A Kinase-Independent Role for the Rad3-Rad26 Complex in Recruitment of Tel1 to Telomeres in Fission Yeast

Summary

ATM and ATR are two redundant checkpoint kinases essential for the stable maintenance of telomeres in eukaryotes. Previous studies have established that MRN (Mre11-Rad50-Nbs1) and ATRIP (ATR Interacting Protein) interact with ATM and ATR, respectively, and recruit their partner kinases to sites of DNA damage. Here, we investigated how Tel1^ATM and Rad3^ATR recruitment to telomeres is regulated in fission yeast. Quantitative chromatin immunoprecipitation (ChIP) assays unexpectedly revealed that the MRN complex could also contribute to the recruitment of Tel1^ATM to telomeres independently of the previously established Nbs1 C-terminal Tel1^ATM interaction domain. Recruitment of Tel1^ATM to telomeres in nbs1-c60Δ cells, which lack the C-terminal 60 amino acid Tel1^ATM interaction domain of Nbs1, was dependent on Rad3^ATR-Rad26^ATRIP, but the kinase domain of Rad3^ATR was dispensable. Thus, our results establish that the Rad3^ATR-Rad26^ATRIP complex contributes to the recruitment of Tel1^ATM independently of Rad3^ATR kinase activity, by a mechanism redundant with the Tel1^ATM interaction domain of Nbs1. Furthermore, we found that the N-terminus of Nbs1 contributes to the recruitment of Rad3^ATR-Rad26^ATRIP to telomeres. In response to replication stress, mammalian ATR–ATRIP also contributes to ATM activation by a mechanism that is dependent on the MRN complex but independent of the C-terminal ATM interaction domain of Nbs1. Since telomere protection and DNA damage response mechanisms are very well conserved between fission yeast and mammalian cells, mammalian ATR–ATRIP may also contribute to the recruitment of ATM to telomeres and to sites of DNA damage independently of ATR kinase activity.

Introduction

ATM (Ataxia Telangiectasia Mutated) and ATR (ATM and Rad3-related), members of the phosphoinositol-3-kinase like kinase (PIKK) family, are central players in coordinating cellular responses to various forms of DNA damage, such as DNA double-stranded breaks (DSBs) and problems that are encountered by DNA replication forks, in eukaryotic cells [1],[2]. ATM and ATR both preferentially recognize and phosphorylate Serine (S) or Threonine (T) amino acid residues followed by Glutamate (Q), and over 900 sites in more than 700 proteins have been identified as potential phosphorylation sites for these two kinases in mammalian cells [3].

Previous studies have identified the Mre11-Rad50-Nbs1 (MRN) DNA repair complex as a key player in the activation of ATM kinase in response to DSBs [1],[4]. The MRN complex interacts with ATM through an evolutionarily conserved C-terminal motif in its Nbs1 subunit, and this interaction is critical for recruitment of ATM to DSBs and phosphorylation of downstream targets by ATM [5],[6]. Likewise, ATRIP (ATR-Interacting Protein) interacts with ATR through its evolutionarily conserved extreme C-terminal motif and promotes recruitment of ATR to sites of DNA damage [6]. RPA (Replication Protein A)-coated single-stranded DNA (ssDNA) serves as a platform for recruitment of the ATR-ATRIP complex, where phosphorylation of various downstream targets can take place [7]. Besides its role in activation of ATM, the MRN complex has also been shown to contribute to ATR signaling in mammalian cells [8].

Initial studies have suggested that ATM is particularly important for recognition of DSBs, while ATR is more important for recognition of replication stress and ultraviolet radiation (UV)-induced DNA damage [1],[2]. However, recent studies have uncovered an intimate crosstalk between the ATM and ATR signaling pathways in response to DNA damage. For example, ATM-MRN can act upstream of ATR-ATRIP in cellular responses to DSBs by promoting recruitment of ATR-ATRIP through its contribution to generate RPA-coated ssDNA at DSBs [9]–[11]. Conversely, ATR contributes to the activation of ATM in response to DNA replication stress by converting inactive ATM dimers into active monomers through direct phosphorylation of ATM [12]. However, it is currently unknown if ATR-ATRIP may also contribute to the recruitment of ATM to stalled replication forks. In contrast to DSB-induced ATM activation, the C-terminal ATM interaction domain of Nbs1 is not required for ATR-dependent activation of ATM in response to DNA replication stress; however, the Nbs1 N-terminus is essential for ATR-dependent activation of ATM [12].

