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Telomere Binding of Checkpoint Sensor and DNA Repair Proteins Contributes to Maintenance of Functional Fission Yeast Telomeres
Toru M. Nakamura1,a, Bettina A. Moser1,a, and Paul Russellaa Departments of Molecular Biology and Cell Biology, The Scripps Research Institute, La Jolla, California 92037
Corresponding author: Toru M. Nakamura, MB3, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037., nakamut{at}scripps.edu (E-mail)
Communicating editor: G. SMITH
| ABSTRACT |
|---|
Telomeres, the ends of linear chromosomes, are DNA double-strand ends that do not trigger a cell cycle arrest and yet require checkpoint and DNA repair proteins for maintenance. Genetic and biochemical studies in the fission yeast Schizosaccharomyces pombe were undertaken to understand how checkpoint and DNA repair proteins contribute to telomere maintenance. On the basis of telomere lengths of mutant combinations of various checkpoint-related proteins (Rad1, Rad3, Rad9, Rad17, Rad26, Hus1, Crb2, Chk1, Cds1), Tel1, a telomere-binding protein (Taz1), and DNA repair proteins (Ku70, Rad32), we conclude that Rad3/Rad26 and Tel1/Rad32 represent two pathways required to maintain telomeres and prevent chromosome circularization. Rad1/Rad9/Hus1/Rad17 and Ku70 are two additional epistasis groups, which act in the Rad3/Rad26 pathway. However, Rad3/Rad26 must have additional target(s), as cells lacking Tel1/Rad32, Rad1/Rad9/Hus1/Rad17, and Ku70 groups did not circularize chromosomes. Cells lacking Rad3/Rad26 and Tel1/Rad32 senesced faster than a telomerase trt1
mutant, suggesting that these pathways may contribute to telomere protection. Deletion of taz1 did not suppress chromosome circularization in cells lacking Rad3/Rad26 and Tel1/Rad32, also suggesting that two pathways protect telomeres. Chromatin immunoprecipitation analyses found that Rad3, Rad1, Rad9, Hus1, Rad17, Rad32, and Ku70 associate with telomeres. Thus, checkpoint sensor and DNA repair proteins contribute to telomere maintenance and protection through their association with telomeres.
CHECKPOINT and DNA repair pathways are crucial to the progression of the normal cell cycle. Without them, cells cannot maintain a stable genome, and genetic instability can lead to cell death, cancer, and other genetic disorders (![]()
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Telomeres, the natural ends of linear chromosomes, are maintained by the specialized reverse transcriptase called telomerase. Many proteins bind telomeric DNA and protect it from degradation and recombination. Telomeres pose special challenges to the DNA repair machinery and checkpoint proteins because these DNA ends must be maintained, unlike other internal DSBs, which must be rejoined (![]()
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Using the fission yeast S. pombe as a model system, we wished to understand how this apparent alteration in the checkpoint signaling pathways at telomeres is achieved to allow the DNA structure checkpoint proteins to recognize telomeres as the unique DNA ends that should not be repaired. Advantages of the fission yeast system include well-characterized DNA damage responses with high structural and functional conservation to the mammalian system; amenability to genetic, biochemical, and cytological studies; and a small number of telomeres per cell. In addition, the ability of fission yeast to bypass the need for a functional telomere maintenance mechanism by circularizing all chromosomes (![]()
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The DNA structure checkpoint responses in S. pombe require a group of six "checkpoint Rad proteins" (Rad1, Rad3, Rad9, Rad17, Rad26, and Hus1), which are thought to function as sensors of DNA replication arrest and DNA damage (![]()
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In addition to Rad3, S. pombe cells have another protein kinase related to ATR and ATM called Tel1, and the phenotype of rad3
tel1
illustrates the importance of ATR and ATM family proteins in telomere maintenance. The double-mutant cells have dramatically shortened telomeres, and the cells often lose their telomeres completely and circularize all chromosomes (![]()
; ![]()
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Studies of telomere length in cells carrying mutations in the DNA damage checkpoint downstream signal transducer proteins support a more direct role for the checkpoint Rad proteins in telomere length maintenance (![]()
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To define the roles that checkpoint and DNA repair proteins play in telomere maintenance, we undertook epistasis analysis of various checkpoint and DNA repair mutants on the basis of steady-state telomere length in a series of multiple mutant combinations. From these studies, we conclude that Rad3/Rad26 and Tel1/Rad32 represent two independent functional pathways required for the maintenance of stable telomeres (Rad32 is an ortholog of the S. cerevisiae and mammalian Mre11 proteins). We also compared senescence rates upon telomerase trt1 deletion in various checkpoint mutant backgrounds and conclude that Rad3/Rad26 and Tel1/Rad32 pathways must also be important for functions other than the recruitment of telomerase to telomeres. In addition, we show that damage-induced phosphorylation of Rad32 is independent of both Rad3 and Tel1 kinases, and we thus implicate other unidentified kinase(s) in phosphorylation of Rad32. We also demonstrate specific association of checkpoint sensor and DNA repair proteins to telomeres by chromatin immunoprecipitation (ChIP) analyses. Through these studies we conclude that checkpoint sensor and DNA repair proteins contribute to maintenance and protection of telomeres through their binding to telomeres.
