. The length of this amplicon should correspond to the regular increase of the aberrant band ladder (~0.9 kb). If this interpretation was true, digesting the aberrant bands with restriction site contained by the amplicon only once should produce a single unit of the amplicon in addition to the band derived from the most distal end (presumably containing telomeric repeats that are heterogeneous in size). This prediction turned out to be correct when we digested the genomic DNA from the pku strain with EcoRI, only a single restriction site of which is present around TAS1 in pNSU70 (Fig. 2B). The results shown in Fig. 2E (middle panel) indicate that two types of signals were obtained with the TAS1 probe in EcoRI-digested pku80 DNAs, both of which were also observed in the wild-type strain. Importantly, the intensity of the band at ~0.9 kb was stronger in the pku80 strain at 10 generations (~2.6-fold increase, when normalized by the internal control signal (ade6)) than that in the wild-type strain and further increased at 230 generations (~3.1-fold increase). Moreover, the length of this band is similar to the length increment in the aberrant band ladder. In contrast, the intensities of the smeared bands appearing above the ~0.9-kb band did not change significantly between the wild-type and pku80 strains and between generations. The smeared bands, but not the 0.9-kb bands, also hybridized with the telomeric probe (Fig. 2E, bottom panel), indicating that the smeared bands were derived from terminal telomere-containing fragments. These results strongly suggest that the aberrant subtelomeric bands resulted from tandem amplification of a specific amplicon that represents the ~0.9-kb TAS1-hybridizing band. We also observed two additional TAS1-hybridizing bands with the sizes of ~1.5 kb (Fig. 2E, asterisk). These bands may represent additional classes of amplicon. Together, we conclude that Pku80 plays a role in inhibiting DNA recombination at telomeres, as proposed for Pku70 previously (26), and more specifically in inhibiting tandem amplification of a unique fragment present at distal TAS. We noticed that some aberrant bands, especially with smaller degrees of amplification, were also observed in the wild-type strain, albeit faintly, suggesting the intrinsic nature of the amplicon sequence to undergo amplification even in the wild-type strain. Indeed, it was suggested that some wild-type-derived subtelomeric clones contain two tandem copies of the 0.9-kb region, whereas others contain a single copy (24). Aberrant bands showing different lengths were also observed in the rad3 strain, with the signal increasing with increasing culture time. Because the genetic background of this mutant (and presumably the subtelomeric structures) is different from those of the other strains, we do not know the precise nature of these bands.

Both Pku70 and Pku80 Are Localized at Telomeres in a Taz1-independent Manner-- In a previous study, Pku70 was found to distribute rather homogeneously in the nucleus, without showing particular association with telomeres (22). We also observed a similar result (data not shown). Therefore, it remains unclear whether Pku70 or Pku80 is localized at telomeres or not. To address this question, we constructed strains expressing Pku70 or Pku80 fused with three copies of the HA epitope at the C terminus using its own promoter at the native locus. We confirmed that Pku70-HA and Pku80-HA were functional in terms of telomere length control (data not shown). These proteins were successfully immunoprecipitated using anti-HA antibodies (Fig. 3B). Next, we tested whether Pku70 and Pku80 are bound to telomeres using the ChIP method. Cross-linked chromatin derived from strains producing Pku70-HA or Pku80-HA was immunoprecipitated with anti-HA antibodies. Precipitated DNA was subjected to PCR using three sets of primers amplifying subtelomeric regions, 70TEL, 701, or 702 (Fig. 3A). 70TEL is localized immediately centromeric to telomeric repeats. 701 and 702 are 2.5 and 6.0 kb centromeric to the repeats. As negative control probes, the DNA was amplified using primer sets for the internal genes, act1+ or ade6+. As a negative control strain, the wild-type strain that did not express HA-tagged protein was used. We found that 70TEL, 701, and 702 DNAs were specifically amplified in the precipitated DNA derived from Pku-HA-producing strains (Fig. 3C) but not from the control strain (Fig. 3C). In contrast, act1+ (Fig. 3C) or ade6+ (Fig. 4A) DNA was not detected in the immunoprecipitated DNA. We carefully controlled the size of the immunoprecipitated DNA to less than 1 kb after ultrasonic fragmentation. Accordingly, it was unlikely that the positive detection of 701 and 702 in the precipitated DNA was caused by immunoprecipitation of long DNA contiguous with the terminal telomeric repeats. We therefore concluded that Pku70 and Pku80 are bound with telomeric repeats and sub-telomeric regions at a distance of at least more than 6 kb (the distance between telomeric repeats and TAS2) from the telomeric end.


