From the 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
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ABSTRACT |
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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.
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.
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).
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( 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, 112gal4 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.
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.
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).
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.
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).
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
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.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Yeast strains used in this study
). 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.
gal80
LYS2::GAL-HIS3 URA3::GAL-lacZ cyhr) was
transformed with each plasmid and assayed for
-galactosidase activity according to the manufacturer's instructions.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (25K):
[in a new window]
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. -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,
pku70, or
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,
pku70 and
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.
View larger version (61K):
[in a new window]
Fig. 2.
Pku70 and Pku80 maintain telomere structure.
A, proficient TPE in the 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
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
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
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.
View larger version (27K):
[in a new window]
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.
View larger version (22K):
[in a new window]
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 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+ (
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.
View larger version (23K):
[in a new window]
Fig. 5.
Telomeric DNA end is essential for Pku
localization at telomeres. A, circular chromosomes used
in this study. Upon the circularization in the 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
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
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
-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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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* 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.
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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.
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