| MATERIALS AND METHODS |
|---|
Yeast strains and general methods:
The fission yeast strains used in this study were constructed by standard techniques (![]()
Mutations were previously described for rad1
(rad1::ura4+; ![]()
(rad9::ura4+; ![]()
(hus1::LEU2; ![]()
(rad17::ura4+; ![]()
(rad3::ura4+; ![]()
(rad26::ura4+; ![]()
(crb2::ura4+; ![]()
(chk1::ura4+; ![]()
(cds1::ura4+; ![]()
(trt1-D2::his3+; ![]()
![]()
![]()
For taz1
, a PCR-based method (![]()
![]()
A PCR-based method (![]()
(rad32-D1::kanMX4), using rad32-KO1 and rad32-KO2 primers; pku70
(pku70-D1::kanMX4), using pku70-KO1 and pku70-KO2 primers; and rad3
(rad3-D2::LEU2), using rad3-LEUT and rad3-LEUB primers.
For tel1
(tel1-D1::kanMX4), the carboxy-terminal untranslated region was amplified by PCR (tel1-T1 and tel1-B2 primers) and then cloned into pBluescript II SK(+) (Stratagene, La Jolla, CA) as a HindIII-XhoI fragment. The amino-terminal untranslated region was subsequently amplified (tel1-T3 and tel1-B4 primers) and cloned into the same plasmid as the SacII-XbaI fragment. This plasmid was then digested with BamHI and EcoRI to clone the BamHI-EcoRI kanMX4 fragment from the pFA6a-kanMX4 plasmid (![]()
(te11-D2::LEU2) was created by a PCR-based method (![]()
A PCR-based method (![]()
![]()
HA-rad3 cells express the amino-terminally 3HA-tagged Rad3 fusion protein from the endogenous rad3+ promoter. It was created by transforming a strain with an integrated ura4+ marker 5' adjacent to the rad3+ gene with the plasmid carrying the 3HA-rad3 fusion construct and then selecting for 5-fluoroorotic acid (5-FOA)-resistant cells (![]()
Pulsed-field gel electrophoresis:
For pulsed-field gel electrophoresis (PFGE), cells were suspended and lysed in agarose plugs as follows: Cells were washed twice in SP1 [50 mM citrate-phosphate (pH 5.6), 40 mM EDTA, 1.2 M sorbitol] and then incubated for 23 hr at 37° in SP1 with 0.6 mg/ml Zymolyase-100T (ICN Biomedicals). The cells were pelleted and resuspended at 67 x 108 cells per ml in TSE [10 mM Tris-HCl (pH 7.5), 0.9 M sorbitol, 45 mM EDTA]. The cell suspension was warmed to 42°, and 11.5 volume of 1% low-melting agarose (Bio-Rad, Richmond, CA) in TSE was added. Aliquots were dispensed into plug molds and allowed to solidify. The gelled plugs were incubated at 55°, first for
90 min in 0.25 M EDTA, 50 mM Tris-HCl (pH 7.5), and 1% SDS and then for 48 hr in 1% lauryl sarcosine, 0.5 M EDTA (pH 9.5), and 1 mg/ml proteinase K. Plugs were washed three times in Tris-EDTA and stored at 4° in Tris-EDTA. For NotI-digested PFGE, plugs were preequilibrated 23 hr at 37° in NEB3 buffer [10 mM NaCl, 5 mM Tris-HCl, 1 mM MgCl2, 0.1 mM dithiothreitol (pH 7.9 at 25°)] plus 100 µg/ml BSA and then digested with NotI restriction endonuclease at 37° overnight. Probes specific for telomeric NotI fragments (C, I, L, and M) were created as previously described (![]()
Liquid culture growth curve:
Heterozygous diploid strains were sporulated and the resulting tetrads were dissected and germinated on yeast extract medium-supplemented (YES) plates (![]()
Immunopurification and Western blot analysis:
Whole-cell extracts from rad32-TAP-tagged (![]()
protein phosphatase (New England Biolabs, Beverly, MA) was used to perform phosphatase treatment.