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Fig. 3.   Pku70 and Pku80 are bound to telomeres. A, schematic presentation of the locations of the 70TEL, 701, and 702 primer sets on pNSU70. B, immunoprecipitation of Pku70-HA and Pku80-HA. Extracts from cells expressing Pku70-HA or Pku80-HA as well as control cells (expressing no tagged protein, Cont.) were immunoprecipitated with anti-HA antibodies. The immunoprecipitates were examined with anti-HA antibodies in the immunoblot. The positions of these proteins and immunoglobulin are shown. C, Pku70-HA and Pku80-HA are bound to TAS in the ChIP assay. ChIP assay was performed with the three subtelomeric primer sets indicated in A, and one for the act1 gene. PCR products from immunoprecipitates (IP) or from whole-cell extracts (WCE) are shown.


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Fig. 4.   Pku70 and Pku80 are bound to telomeres in a Taz1-independent manner. A, the presence of Pku70-HA and Pku80-HA at 70TEL was analyzed in the Delta taz1 strain by the ChIP assay using primer sets for 70TEL and the control internal gene, ade6. B, Taz1 is not required for the localization of Pku70-HA and Pku80-HA at telomeres. Genomic DNAs present in the immunoprecipitates (IP) and in whole-cell extracts (WCE), derived from cells expressing Pku70-HA or Pku80-HA or not expressing a tagged protein (non-tag), were examined for telomeric repeats using dot blot hybridization. After hybridization with telomeric repeats (Telomere), the same membrane was hybridized with the control gene, act1+ (act1). Strains with the wild-type taz1+ (taz1+) or without taz1+ (Delta taz1) were examined in each case. C, Pku70 is delocalized from telomeres in the absence of Pku80. The presence of Pku70 at telomeric repeats was examined as in B, in the presence or absence of the pku80+ gene.

TRF1 and TRF2 are human telomeric DNA-binding proteins and are supposed to be Taz1 homologues (35-37). Specifically, both Taz1 and TRF2 distinguish and protect the natural ends of chromosomes (37, 38). Interestingly, it was reported that human Ku80 and Ku70 directly bind to TRF1 and TRF2, respectively (39, 40), suggesting that the localization of Ku proteins at telomeres is dependent on their interactions with TRF1 or TRF2. We were therefore interested in whether the physical interactions between Pku proteins and telomeres in fission yeast were Taz1-dependent or not. When the taz1+ gene was disrupted in the Pku70-HA- or Pku80-HA-expressing strains, the amount of 70TEL present in the anti-HA-immunoprecipitated DNA in the ChIP assay was significantly reduced (Fig. 4A). However, this result should be interpreted cautiously, because it is known that the telomeric repeats in the taz1 strain are elongated to ~2.5-4.5 kb (31). Because genomic DNA co-immunoprecipitated with the antibody was fragmented with an average length of 500-1000 bp in this study, it was possible that this apparent failure of detection of telomeric DNA was due to the association of most Pku proteins with the distal telomeric DNA that was more than 1 kb distant from 70TEL. To examine this possibility, DNAs were similarly recovered from immunoprecipitated fractions as in the ChIP assay and blotted onto membrane without PCR amplification. The blot was then probed with telomeric DNA (not 70TEL DNA). As shown in Fig. 4B, strong telomeric signals were detected in the immunoprecipitated DNA derived from the Pku70-HA- or Pku80-HA-expressing strains but not from the strains that did not express HA proteins. In all cases, the internally positioned control DNA act1 was detected not in the immunoprecipitated DNA but in the whole-cell extracts at nearly equal levels. This result indicates that the immunoprecipitates were specifically obtained and that almost equal amounts of genomic DNA were examined among different samples. Consistently large amounts of telomeric DNA were observed in the whole-cell extracts derived from the taz1 strains, compared with those in the wild-type strains, in agreement with the longer telomeres in the taz1 strain. This result suggests that the experiments were performed under semi-quantitative conditions. Under these conditions, we found that similar amounts of telomeric DNA were detected in the immunoprecipitated DNAs from the Pku-HA-expressing wild-type and taz1 strains, indicating that Pku70 and Pku80 associate with telomeric DNA in a similar manner irrespective of the presence of Taz1.