ATM-MRN and ATR-ATRIP are also redundantly required for the maintenance of telomeres, stable DSBs at ends of chromosomes, in a wide variety of eukaryotic species [13] (Figure 1A). Studies in yeasts and mammalian cells have shown that ATM-MRN and ATR-ATRIP are recruited to functional telomeres during S/G₂ phases of the cell cycle [14]–[16]. In budding yeast, Tel1^ATM and Mec1^ATR have been shown to phosphorylate several Serine residues within the telomerase-recruitment domain of the telomere capping protein Cdc13, and these phosphorylation events have been proposed to promote efficient telomerase recruitment to telomeres [14],[17]. In fission yeast, we have recently shown that Tel1^ATM and Rad3^ATR redundantly promote interaction between the Pot1 telomere capping complex (consisting of Pot1, Tpz1, Poz1 and Ccq1 subunits) and telomerase, and thereby help to recruit telomerase to telomeres [18]. On the other hand, the telomeric GT-rich repeat DNA binding factors TRF2 and POT1, essential for protection of telomeres against degradation and recombination, play critical roles in attenuating DNA damage checkpoint activation mediated by ATM and ATR in mammalian cells [19]–[21]. Therefore, it has been suggested that telomeres transiently become de-protected during S- and G₂-phases, and can thus be recognized as DSBs by ATM/ATR to allow the timely recruitment of telomerase [22].

On the other hand, we have recently found that the arrival of lagging strand DNA polymerases (α and δ) to telomeres is significantly delayed compared to the arrival of the leading strand DNA polymerase ε in fission yeast, and that significant quantities of RPA and Rad26^ATRIP transiently accumulate at replicating telomeres [15]. These observations thus raised the possibility that replicating telomeres may be primarily recognized as unusual/stressed replication forks by Rad3^ATR-Rad26^ATRIP rather than being recognized as DSBs in fission yeast [15]. In mammalian cells, recruitment of ATR and MRN to telomeres precedes recruitment of ATM to telomeres [16], and replication of lagging-strand telomeres also appears to be significantly delayed compared to leading-strand telomeres [23]. Thus, replicating mammalian telomeres might also accumulate unusually high levels of RPA on the lagging strand and activate ATR, and ATR might subsequently contribute to the activation of ATM signaling at telomeres to promote stable telomere maintenance.

Here, we investigated inter-dependencies among Tel1^ATM, Nbs1, and Rad3^ATR-Rad26^ATRIP for their recruitment to telomeres in fission yeast. Our studies uncovered a surprising kinase-independent role of the Rad3^ATR-Rad26^ATRIP complex, which is redundant with the C-terminal Tel1^ATM-interacting domain of Nbs1, for the recruitment of Tel1^ATM to telomeres. We also demonstrate that the N-terminal domain of Nbs1 contributes to Rad3^ATR-Rad26^ATRIP function by promoting recruitment of Rad3^ATR-Rad26^ATRIP to telomeres. Thus, our findings provide mechanistic insights into how recruitment of Tel1^ATM and Rad3^ATR to telomeres is regulated, and define a novel molecular crosstalk between Rad3^ATR and Tel1^ATM in recognition of fission yeast telomeres, potentially resembling the ATR-ATM crosstalk in response to DNA replication stress in mammalian cells.

Results

Tel1^ATM interaction domain of Nbs1 is dispensable for telomere maintenance in rad3-kdΔ cells

The C-terminus of Nbs1 is important for its interaction with Tel1^ATM in fission yeast Schizosaccharomyces pombe [5] (Figure 1B). This interaction is evolutionarily conserved, since the corresponding region of Nbs1 is also required for its interaction with ATM in Xenopus and humans [5],[6]. Mutating or truncating the Nbs1 C-terminus in fission yeast cells lacking functional Rad3^ATR eliminated DNA damage-induced histone H2A phosphorylation (γ-H2A) and foci formation of the checkpoint mediator protein Crb2/Rhp9 [5], since Tel1^ATM and Rad3^ATR are redundantly required for DNA damage-induced histone H2A phosphorylation and Crb2 foci formation [24]. Tel1^ATM-MRN (Rad32^Mre11-Rad50-Nbs1) and Rad3^ATR-Rad26^ATRIP pathways are also redundantly required for stable telomere maintenance, and simultaneous inactivation of these two pathways results in chromosome circularization in fission yeast [25] (Figure 1A). Thus, it was previously hypothesized that disruption of Tel1^ATM-Nbs1 interaction in fission yeast cells lacking functional Rad3^ATR would lead to telomere dysfunction and chromosome circularization [5].