ChIP assays:
ChIP assays were performed as described (![]()
![]()
| RESULTS |
|---|
Checkpoint sensor mutants all have shorter telomeres:
Previous studies in S. pombe reported that rad1, rad17, rad3, and rad26 mutant cells have shorter telomeres, while rad9 and hus1 mutant cells have normal telomere length (![]()
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In contrast to previous reports, we found that rad1
, rad9
, and hus1
strains all had shorter telomeres (Fig 1A, lanes 24; Fig 2A). In addition, the rad17
mutant strain had shorter telomere length and the extent of shortening was similar to that of rad1
, rad9
, and hus1
strains (Fig 1A and Fig 2A). rad3
and rad26
cells had the shortest telomere lengths among the six checkpoint sensor mutants (Fig 1A, lanes 7 and 8; Fig 2A). Mutations in other checkpoint-related proteins (crb2
, chk1
, cds1
, and chk1
cds1
) that are thought to function downstream of the six checkpoint sensor proteins had little or no effect on telomere length (Fig 1A, lanes 913; Fig 2A).
|
|
We further analyzed telomere length in various double-mutant combinations among checkpoint sensor proteins (Fig 1B) and found that rad1
hus1
, rad9
hus1
, and rad17
hus1
mutant combinations have the same telomere length as the single mutants (Fig 1B, lanes 28; Fig 2B). These results suggest that rad1
, rad9
, hus1
, and rad17
function in a single pathway for telomere maintenance, consistent with their function in the checkpoint response (![]()
rad26
double mutant had the same telomere length as the single mutants (Fig 1B, lanes 911; Fig 2B; ![]()
rad1
, rad3
rad9
, rad3
hus1
, rad3
rad17
, and rad26
hus1
all showed no additional telomere shortening compared to rad3
or rad26
single mutants (Fig 1B, lanes 1017; Fig 2B). These results thus suggest that Rad1, Rad9, Hus1, Rad17, Rad3, and Rad26 contribute to telomere maintenance in a single pathway, but that Rad3 and Rad26 are more important in maintenance of telomeres in fission yeast.
Tel1 and Rad32 function in the same pathway for telomere maintenance:
We next examined how checkpoint proteins interact with Tel1 and Rad32 proteins in S. pombe. Rad32 is an ortholog of the S. cerevisiae and mammalian Mre11 proteins. Studies in S. cerevisiae have shown that the Mre11-Rad50-Xrs2 complex and Tel1 function in a single pathway for telomere maintenance (![]()
![]()
We found that tel1
mutant cells had normal telomere length. We observed synergistic loss of telomeres in tel1
rad3
and tel1
rad26
cells (Fig 1C, lanes 7 and 8), in agreement with previous studies (![]()
![]()
rad3
cells was previously reported, using PFGE analysis and microscopic observations (![]()
rad26
cells has not been reported. As shown in Fig 3B, we observed that both tel1
rad3
and tel1
rad26
cells have fused C, I, L, and M NotI telomeric fragments to generate I + L and C + M bands that are specific to circularized chromosome I and chromosome II, respectively (lanes 7 and 8), like trt1
telomerase mutant survivors (lane 11; ![]()
, rad9
, hus1
, and rad17
) showed only slight telomere shortening compared to single mutants when combined with tel1
mutation (Fig 1C, lanes 36). Mutants of downstream effectors of the checkpoint pathway (crb2
, chk1
, cds1
, and chk1
cds1
) showed wild-type telomere length even in combination with a tel1
mutation (Fig 1C, lanes 912).