We next examined whether Pku70 is bound to telomeric DNA in the absence of Pku80. When pku80+ was disrupted, the amounts of telomeric DNA were undetectable in the immunoprecipitates obtained by the addition of anti-HA antibodies from the Pku70-HA-expressing strain (Fig. 4C). We found that the abundance of Pku70 was reduced to ~1/3 in the pku80 strain compared with that in the wild-type strain (data not shown). Because it is unlikely that this reduction of the total Pku70 caused the complete loss of telomeric signals in the pku80-derived immunoprecipitates, it was suggested that the association of Pku proteins with telomeric repeats depends on the Pku70-Pku80 heterodimer formation.

Telomeric DNA Ends Are Essential for Telomere Binding of Pku80-- Previously, we and others (33, 34) reported that fission yeast survives in the absence of telomeric repeats by rendering all three chromosomes circular. All the telomeric repeats and at least several kilobases of the subtelomeric regions are lost in these circular chromosomes. Therefore, 70TEL is lost at its native loci in the circular chromosomes. We integrated telomeric repeats and the TAS sequence (including the 70TEL sequence) into the ade6 locus of the circular chromosomes in cells deleted of the trt1+ gene that encodes the fission yeast telomerase catalytic component. The ura4+ reporter gene was inserted into the ectopically integrated TAS sequence (Fig. 5A). When Taz1-HA was expressed from its endogenous locus, we found that Taz1-HA is bound to the internal telomeric repeats in the ChIP assay using the 70TEL primers (Fig. 5B, lane 5). Indeed, the ura4+ gene was transcriptionally repressed in a Taz1-dependent manner, suggesting that the internal Taz1 is functional in terms of heterochromatin formation.2 When Pku80-HA was expressed, we found that Pku80-HA is not bound to telomeric repeats in circular chromosomes (Fig. 5B, lane 3), whereas it is bound to native telomeric repeats in linear chromosomes (Fig. 5B, lane 1). We confirmed that Pku80-HA was equally present in both cell extracts (data not shown). Taken together, we concluded that in fission yeast, Ku proteins associate with telomeric DNA in a manner dependent on the physical DNA ends and not on the telomeric DNA-binding protein, Taz1 (Fig. 5C).