We tested this hypothesis by combining the most commonly used rad3 deletion allele (rad3Δ::ura4⁺, hereafter referred to as rad3-kdΔ) with the Nbs1 C-terminal 60 amino acid deletion allele nbs1-c60Δ that disrupts Tel1^ATM-Nbs1 interaction [5],[26] (Figure 1B and 1C). After extensive restreaking of fission yeast cells on agar plates, we examined the status of telomeres by separating NotI-digested chromosomal DNA by pulsed-field gel electrophoresis (PFGE), and then performing Southern blot analysis with telomeric NotI fragment-specific probes C, I, L and M (Figure 1D). In this assay, cells carrying circular chromosomes lose individual telomeric NotI fragments and show I+L and C+M bands, corresponding to the fused telomeric fragments from chromosomes I and II.

We were surprised to find that the double mutant nbs1-c60Δ rad3-kdΔ cells stably maintained telomeres (Figure 1D). By contrast, combining tel1Δ or nbs1Δ with rad3-kdΔ caused complete loss of telomeres and chromosome circularization, as previously shown [25],[27] (Figure 1D). Thus, while Nbs1 and Tel1^ATM are both essential for telomere maintenance in rad3-kdΔ cells, the Nbs1-Tel1^ATM interaction is dispensable for telomere maintenance in rad3-kdΔ cells. The mutant Nbs1-c60Δ protein retains its ability to interact with Rad32^Mre11, and both rad32Δ rad3-kdΔ and rad50Δ rad3-kdΔ cells are unable to maintain telomeres [5],[27]. Thus, the N-terminus of Nbs1 is required for the MRN (Rad32^Mre11-Rad50-Nbs1) complex to fulfill its essential telomere maintenance function(s) in rad3-kdΔ cells.

The N-terminal non-kinase domain of Rad3^ATR and its regulatory subunit Rad26^ATRIP are both required for telomere maintenance in the absence of Nbs1-Tel1^ATM interaction domain

The rad3-kdΔ allele does not completely delete the rad3⁺ coding region and only removes a C-terminal portion of the Rad3^ATR protein including its kinase domain [26] (Figure 1C). We found that the truncated Rad3^ATR protein (Rad3-kdΔ^ATR) expressed stably, and retained its ability to interact with its regulatory subunit Rad26^ATRIP in co-immunoprecipitation (co-IP) experiments and yeast 2-hybrid assays (Figure 1E and 1F). Similarly, the N-terminal regions of mammalian ATR and budding yeast Mec1, outside of their C-terminal kinase domains, interact with their respective regulatory subunits ATRIP and Ddc2 [28],[29]. Furthermore, when the fission yeast rad3⁺ gene was originally cloned, a truncated Rad3^ATR protein lacking its kinase domain was found to fully suppress UV, IR and HU hypersensitivities and a G₂-checkpoint defect of rad3-136 cells [30]. In addition, over-expression of the N-terminal fragments of Xenopus ATR or budding yeast Mec1^ATR lacking the kinase domain has been shown to partially suppress the DNA damage sensitivity of mec1-1 budding yeast cells by activating the spindle checkpoint [31]. Therefore, we next examined if a complete deletion allele of rad3⁺ (rad3Δ::LEU2, hereafter referred to as rad3Δ, deletes genomic DNA corresponding to amino acids 1-2363 of Rad3^ATR [25]) would show a different genetic interaction with nbs1-c60Δ with respect to DNA damage sensitivity and telomere maintenance, even though we saw no difference between rad3-kdΔ and rad3Δ cells for their average telomere lengths in nbs1⁺ background (Figure S1A).