|
Rad32 mutant cells have previously been reported to have shorter than wild-type telomere length in S. pombe (![]()
![]()
mutant (Fig 1D and Fig 2D). Since S. pombe Rad32 and Rad50 are expected to be in a complex analogous to the S. cerevisiae Mre11-Rad50-Xrs2 complex, both mutations might be expected to show similar effects on telomere length. S. pombe rad50
cells have also been reported to have short telomeres (![]()
cells (data not shown), much like in rad32
cells. It was also suggested that rad32
mutation is synthetic lethal with rad3 mutation (![]()
and rad50
strains grow poorly and appear to accumulate DNA damage, as many cells appear to be arrested by the checkpoint.
When the rad32
mutation was combined with the tel1
mutation, we found that the rad32
tel1
double mutant still had normal telomere length (Fig 1D, lanes 14). When the rad32
mutation was combined with rad1
, rad9
, hus1
, or rad17
mutations, double mutants showed only slight shortening of telomere lengths compared to single mutants in rad1
, rad9
, hus1
, or rad17
strains (Fig 1D, lanes 58). Combination of the rad32
mutation with either rad3
or rad26
, on the other hand, caused total loss of the telomere hybridization signal (Fig 1D, lanes 9 and 10). This is due to circularization of chromosomes, as PFGE analysis showed a shift of C, I, L, and M telomeric NotI fragments into two bands corresponding to I + L and C + M bands (Fig 3B, lanes 9 and 10). Combination of the rad32
mutation and mutations of downstream effectors of the checkpoint pathway (crb2
, chk1
, cds1
, and chk1
cds1
) showed wild-type telomere length (Fig 1D, lanes 1116). Therefore, rad32
and tel1
mutations caused identical phenotypes in terms of telomere length in all checkpoint mutant backgrounds we tested. Taken together, these results are consistent with the idea that Tel1 and Rad32 function in the same pathway for telomere maintenance much like S. cerevisiae Tel1 and Mre11-Rad50-Xrs2. The above data also indicate that Rad3/Rad26 and Tel1/Rad32 represent two functional groups required for telomere maintenance in S. pombe.
Interaction between Ku70 and checkpoint proteins:
Next, we tested how telomere length is affected by combining the pku70
mutation with mutations in checkpoint genes tel1
and rad32
. In S. pombe, pku70
makes telomeres shorter and the telomere-associated sequences (TAS) more recombinogenic (![]()
![]()
cells compared to wild-type cells. We also found that double mutants of pku70
and checkpoint sensor protein mutations have the telomere lengths of checkpoint sensor single mutants for rad1
, rad9
, hus1
, rad17
, rad3
, and rad26
and also made telomere length more homogeneous compared to a pku70
strain (Fig 1E, lanes 111; Fig 2C and Fig D). Therefore, mutations in checkpoint sensor genes are epistatic to pku70
in maintenance of telomere length. For combinations of pku70
and downstream protein mutations (crb2
, chk1
, cds1
, and chk1
cds1
), telomere lengths were like that of the pku70
single mutant (Fig 1E, lanes 1217). Telomere lengths in pku70
tel1
and pku70
rad32
cells were also the same as in the pku70
single mutant (Fig 1F, lanes 46; Fig 2D).