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Fig. 5.   Telomeric DNA end is essential for Pku localization at telomeres. A, circular chromosomes used in this study. Upon the circularization in the Delta trt1 strain, all telomeric repeats and at least several kilobases of the subtelomeric region (including 70TEL) were lost from their native loci. A cassette containing TAS- ura4+-telomeric repeats was inserted into the ade6 locus of chromosome III. B, Taz1 is bound to the internal telomeric repeats, but Pku80 is not. The interaction of the indicated proteins with the ectopically integrated 70TEL was examined by ChIP assay, as in Fig. 4A. MS816 (all three chromosomes circularized, expressing no tagged protein; Cont.), isogenic pku80-3HA, pku80-3HA Delta taz1, or taz1-3HA strain was analyzed. L, linear chromosomes; C, circular chromosomes. C, model for Pku70 and Pku80 binding to telomeres. Pku70 and Pku80 are bound to telomeric ends only in the form of a heterodimer and independently of Taz1. In the Delta taz1 strain, the Pku70-Pku80 heterodimer binds to telomeric ends that are far from the subtelomeric region, leading to the inability of detection by ChIP using 70TEL primers.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this study, we showed that the fission yeast Ku80 orthologue, Pku80, is involved in DNA repair, telomere length control, and inhibition of the tandem amplification of subtelomeric regions. We also demonstrated that Pku70 and Pku80 physically associate with telomeric DNA using the ChIP assay. Unexpectedly, the telomeric DNA-binding protein, Taz1, is not required for the localization of Pku proteins at telomeres. Furthermore, Pku80 is not bound to internal telomeric repeats that are ectopically integrated in circular chromosomes. Finally, it was suggested that Pku proteins associate with telomeres as a heterodimer. These results indicate that the physical end of DNA at telomeres is required for the localization of Ku proteins at telomeres in fission yeast.

Roles of Pku Proteins in DNA Repair-- This study, together with previous studies (22, 26), demonstrated that both pku70+ and pku80+ are necessary for the efficient NHEJ measured by the in vivo plasmid DSB repair assay. However, it was reported in a previous study that the pku70 and pku80 strains are not sensitive to MMS or to bleomycin and gamma -irradiation, both of which induce DSB (22). This result was interpreted as follows. In fission yeast, DSB is predominantly repaired by HR and not by NHEJ, because the G2 phase, in which HR is proficient because of the presence of sister chromatids, is longer than the other phases in this organism. In this study, we found that both the pku70 and pku80 strains show mild but significant sensitivities to MMS and HU (Fig. 1). In the previous study (22), low doses of MMS were used compared with those used in this study. We also observed that the pku70 or pku80 strain did not show any sensitivity to low doses of MMS, a finding consistent with the previous study. Therefore, the pku70 and pku80 strains are sensitive to relatively high doses of MMS.

MMS is an alkylating agent that methylates nitrogen ring atoms on DNA (41). It is believed that DSBs occur at the alkylated bases in the S phase (42, 43). HU inhibits the elongation step of DNA replication by depleting the deoxyribonucleotide pool through inactivation of ribonucleotide reductase. Because Pku proteins are important for NHEJ that repairs DSBs, it is possible that the pku70 and pku80 strains are sensitive to MMS and HU because of their inability to repair DSBs secondarily produced in the S phase from the primary lesions. This interpretation suggests that NHEJ may be particularly important in the S phase in the normal cell cycle of fission yeast. A similar notion was recently reported in vertebrate cells (44, 45).

Roles of Pku Proteins in Telomere Metabolism-- It is well established that Ku proteins are involved in telomere metabolism in several model organisms. In budding yeast, mutant strains defective in the Ku-encoding gene (YKU70/HDF1 or YKU80/HDF2) show telomere shortening, constitutively overhanging telomeric 3'-ends, defective TPE, defective clustering of telomeres in the nucleus, and possibly hyper-recombination of telomeric sequences (6, 13-19). In mammals, Ku proteins are bound to telomeres, thereby protecting the telomeres from end-to-end fusion (7, 8, 39, 46-48). However, the single-stranded 3'-tail does not appear to be altered in Ku-defective cells (47, 48). It is controversial whether telomeric DNA is shortened in Ku-defective cells or not (47, 48). Taken together, Ku proteins are conservatively involved in telomere maintenance from yeast to human, although their precise roles may differ among organisms. Interestingly, although XRCC4 and DNA ligase IV proteins are as essential for NHEJ as Ku proteins, telomere shortening is not observed in XRCC4- or DNA ligase IV-defective cells in all cases examined in budding yeast, fission yeast, and mammals (22, 48-50). Furthermore, telomere fusions were frequently observed in both Ku70-defective ES cells and XRCC4-defective ES cells, but the instability was more profound in the former cells (48). Therefore, it was suggested that Ku plays dual roles in telomere maintenance as follows: first, as a component of the NHEJ pathway, and second, as a component independent of NHEJ.