While we detected no differences in sensitivity towards various DNA damaging agents between rad3-kdΔ and rad3Δ alleles in nbs1⁺, nbs1-c60Δ or nbs1Δ backgrounds (Figure S1), we found that rad3Δ nbs1-c60Δ cells cannot maintain stable telomeres, unlike rad3-kdΔ nbs1-c60Δ cells (Figure 1D). Thus, the N-terminal domain of Rad3^ATR contributes to Tel1^ATM-dependent telomere maintenance in nbs1-c60Δ cells. Additionally, we found that rad26Δ nbs1-c60Δ cells cannot maintain telomeres (Figure 1D), establishing that Rad26^ATRIP is essential for telomere maintenance in nbs1-c60Δ cells. Thus, these results indicated that the Rad3-kdΔ^ATR-Rad26^ATRIP complex has a kinase-independent function for telomere maintenance in cells lacking the C-terminal Tel1^ATM interaction domain of Nbs1. The apparent discrepancy in absolute requirement for the Rad3^ATR kinase domain for DNA damage sensitivity versus telomere maintenance is plausible, since previous studies have established that the downstream checkpoint kinases Chk1 and Cds1^CHK2 are not involved in telomere maintenance in fission yeast, while these kinases play critical roles in cell survival in response to DNA damage [25],[32].

The N-terminal domain of Rad3^ATR is required for recruitment of Rad26^ATRIP to telomeres

To gain insights into how the Rad3-kdΔ^ATR-Rad26^ATRIP complex contributes to telomere maintenance in nbs1-c60Δ cells, we next examined the association of the Rad3^ATR-Rad26^ATRIP complex with fission yeast telomeres by quantitative chromatin immunoprecipitation (ChIP) assays. While a significant amount of telomeric DNA was found to associate with Rad26^ATRIP in rad3⁺ and rad3-kdΔ cells, the amount of telomeric DNA precipitated by Rad26^ATRIP in rad3Δ cells was reduced to background levels (Figure 2A). Conversely, both wild-type Rad3^ATR and Rad3-kdΔ^ATR proteins associate with telomeric DNA in a Rad26-dependent manner, although the precipitation efficiency for Rad3-kdΔ^ATR was reduced compared to wild-type Rad3^ATR (Figure 2B). Taken together, these results are consistent with the notion that Rad26^ATRIP is essential for recruitment of Rad3^ATR to telomeres and that the N-terminal domain of Rad3^ATR contributes to the recruitment of Rad26^ATRIP to telomeres.

We also examined the recruitment of wild-type Nbs1 and Nbs1-c60Δ to telomeres in rad3⁺, rad3-kdΔ, and rad3Δ background by ChIP assays (Figure 2C). Since we were able to still detect a robust association of Nbs1-c60Δ with telomeres even in rad3Δ cells, we could exclude the possibility that the Rad3-kdΔ^ATR-Rad26^ATRIP complex contributes to telomere maintenance by promoting recruitment of Nbs1-c60Δ to telomeres.

The Rad3-kdΔ^ATR-Rad26^ATRIP complex and the N-terminal domain of Nbs1 are both required for recruitment of Tel1^ATM to telomeres in the absence of Nbs1-Tel1^ATM interaction

Since expression of Rad3-kdΔ^ATR allowed cells to maintain stable telomeres in the absence of Nbs1-Tel1^ATM interaction but not in tel1Δ and nbs1Δ cells (Figure 1D), we hypothesized that Nbs1-c60Δ protein and the Rad3-kdΔ^ATR-Rad26^ATRIP complex may function collaboratively to recruit Tel1^ATM to telomeres and maintain telomeres. Indeed, we detected robust Tel1^ATM recruitment to telomeres in nbs1-c60Δ rad3-kdΔ cells, but failed to detect Tel1^ATM at telomeres in nbs1-c60Δ rad3Δ, nbs1Δ rad3-kdΔ, and nbs1Δ rad3Δ cells (Figure 2D). The ability to recruit Tel1^ATM to telomeres in rad3 mutant backgrounds (either rad3-kdΔ or rad3Δ) correlated perfectly with the telomere maintenance phenotypes of these mutant cells (Figure 1D and Figure 2D). Thus, our ChIP data establish that the N-terminal domain of Nbs1 and the telomere-bound Rad3-kdΔ^ATR-Rad26^ATRIP complex must both be present, in order for Tel1^ATM to be recruited independently of the C-terminal Tel1^ATM-interaction domain of Nbs1.