We also created triple mutants in which a checkpoint sensor was deleted along with tel1
and pku70
. We hypothesized that the Rad3-Rad26 complex may contribute positively to telomere maintenance both through a pathway involving Ku70 and through another pathway involving the Rad1/Rad9/Hus1/Rad17 proteins since mutations in rad3 and rad26 were found to be epistatic to mutations in rad1, rad9, hus1, rad17, and pku70. If this were true, deletion of both pathways in combination with the tel1
mutation might cause chromosomes to circularize as they do in tel1
rad3
or tel1
rad26
cells. However, we found that pku70
tel1
rad1
, pku70
tel1
rad9
, pku70
tel1
hus1
, and pku70
tel1
rad17
cells all maintained short but stable telomeres (Fig 1F, lanes 811). Telomere lengths in these triple-mutant cells were slightly reduced compared to single checkpoint mutant cells (rad1
, rad9
, hus1
, rad17
), pku70
checkpoint double-mutant cells (pku70
rad1
, pku70
rad9
, pku70
hus1
, pku70
rad17
), or pku70
tel1
cells (Fig 1F and Fig 2D). PFGE analysis found no evidence of chromosome circularization in those triple-mutant cells (Fig 3C, lanes 25). Therefore, the Rad3-Rad26 complex must have additional telomere-associated targets, outside the Rad1/Rad9/Hus1/Rad17 and Ku70 epistasis groups, which confer protection from chromosome circularization in tel1
and rad32
backgrounds.
We also tested the possibility that synergistic chromosome circularization observed in tel1
rad3
cells might be suppressed by pku70
mutation. This might be the case because pku70
cells were reported to have elevated TAS recombination (![]()
rad3
background. Alternatively, the Rad3 and Tel1 kinase pathways may be necessary to specifically inhibit the NHEJ pathway from fusing chromosome ends. In that case, elimination of NHEJ by removal of Ku protein may allow cells to avoid fusing their telomeres. Indeed, telomere fusions observed in nitrogen-starved taz1
cells can be suppressed by pku70
or lig4
mutation (![]()
tel1
rad3
cells again completely lost telomeric hybridization (Fig 1F, lane 13) and have circular chromosomes (Fig 3C, lane 6). Therefore, pku70
mutation cannot suppress chromosome circularization in tel1
rad3
cells.
Interaction between Taz1 and checkpoint proteins:
In S. cerevisiae, the telomere shortening phenotype of a tel1 mutation is epistatic over the telomere elongation phenotype of the rap1-17 mutation (![]()
![]()
![]()
![]()
rap1-17 double mutant has a short telomere length, much like the tel1
mutant, suggests that in S. cerevisiae telomerase recruitment/activation is still largely dependent on Tel1 kinase even in the absence of negative regulators of telomerase (![]()
![]()
As deletion of S. pombe Taz1 telomere-binding protein leads to extreme elongation of the telomere tract, which is reminiscent of the S. cerevisiae rap1-17 phenotype (![]()
required Tel1, Rad32, Ku70, or other checkpoint proteins. We created double-mutant combinations by individually deleting the taz1 gene from single-mutant cells of rad1
, rad9
, hus1
, rad17
, rad3
, rad26
, crb2
, chk1
, cds1
, chk1
cds1
, tel1
, rad32
, and pku70
. We used this sequential procedure to eliminate the possibility that starting with highly elongated taz1
telomeres would mask the effects of the checkpoint mutations. The resulting double-mutant cells were then restreaked multiple times on rich media to allow cells to achieve equilibrium telomere length. As shown in Fig 1G, we found that telomeres are still elongated in all double-mutant cells. In taz1
rad3
and taz1
rad26
, telomere elongation was slightly reduced compared to taz1
cells, but they were still extremely elongated compared to wild-type telomere length (Fig 1G, lanes 1, 7, and 8). These results indicate that telomere elongation in the taz1
mutant is epistatic to mutations in the Tel1/Rad32, checkpoint sensors (Rad1/Rad9/Hus1/Rad17 and Rad3/Rad26), or Ku70 epistasis groups.