In this study, we found that the fission yeast Ku80 protein is bound to telomeric DNA and is involved in telomere length regulation but apparently not in TPE, a finding distinct from budding yeast Ku proteins. These properties of Pku80 agree with those of Pku70 that were reported previously (22, 26), and together they underscore the conserved functions of Ku proteins in telomere maintenance. Both Pku70 (26) and Pku80 (this study) were suggested to inhibit DNA rearrangements in the fission yeast subtelomeric regions. Similar observations were also reported in budding yeast (17). In this study, we found that Pku70 or Pku80 is not bound to subtelomeric regions in the taz1 strain. If the Pku proteins are involved in inhibiting DNA recombination, one would expect that the subtelomeric region and the telomeric DNA close to the subtelomeric region in the taz1 strain would be highly unstable because of the lack of Pku proteins. This is indeed the case, because it was suggested previously that subtelomeric sequences and telomeric DNA are highly amplified in the taz1 background (34).

ChIP analyses suggested that Pku70 and Pku80 are bound to telomeric DNA and subtelomeric regions in the wild-type strains. The interaction between the telomeric repeats and Pku proteins was further supported by analyzing the taz1 strain. In the taz1 strain, which contains ~2.5-4.5 kb of telomeric repeats, we observed the interaction of Pku proteins with telomeric repeats in blot hybridization assay but not with the subtelomeric 70TEL sequence in the ChIP assay. This result is consistent with the hypothesis that the primary target of Pku proteins is not subtelomeric sequences but telomeric repeats. This hypothesis also agrees with our observation that the Pku proteins interact with telomeres independently of Taz1 and appear to be targeted at the DNA ends. Therefore, we propose that Pku proteins are primarily bound to the DNA ends at telomeres. We, however, do not exclude the possibility that Pku is recruited to the telomeric ends by other telomere proteins, such as Pot1 (51).

In the ChIP assay, we demonstrated that Pku70 and Pku80 interact with subtelomeric sequences ~6 kb distant from telomeric repeats. If Pku proteins are primarily bound to telomeric ends, how could this interaction happen? It was demonstrated recently (52) that human Ku heterodimers do not bind to DNA cooperatively in vitro. Therefore, it appears unlikely that Pku proteins are first nucleated at the telomeric ends and then spread over non-telomeric DNA cooperatively by their own. However, it is possible that this spreading could occur with the aid of interactions with other proteins. Alternatively, it is possible that the apparent interaction between Pku proteins and subtelomeric sequences is caused by the folding back of telomere-associated Pku proteins onto the subtelomeric region, as proposed in budding yeast (53, 54). Finally, it was recognized that once Ku proteins bind to the telomeric DNA ends, they can slide on DNA internally (55-57). A molecular explanation of this sliding model was recently given by determining the three-dimensional structure of the Ku heterodimer-DNA complex (58). According to the structure, the Ku heterodimer forms a ring-like structure, thereby providing a hole through which DNA passes. It is therefore possible that the Ku heterodimer binds to the telomeric ends and then slides on DNA into the subtelomeric region as embracing DNA.

The finding that the Pku proteins are bound to telomeres independently of Taz1 implies a link between Taz1 and Pku. It was reported that end-to-end fusion frequently occurs in nitrogen-starved taz1 haploid cells. The frequency of the end-to-end fusion was significantly reduced when additional deletion of pku70+ or lig4+ was generated (37). Because Pku proteins remain associated with telomeres when taz1+ is absent (this study), it is possible that Pku-bound telomeric DNA is recognized as a non-physiological DNA end in the absence of Taz1. In this sense, the protective role of Taz1 at telomeres (37) may be to sequester the telomeric Pku proteins from the NHEJ pathway.