It should be noted that for those mutant strains that are defective in telomere maintenance and ultimately circularize their chromosomes, ChIP assays were performed with early generation cells prior to chromosome circularization by utilizing our recently described Rad3 plasmid loss system [18]. This system allows us to harvest cells immediately after the loss of Rad3^ATR, when the average telomere length is comparable or slightly longer than in rad3Δ or rad3-kdΔ cells. Furthermore, by monitoring real-time PCR amplification cycle numbers for all ChIP input samples, we have ensured that mutant cells utilized in our experiments have not yet circularized their chromosomes, since the PCR primer-annealing sites would be completely lost upon chromosome circularization, resulting in a significant delay in PCR amplification.

In contrast to nbs1-c60Δ cells, nbs1⁺ cells were able to recruit Tel1^ATM to telomeres in both rad3-kdΔ and rad3Δ backgrounds (Figure 2D). Thus, our ChIP data also support the notion that the C-terminal Tel1^ATM interaction domain of Nbs1 and the Rad3-kdΔ^ATR-Rad26^ATRIP complex represent two pathways that are redundantly required for recruitment of Tel1^ATM to telomeres. Since we detect Tel1^ATM at telomeres only in cells carrying very short telomere-repeats (∼180 bp in rad3-kdΔ and rad3Δ cells), but not in cells with wild-type telomere-repeat length (∼280 bp in rad3⁺ cells) (Figure 2D, Figure S1A), fission yeast Tel1^ATM may be preferentially recruited to short telomeres. Alternatively, active Rad3^ATR kinase might prevent association of Tel1^ATM with telomeres. In any case, our results establish that the C-terminal 60 amino acids of fission yeast Nbs1 are not essential for the recruitment of Tel1^ATM to critically short telomeres, due to a redundant non-kinase contribution of Rad3^ATR-Rad26^ATRIP to Tel1^ATM recruitment.

The N-terminal domain of Nbs1 contributes to the recruitment of Rad3^ATR-Rad26^ATRIP to telomeres

In order to better understand how the N-terminal domain of Nbs1 contributes to the Rad3-kdΔ^ATR-dependent maintenance of telomeres in nbs1-c60Δ cells, we next examined the recruitment of Rad3^ATR or Rad3-kdΔ^ATR to telomeres in nbs1⁺, nbs1-c60Δ or nbs1Δ backgrounds by ChIP assays. As shown in Figure 3A, we found that nbs1Δ cells recruit significantly less wild-type Rad3 or Rad3-kdΔ^ATR proteins to telomeres compared to nbs1⁺ or nbs1-c60Δ cells, suggesting that Nbs1-c60Δ protein contributes to the efficient recruitment of the Rad3^ATR-Rad26^ATRIP complex to telomeres. Accordingly, the loss of the Rad3-kdΔ^ATR-Rad26^ATRIP complex from telomeres in nbs1Δ cells might explain why rad3-kdΔ nbs1Δ cells fail to recruit Tel1^ATM to telomeres and circularize chromosomes (Figure 1D, Figure 2D, and Figure 3A). It should be noted that a substantial amount of wild-type Rad3^ATR could still be recruited to telomeres in nbs1Δ cells. In fact, since nbs1Δ, rad50Δ, rad32Δ^mre11 and tel1Δ cells all maintain essentially wild-type length or only slightly shorter telomeres in fission yeast [27],[33], it appears that the MRN complex and Tel1^ATM do not make significant contributions to telomere maintenance as long as cells express the wild-type Rad3^ATR-Rad26^ATRIP complex.

The ATR-ATRIP complex preferentially binds to RPA-coated ssDNA, and the MRN complex promotes formation of 3′ ssDNA at DSBs and telomeres [10], [34]–[36]. Thus, we considered the possibility that the fission yeast MRN complex, independently of its Nbs1 C-terminal Tel1^ATM interaction domain, might promote recruitment of Rad3^ATR-Rad26^ATRIP to telomeres by promoting accumulation of RPA-coated ssDNA at telomeres. However, we found comparable level of RPA (Rad11) associated with telomeric DNA in rad3-kdΔ nbs1-c60Δ and rad3-kdΔ nbs1Δ cells (Figure 3B). We also considered the possibility that the N-terminus of Nbs1 might contribute to the recruitment of Rad3^ATR-Rad26^ATRIP to telomeres by promoting association between the MRN complex and the Rad3^ATR-Rad26^ATRIP complex, since it was previously shown that mammalian MRN interacts with ATR-ATRIP [37]. However, we could not detect a direct interaction between Nbs1 and Rad3^ATR or between Nbs1 and Rad26^ATRIP by yeast 2-hybrid assays, and failed to detect an interaction between Nbs1 and Rad26^ATRIP by co-IP experiments (data not shown). Thus, further investigations are necessary to fully understand how the N-terminal domain of Nbs1 contributes to efficient recruitment of the Rad3^ATR-Rad26^ATRIP complex to telomeres.