If telomere elongation in taz1
cells is independent of Tel1 and Rad3 kinases, the elongation induced by the taz1 deletion might be expected to suppress the rapid telomere loss and circularization of tel1
rad3
cells. To test this possibility, we also created taz1
tel1
rad3
, taz1
tel1
rad26
, taz1
rad32
rad3
, and taz1
rad32
rad26
cells. These triple-mutant strains were created by deleting the tel1 or rad32 gene from the taz1
rad3
or taz1
rad26
cells. Therefore, these cells originally had highly elongated telomeres prior to the deletions. We found that the triple-mutant cells still completely lost their telomere hybridization signal (Fig 1H, lanes 25) and circularized their chromosomes (Fig 3C, lanes 912), indicating that even in the absence of Taz1 protein, telomeres cannot be maintained in tel1
rad3
, tel1
rad26
, rad32
rad3
, or rad32
rad26
backgrounds. In contrast, chromosome circularization observed in telomerase trt1
mutants (Fig 3B, lane 11) was suppressed and the cells maintained stable linear chromosomes indefinitely if the trt1 gene was deleted in cells that were already deleted for taz1 (Fig 1H, lane 6; Fig 3C, lane 13; ![]()
trt1
cells, telomeres are presumably maintained by recombination (![]()
mutation could not overcome elimination of the Rad3/Rad26 and Tel1/Rad32 pathways suggests that these pathways are necessary for both telomerase-based and recombination-based maintenance of telomeres.
Rad3 and Tel1 kinases have additional roles other than recruitment of telomerase:
In S. pombe, careful analysis of how tel1
or checkpoint mutants affect the rate of senescence in telomerase mutant cells has not yet been carried out, nor has direct comparison of the rate of senescence for telomerase vs. tel1
rad3
mutants. Therefore, we undertook such analyses to gain insight into the contribution of Rad3/Rad26 and Tel1/Rad32 pathways to telomere maintenance.
We performed a series of growth curve experiments in which heterozygous diploid cells were sporulated and dissected, and then cultures of cells with appropriate genotypes were serially diluted (Fig 4). As previously reported (![]()
![]()
cells gradually declined in a reproducible manner from day 2 to day 10 in independent liquid cultures (Fig 4A and data not shown). On the other hand, different trt1
cultures displayed different patterns of recovery in growth rate in the phase when survivor cells start to take over the cultures. We did not observe a delayed decline in growth rate for tel1
trt1
cells compared to trt1
cells (Fig 4B). For rad3
trt1
and rad26
trt1
cells, the rate at which growth rates declined among independent cultures became much less reproducible compared to trt1
cells (Fig 4C and Fig D). This effect presumably is related to the checkpoint-related functions of Rad3 and Rad26, as chk1
trt1
cells showed similarly wide-ranging variability in decline of growth rate among independent cultures (data not shown).
|
As trt1
cells undergo senescence, an increasingly large fraction of cells becomes highly elongated (![]()
trt1
, rad26
trt1
, or chk1
trt1
cells, suggesting that senescing trt1
cells show checkpoint-dependent cell cycle arrest as the cells lose their telomeric DNA (data not shown). Interestingly, cds1
trt1
cells still elongated as they senesced, and growth rate decline was similar to that in trt1
cells and without wide-ranging variations among independent cultures (data not shown). Therefore, defective telomeres in trt1
cells appear to be recognized as DSBs and trigger G2 checkpoint cell cycle arrest.