    ACKNOWLEDGEMENTS

We thank A. Nabetani and M. Shimoseki for useful discussions. We also thank Dr. R. Allshire for the FY1862 strain and Dr. A. Carr for the pAL19 plasmid. The excellent secretarial work of F. Nakayama, A. Orii, K. Saito, K. Yokoyama, and A. Katayama is also acknowledged.

    FOOTNOTES

* This work was supported by a Center of Excellence grant (to F. I.), a grant-in-aid for Cancer Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (to F. I.), a Health and Labor Sciences Research grant from the Ministry of Health, Labor and Welfare, Japan (to F. I.), and a grant-in-aid from the Ministry of Education, Culture, Sports, Science and Technology, Japan (to J. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 81-75-753-4195; Fax: 81-75-753-4197; E-mail: fishikaw@lif.kyoto-u.ac.jp.

Published, JBC Papers in Press, November 6, 2002, DOI 10.1074/jbc.M208813200

2 M. Sadaie and F. Ishikawa, unpublished data.

    ABBREVIATIONS

The abbreviations used are: DSBs, double strand breaks; NHEJ, non-homologous end-joining; MMS, methyl methanesulfonate; HU, hydroxyurea; TPE, telomere position effect; ChIP, chromatin immunoprecipitation; DNA-PK, DNA-dependent protein kinase; ORF, open reading frame; HA, hemagglutinin; TAS, telomere-associated sequence.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Sandell, L. L., and Zakian, V. A. (1993) Cell 75, 729-739[Medline] [Order article via Infotrieve]
2. Haber, J. E. (2000) Trends Genet. 16, 259-264[CrossRef][Medline] [Order article via Infotrieve]
3. Moshous, D., Callebaut, I., de Chasseval, R., Corneo, B., Cavazzana-Calvo, M., Le, Deist, F., Tezcan, I., Sanal, O., Bertrand, Y., Philippe, N., Fischer, A., and de Villartay, J. P. (2001) Cell 105, 177-186[CrossRef][Medline] [Order article via Infotrieve]
4. Ma, Y., Pannicke, U., Schwarz, K., and Lieber, R. M. (2002) Cell 108, 781-794[Medline] [Order article via Infotrieve]
5. Smith, G. C., and Jackson, S. P. (1999) Genes Dev. 13, 916-934[Free Full Text]
6. Gravel, S., Larrivee, M., Labrecque, P., and Wellinger, R. J. (1998) Science 280, 741-744[Abstract/Free Full Text]
7. Bianchi, A., and de Lange, T. (1999) J. Biol. Chem. 274, 21223-21227[Abstract/Free Full Text]
8. Hsu, H. L., Gilley, D., Blackburn, E. H., and Chen, D. J. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 12454-12458[Abstract/Free Full Text]
9. Cooper, J. P. (2000) Curr. Opin. Genet. & Dev. 10, 169-177[CrossRef][Medline] [Order article via Infotrieve]
10. de Lange, T. (2002) Oncogene 21, 532-540[CrossRef][Medline] [Order article via Infotrieve]
11. Funabiki, H., Hagan, I., Uzawa, S., and Yanagida, M. (1993) J. Cell Biol. 121, 961-976[Abstract]
12. Gotta, M., Laroche, T., Formenton, A., Maillet, L., Scherthan, H., and Gasser, S. M. (1996) J. Cell Biol. 134, 1349-1363[Abstract]
13. Porter, S. E., Greenwell, P. W., Ritchie, K. B., and Petes, T. D. (1996) Nucleic Acids Res. 24, 582-585[Abstract/Free Full Text]