Over-expression of Tel1^ATM can bypass the requirement for the Nbs1 C-terminal domain and Rad3^ATR-Rad26^ATRIP complex in telomere maintenance

Over-expression of Tel1^ATM results in MRX (Mre11-Rad50-Xrs2) complex-dependent elongation of telomeres in budding yeast [14],[38],[39], consistent with the notion that recruitment of Tel1^ATM to telomeres represents the critical rate limiting step in regulating telomere length in budding yeast. By contrast, over-expression of Tel1^ATM was unable to increase telomere length in wild-type fission yeast cells (Figure 4A), even though over-expressed Tel1^ATM can be detected at telomeres by ChIP assays (Figure 4D). However, we did observe a partial suppression of telomere shortening in rad3-kdΔ and rad3Δ cells upon Tel1^ATM over-expression (Figure 4A), suggesting that the level of Tel1^ATM recruitment to short telomeres becomes a critical limiting factor in telomere length determination in the absence of the functional Rad3^ATR-Rad26^ATRIP complex. Interestingly, we also found that the Nbs1 C-terminal Tel1^ATM interaction domain is essential for over-expressed Tel1^ATM to associate with telomeres in rad3⁺ cells, but dispensable in rad3-kdΔ and rad3Δ cells (Figure 4D). Thus, it appears that the presence of the kinase active Rad3^ATR-Rad26^ATRIP complex can prevent recruitment of over-expressed Tel1^ATM to telomeres if cells are missing the C-terminal Tel1^ATM interaction domain of Nbs1.

We reasoned that over-expression of Tel1^ATM might be able to suppress chromosome circularization caused by simultaneous mutations in Nbs1 and Rad3^ATR-Rad26^ATRIP, if the essential telomere maintenance function, redundantly provided by the Nbs1 C-terminal Tel1^ATM interaction domain and the kinase-inactive Rad3^ATR-Rad26^ATRIP complex, were to promote efficient recruitment of Tel1^ATM to telomeres. Indeed, over-expression of Tel1^ATM allowed recruitment of Tel1^ATM in nbs1-c60Δ rad3Δ cells (Figure 4D) and suppressed chromosome circularization in nbs1-c60Δ rad3Δ and nbs1-c60Δ rad26Δ cells (Figure 4B and 4C). However, over-expression of Tel1^ATM was unable to suppress chromosome circularization in nbs1Δ rad3-kdΔ, nbs1Δ rad3Δ and nbs1Δ rad26Δ cells, indicating that the expression of Nbs1 (and likely other subunits of the MRN complex) is essential for telomere maintenance in the absence of kinase-active Rad3^ATR-Rad26^ATRIP even when Tel1^ATM is over-expressed. Taken together, our results indicate that the Nbs1 C-terminal Tel1^ATM interaction domain and the kinase-independent function of the Rad3-kdΔ^ATR-Rad26^ATRIP complex redundantly contribute to efficient recruitment of Tel1^ATM to telomeres, when Tel1^ATM is expressed at endogenous level. Our results further demonstrate that when Tel1^ATM is over-expressed, the MRN complex can contribute to telomere maintenance even in the absence of both the C-terminal Tel1^ATM interaction domain of Nbs1 and the Rad3^ATR-Rad26^ATRIP complex.