We next compared growth characteristics among tel1
rad3
, tel1
rad3
trt1
, tel1
rad26
, and tel1
rad26
trt1
cells after germination of meiotic spores from heterozygous diploid cells. We observed that both tel1
rad3
and tel1
rad26
reached the point of lowest viability much earlier (
5 days) than trt1
cells did (
10 days), and survivors grew more slowly than trt1
survivor cells (Fig 4E and Fig F). Moreover, tel1
rad3
trt1
and tel1
rad26
trt1
cells did not show any additional loss of growth rate compared to tel1
rad3
and tel1
rad26
. Therefore, the presence of functional telomerase did not help to delay senescence. The accelerated senescence phenotype observed for tel1
rad3
and tel1
rad26
cells is reminiscent of those seen in pku70
trt1
cells and cells lacking the proposed telomere capping protein, Pot1 (![]()
Phosphorylation of Rad32 is independent of Rad3 and Tel1:
Previous studies have shown that Rad32 is phosphorylated in a cell-cycle-dependent manner. Rad32 phosphorylation accumulates in S-phase and this phosphorylation is independent of Rad3 (![]()
Rad32 phosphorylation was detected by the appearance of a slow mobility species in SDS-PAGE that can be converted to a faster mobility species by treatment with phosphatase (Fig 5A). Asynchronous S. pombe cells showed a small amount of Rad32 phosphorylation, which is probably due to a small percentage of cells that are in S-phase (Fig 5B). In contrast, when cells were arrested in S-phase through the addition of hydroxyurea (HU) or when cells were exposed to the DNA-damaging agent methyl methanesulfonate (MMS), increased phosphorylation of Rad32 was observed. Phosphorylation of Rad32 was still observed in rad1
, rad9
, hus1
, rad17
, crb2
, chk1
, cds1
, and chk1
cds1
cells (data not shown) and, surprisingly, in tel1
, tel1
rad3
(Fig 5B), and tel1
rad26
cells (data not shown). These results showed that Rad3 and Tel1 are not the kinases responsible for the observed Rad32 phosphorylation and suggest that there must be other kinase(s) that can phosphorylate Rad32. However, it is possible that Rad3 or Tel1 carries out phosphorylation of Rad32 that does not alter its mobility on SDS-PAGE. Whether Rad32 phosphorylation is actually required to maintain telomeres has to be resolved. We observed more prominent phosphorylation of Rad32 in asynchronous tel1
rad3
and tel1
rad26
cells (Fig 5B and data not shown). These cells are extremely sick and have circular chromosomes (Fig 3B, lanes 7 and 8). We suggest that these cells have problems in either DNA replication or DNA segregation and therefore accumulate DNA damage, which may explain why these cells have elevated Rad32 phosphorylation.
|
Checkpoint sensor and DNA repair proteins are bound to telomeric DNA:
Recent studies in S. cerevisiae showed that Mec1, Ddc2, Rad24, Rad17, Ddc1, and Mec3 (homologs of S. pombe Rad3, Rad26, Rad17, Rad1, Rad9, and Hus1, respectively) are recruited to sites of DNA breaks upon induced DNA damage (![]()
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|
We were unable to detect HA-Rad3 at the telomere when it was expressed from its endogenous promoter, possibly because of its low abundance. On the other hand, HA-Rad3 overexpressed from the nmt promoter was able to specifically enrich telomeric DNA, but not the control ade6+ DNA, suggesting that Rad3 binds specifically to telomeres (Fig 6B). However, we cannot exclude the possibility that overexpressed Rad3 associates with telomeres in a nonphysiological manner. We also observed enrichment of telomeric DNA over ade6+ DNA in immunoprecipitates from Rad17-myc and to a lesser extent from Rad1-myc, Rad9-myc, and Hus1-myc (Fig 6C). Although the signals we obtained were weaker than those for Ku70-myc, they were reproducible. Differences in signal intensity are most likely due to differences in immunoprecipitation efficiency and protein abundance at the telomere. Taken together, these ChIP assays show that Rad3 and Rad17 and most likely Rad1, Rad9, and Hus1 bind to telomeres. We also obtained a low, but significant signal for telomeric DNA in immunoprecipitates from Rad32-myc cells (Fig 6B). Therefore, our data show that Rad32 also binds to telomeres.
Ku70 binding to telomeric DNA is independent of checkpoint sensor proteins but dependent on Taz1 protein:
In our genetic analysis we found that the mutations eliminating checkpoint sensor proteins are epistatic to pku70
in maintaining stable telomere length, indicating that these proteins may function in the same pathway. To investigate whether Ku70 binding to telomeres might be dependent on the checkpoint sensor proteins, we undertook ChIP analyses (Fig 6D). We observed no change in Ku70 binding in either rad17
or rad3
mutants, indicating that the checkpoint sensor proteins do not function through regulating binding of Ku70 to telomeres.