14. Boulton, S. J., and Jackson, S. P. (1996) Nucleic Acids Res. 24, 4639-4648[Abstract/Free Full Text]
15. Laroche, T., Martin, S. G., Gotta, M., Gorham, H. C., Pryde, F. E., Louis, E. J., and Gasser, S. M. (1998) Curr. Biol. 8, 653-656[Medline] [Order article via Infotrieve]
16. Nugent, C. I., Bosco, G., Ross, L. O., Evans, S. K., Salinger, A. P., Moore, J. K., Haber, J. E., and Lundblad, V. (1998) Curr. Biol. 8, 657-660[Medline] [Order article via Infotrieve]
17. Polotnianka, R. M., Li, J., and Lustig, A. J. (1998) Curr. Biol. 8, 831-834[Medline] [Order article via Infotrieve]
18. Boulton, S. J., and Jackson, S. P. (1998) EMBO J. 17, 1819-1828[Abstract/Free Full Text]
19. Galy, V., Olivo-Marin, J. C., Scherthan, H., Doye, V., Rascalou, N., and Nehrbass, U. (2000) Nature 403, 108-112[CrossRef][Medline] [Order article via Infotrieve]
20. Alfa, C., Fantes, P., Hyams, J., McLeod, M., and Warbrick, E. (1993) Experiments with Fission Yeast , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
21. Kanoh, J., and Ishikawa, F. (2001) Curr. Biol. 11, 1624-1630[CrossRef][Medline] [Order article via Infotrieve]
22. Manolis, K. G., Nimmo, E. R., Hartsuiker, E., Carr, A. M., Jeggo, P. A., and Allshire, R. C. (2001) EMBO J. 20, 210-221[Abstract/Free Full Text]
23. Kaiser, C., Michaelis, S., and Mitchell, A. E. (1994) Methods in Yeast Genetics , pp. 137-147, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
24. Sugawara, N. (1988) DNA Sequences at the Telomeres of the Fission Yeast S. pombePh.D. thesis , Harvard University, Cambridge, MA
25. Saitoh, S., Takahashi, K., and Yanagida, M. (1997) Cell 90, 131-143[Medline] [Order article via Infotrieve]
26. Baumann, P., and Cech, T. R. (2000) Mol. Biol. Cell 11, 3265-3275[Abstract/Free Full Text]
27. Milne, G. T., Jin, S., Shannon, K. B., and Weaver, D. T. (1996) Mol. Cell. Biol. 16, 4189-4198[Abstract]
28. Wilson, S., Warr, N., Taylor, D. L., and Watts, F. Z. (1999) Nucleic Acids Res. 27, 2655-2661[Abstract/Free Full Text]
29. Bentley, N. J., Holtzman, D. A., Flaggs, G., Keegan, K. S., DeMaggio, A., Ford, J. C., Hoekstra, M., and Carr, A. M. (1996) EMBO J. 15, 6641-6651[Abstract]
30. Nimmo, E. R., Pidoux, A. L., Perry, P. E., and Allshire, R. C. (1998) Nature 392, 825-828[CrossRef][Medline] [Order article via Infotrieve]
31. Cooper, J. P., Nimmo, E. R., Allshire, R. C., and Cech, T. R. (1997) Nature 385, 744-747[CrossRef][Medline] [Order article via Infotrieve]
32. Dahlen, M., Olsson, T., Kanter-Smoler, G., Ramne, A., and Sunnerhagen, P. (1998) Mol. Biol. Cell 9, 611-621[Abstract/Free Full Text]
33. Naito, T., Matsuura, A., and Ishikawa, F. (1998) Nat. Genet. 20, 203-206[CrossRef][Medline] [Order article via Infotrieve]
34. Nakamura, T. M., Cooper, J. P., and Cech, T. R. (1998) Science 282, 493-496[Abstract/Free Full Text]
35. Broccoli, D., Smogorzewska, A., Chong, L., and de Lange, T. (1997) Nat. Genet. 17, 231-235[Medline] [Order article via Infotrieve]
36. Li, B., Oestreich, S., and de Lange, T. (2000) Cell 101, 471-483[Medline] [Order article via Infotrieve]