Over-expression of Rad3^ATR can bypass the requirement for Rad26^ATRIP and Nbs1, but not Tel1^ATM in telomere maintenance

Our ChIP data indicated that both the Rad26^ATRIP regulatory subunit and the N-terminal domain of Nbs1 play critical roles in promoting recruitment of Rad3^ATR to telomeres (Figure 2B, Figure 3A). Therefore, we next investigated if over-expression of Rad3^ATR might be able to bypass the requirement for Rad26^ATR and/or Nbs1 in telomere maintenance. Based on ChIP analysis, over-expression of Rad3^ATR bypassed the requirement for Rad26^ATRIP in recruitment of Rad3^ATR to telomeres (Figure 5C). However, over-expression of Rad3^ATR was not able to suppress telomere shortening observed in rad26Δ cells (Figure 5A). Thus, in the presence of wild-type Nbs1 (and thus intact Nbs1-Tel1^ATM interaction), forced recruitment of Rad3^ATR to telomeres by over-expression is not sufficient to elongate telomeres in rad26Δ cells. On the other hand, we found that Rad3^ATR over-expression completely suppressed chromosome circularization in rad26Δ nbs1-c60Δ cells (Figure 5B), consistent with the notion that the essential telomere maintenance function contributed by Rad26^ATRIP in nbs1-c60Δ is to promote recruitment of Rad3^ATR to telomeres.

Even when Rad3^ATR is over-expressed, the N-terminus of Nbs1 still appears to contribute to telomere maintenance in rad26Δ cells, since suppression of chromosome circularization by over-expression of Rad3^ATR was much more complete in rad26Δ nbs1-c60Δ cells than in rad26Δ nbs1Δ cells (Figure 5B). Since we observed a mixed telomere phenotype among survivor cells after extensive restreaking of nbs1Δ rad26Δ cells over-expressing Rad3^ATR on agar plates, we concluded that over-expression of Rad3^ATR can partially bypass the essential telomere function of Nbs1 in the absence of Rad26^ATRIP (Figure 5B). The observed partial suppression of chromosome circularization in nbs1Δ rad26Δ by over-expression of Rad3^ATR is consistent with the notion that the N-terminal domain of Nbs1 contributes to telomere maintenance by promoting recruitment of Rad3^ATR to telomeres.

By contrast, Rad3^ATR over-expression was unable to suppress chromosome circularization observed in rad26Δ tel1Δ cells (Figure 5B). Thus, it appears that over-expression of Rad3^ATR allows the Tel1^ATM-dependent mechanism to maintain telomeres in rad26Δ nbs1-c60Δ or rad26Δ nbs1Δ cells. One possible mechanism by which Rad3^ATR over-expression might promote Tel1^ATM-dependent telomere maintenance in rad26Δ nbs1-c60Δ or rad26Δ nbs1Δ cells is to promote recruitment of Tel1^ATM to telomeres. Therefore, we examined changes in Tel1^ATM recruitment to telomeres in nbs1-c60Δ rad26Δ cells with or without over-expression of Rad3^ATR by ChIP analyses. However, we were unable to detect Rad3^ATR over-expression dependent recruitment of Tel1^ATM to telomeres (Figure 5D). Thus, further investigations are necessary to fully understand how over-expression of Rad3^ATR contributes to the Tel1^ATM-dependent suppression of chromosome circularization in fission yeast cells simultaneously lacking functional Rad26^ATRIP and Nbs1.

Discussion

In the current study, we investigated how two PIKK-containing complexes, Tel1^ATM-MRN and Rad3^ATR-Rad26^ATRIP, contribute to telomere length regulation in fission yeast (summarized in Figure 6). We have demonstrated that fission yeast Tel1^ATM can be recruited to telomeres by two alternative and redundant mechanisms, which are either dependent or independent of the Nbs1 C-terminal Tel1^ATM interaction domain (Figure 6A and 6B). Our analyses indicated that the Nbs1 C-terminus dependent mode of Tel1^ATM recruitment to telomeres does not require the Rad3^ATR-Rad26^ATRIP complex, and in fact, it might be inhibited by the presence of wild-type Rad3^ATR-Rad26^ATRIP (Figure 2D). On the other hand, the Nbs1 C-terminus independent mode of Tel1^ATM recruitment requires the presence of telomere bound kinase-inactive Rad3-kdΔ^ATR-Rad26^ATRIP complex (Figure 2A, 2B, and 2D). Since the N-terminal domain of Nbs1 is essential for recruitment of the Rad3-kdΔ^ATR-Rad26^ATRIP complex to telomeres (Figure 3A), all our results are consistent with the notion that association of Rad3-kdΔ^ATR-Rad26^ATRIP to telomeres is the crucial determinant that allows recruitment of Tel1^ATM to telomeres in the absence of the Nbs1 C-terminal Tel1^ATM interaction domain. The notion that the C-terminus of Nbs1 and the Rad3-kdΔ^ATR-Rad26^ATRIP complex redundantly contribute to the recruitment of Tel1^ATM is also supported by our finding that over-expression of Tel1^ATM can entirely bypass the requirement for Rad3^ATR-Rad26^ATRIP for telomere maintenance and Tel1^ATM recruitment to telomeres in nbs1-c60Δ cells (Figure 4, Figure 6C). Moreover, the finding that over-expression of Rad3^ATR was able to at least partially suppress the loss of Nbs1 protein (Figure 5B, Figure 6D) provided further support for the notion that the N-terminal domain of Nbs1 also contributes to the recruitment of Rad3^ATR to telomeres.