We also investigated Ku70 binding to telomeres in tel1
and taz1
mutants. Again, no change in telomere binding was found in the tel1
strain. In contrast, in the absence of Taz1 protein, Ku70 binding was greatly diminished. This datum is consistent with data from mammalian cells in which Ku70 is found to bind the Taz1 homologs TRF1 and TRF2 (![]()
![]()
![]()
cells, since our ChIP assay is designed to detect proteins bound to sites close to TAS (
5001000 bp).
| DISCUSSION |
|---|
Checkpoint sensor proteins have alternative targets for telomere maintenance:
In this study, we extensively tested the relative contributions of S. pombe checkpoint and DNA repair proteins in telomere maintenance by creating cells carrying various mutant combinations and examining average telomere length and chromosome circularization in the resulting cells. Our results are summarized in Fig 2E. One of the conclusions we draw from such analyses is that downstream effectors of the checkpoint (Crb2, Chk1, and Cds1) are not important for telomere maintenance in S. pombe, even though checkpoint sensor proteins (Rad1, Rad9, Hus1, Rad17, Rad3, and Rad26) are required for proper telomere maintenance. Therefore, checkpoint sensor proteins must contribute to telomere maintenance through alternative telomere target(s) that are unrelated to these checkpoint effectors.
Previous studies in S. pombe also found Chk1 and Cds1 to be not important for telomere maintenance (![]()
![]()
(rhp9
) cells have shorter telomere length (![]()
strains are generated by backcrossing a crb2
strain obtained from a laboratory different (![]()
cells. In comparison, S. cerevisiae rad53 (S. pombe Cds1 homolog) mutants have been reported to have short telomeres (![]()
(S. pombe Crb2 homolog) cells were variously reported to have short (![]()
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Checkpoint sensors Rad1, Rad9, Hus1, and Rad17 function in a single pathway for telomere maintenance and associate with telomeres:
Our studies indicate that checkpoint proteins with PCNA homology (Rad1, Rad9, and Hus1) as well as the RFC-like protein Rad17, which has been proposed to recruit the Rad1-Rad9-Hus1 complex to sites of DNA damage, function in the same pathway for maintenance of telomere length. This conclusion is based on the observation that mutant combinations among these proteins did not lead to additional telomere shortening and mutants lacking these four proteins showed identical telomere lengths under all different mutant backgrounds (tel1
, rad32
, pku70
, pku70
tel1
, taz1
) that we tested.
Our results are consistent with results from previous studies for rad1 and rad17 mutants (![]()
![]()
mutations were found not to affect telomere length (![]()
cells, the difference between the two results may be explained by partial retention of function of the rad9-192 allele with respect to telomere length maintenance, although rad9-192 is as sensitive to UV and ionizing radiation as a rad9
mutant strain (![]()
![]()
In S. cerevisiae, rad17
(S. pombe rad1 homolog) and ddc1
(S. pombe rad9 homolog) cells were reported to have short telomeres, and they were considered to be in the same pathway, since rad17
ddc1
double-mutant cells showed no additional telomere shortening (![]()
cells (S. pombe hus1 homolog) were reported to have longer (![]()
![]()
![]()
(S. pombe rad17 homolog) was reported to have wild-type telomere length (![]()
![]()
![]()
Our ChIP analyses showed robust binding of S. pombe Rad17 to telomeres, while the PCNA-like checkpoint proteins (Rad1, Rad9, and Hus1) bound weakly. Therefore, we suggest that the checkpoint proteins with RFC and PCNA homology contribute to telomere maintenance through their binding to telomeres. As telomere shortening in this class of checkpoint proteins is also observed in S. cerevisiae and C. elegans (![]()
![]()
The Rad3-Rad26 complex has additional roles that are independent of other checkpoint sensor proteins and that function through its association with telomeres:
Our data indicated that Rad3 kinase and its proposed regulatory subunit Rad26 together form a separate epistasis group for telomere maintenance from other checkpoint sensor proteins, as these two proteins had the shortest telomere lengths among checkpoint sensor proteins and the rad3
rad26
double-mutant cells had the same telomere length as single-mutant cells. Studies by other