37. Ferreira, M. G., and Cooper, J. P. (2001) Mol. Cell 7, 55-63[Medline] [Order article via Infotrieve]
38. van Steensel, B., Smogorzewska, A., and de Lange, T. (1998) Cell 92, 401-413[Medline] [Order article via Infotrieve]
39. Hsu, H. L., Gilley, D., Galande, S. A., Hande, M. P., Allen, B., Kim, S. H., Li, G. C., Campisi, J., Kohwi-Shigematsu, T., and Chen, D. J. (2000) Genes Dev. 14, 2807-2812[Abstract/Free Full Text]
40. Song, K., Jung, D., Jung, Y., Lee, S. G., and Lee, I. (2000) FEBS Lett. 481, 81-85[CrossRef][Medline] [Order article via Infotrieve]
41. Beranek, D. T. (1990) Mutat. Res. 231, 11-30[Medline] [Order article via Infotrieve]
42. Chlebowicz, E., and Jachymczyk, W. J. (1979) Mol. Gen. Genet. 167, 279-286[Medline] [Order article via Infotrieve]
43. Schwartz, J. L. (1989) Mutat. Res. 216, 111-118[CrossRef][Medline] [Order article via Infotrieve]
44. Takata, M., Sasaki, M. S., Sonoda, E., Morrison, C., Hashimoto, M., Utsumi, H., Yamaguchi-Iwai, Y., Shinohara, A., and Takeda, S. (1998) EMBO J. 17, 5497-5508[Abstract/Free Full Text]
45. Saintigny, Y., Delacote, F., Vares, G., Petitot, F., Lambert, S., Averbeck, D., and Lopez, B. S. (2001) EMBO J. 20, 3861-3870[Abstract/Free Full Text]
46. Bailey, S. M., Meyne, J., Chen, D. J., Kurimasa, A., Li, G. C., Lehnert, B. E., and Goodwin, E. H. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 14899-14904[Abstract/Free Full Text]
47. Samper, E., Goytisolo, F. A., Slijepcevic, P., van Buul, P. P., and Blasco, M. A. (2000) EMBO Rep. 1, 244-252[Abstract/Free Full Text]
48. d'Adda di Fagagna, F., Hande, M. P., Tong, W. M., Roth, D., Lansdorp, P. M., Wang, Z. Q., and Jackson, S. P. (2001) Curr. Biol. 11, 1192-1196[CrossRef][Medline] [Order article via Infotrieve]
49. Teo, S. H., and Jackson, S. P. (1997) EMBO J. 16, 4788-4795[Abstract/Free Full Text]
50. Herrmann, G., Lindahl, T., and Schar, P. (1998) EMBO J. 17, 4188-4198[Abstract/Free Full Text]
51. Baumann, P., and Cech, T. R. (2001) Science 292, 1171-1175[Abstract/Free Full Text]
52. Arosio, D., Cui, S., Ortega, C., Chovanec, M., Di, Marco, S., Baldini, G., Falaschi, A., and Vindigni, A. (2002) J. Biol. Chem. 277, 9741-9748[Abstract/Free Full Text]
53. Strahl-Bolsinger, S., Hecht, A., Luo, K., and Grunstein, M. (1997) Genes Dev. 11, 83-93[Abstract]
54. de Bruin, D., Zaman, Z., Liberatore, R. A., and Ptashne, M. (2001) Nature 409, 109-113[CrossRef][Medline] [Order article via Infotrieve]
55. de Vries, E., van Driel, W., Bergsma, W. G., Arnberg, A. C., and van der Vliet, P. C. (1989) J. Mol. Biol. 208, 65-78[Medline] [Order article via Infotrieve]
56. Paillard, S., and Strauss, F. (1991) Nucleic Acids Res. 19, 5619-5624[Abstract]
57. Bliss, T. M., and Lane, D. P. (1997) J. Biol. Chem. 272, 5765-5773[Abstract/Free Full Text]
58. Walker, J. R., Corpina, R. A., and Goldberg, J. (2001) Nature 412, 607-614[CrossRef][Medline] [Order article via Infotrieve]


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