Thus, our findings are generally consistent with the model that at least one PIKK activity needs to be localized at telomeres, in order to stably maintain telomeres in fission yeast, although we have not yet tested if Tel1^ATM kinase activity is indeed required for telomere maintenance in nbs1-c60Δ rad3-kdΔ cells. It is currently unknown which telomere-associated protein(s) represent critical substrate(s) of Tel1/Rad3 that are essential for telomere maintenance. However, we were able to demonstrate recently that Tel1^ATM and Rad3^ATR are redundantly required to prevent accumulation of homologous recombination DNA repair factors and RPA at telomeres, to promote efficient telomere recruitment of Tpz1 and Ccq1 (subunits of the Pot1 telomere capping complex), and to recruit telomerase to telomeres [18].

Our results also indicate that fission yeast Tel1^ATM is recruited preferentially to short telomeres (Figure 2D). Due to the kinase-independent function of the Rad3^ATR-Rad26^ATRIP complex, the Nbs1 C-terminal 60 amino acid Tel1^ATM interaction domain is dispensable for recruitment of Tel1^ATM to short telomeres. Tel1^ATM is also recruited preferentially to short telomeres in budding yeast [14],[38],[40]. Based on studies utilizing xrs2-664 mutant cells, which express the truncated Xrs2^Nbs1 protein lacking the C-terminal 190 amino acids (full length 884 amino acids), it was suggested that the Xrs2^Nbs1 C-terminal Tel1^ATM interaction domain is essential for recruitment of Tel1^ATM to critically short telomeres [14]. However, a recent study has shown that, while deleting as little as 20 amino acids from C-terminus of Xrs2^Nbs1 is sufficient to disrupt the interaction between Tel1^ATM and Xrs2^Nbs1, xrs2 mutant cells lacking the C-terminal 20 amino acids maintain significantly longer telomeres than xrs2-664 or xrs2Δ cells [41]. Therefore, budding yeast Xrs2^Nbs1 also appears to contribute to telomere maintenance independently of its C-terminal Tel1^ATM interaction domain. Thus, it would be interesting to test if the N-terminal domain of Xrs2^Nbs1 might also collaborate with the Mec1^ATR-Ddc2^ATRIP complex, perhaps independently of Mec1^ATR kinase activity, in regulating Tel1^ATM recruitment to telomeres in budding yeast.

Our ChIP data indicate that very little or no Tel1^ATM is recruited to telomeres in wild-type fission yeast cells. On the other hand, Rad3^ATR-Rad26^ATRIP is specifically recruited to replicating telomeres and appears to act as the primary sensor of transiently “open” telomeres during S-phase [15]. Therefore, it makes sense that rad3Δ or rad26Δ cause a more severe shortening of telomeres than tel1Δ in fission yeast [25],[27],[42]. Thus, Tel1^ATM is likely to function as a back-up mechanism to extend critically short telomeres in fission yeast. In contrast, budding yeast mec1Δ cells show very little shortening of telomeres [43], while tel1Δ or xrs2Δ cells carry extremely short telomeres [44],[45]. It has also been reported that budding yeast Mec1^ATR cannot be detected at telomeres by ChIP in wild-type cells [13]. However, simultaneous loss of Mec1^ATR-Ddc2^ATRIP and Tel1^ATM-MRX pathways results in a severe defect in telomere maintenance [43],[46], much like in fission yeast cells. Thus, available data suggest that budding yeast telomeres are primarily regulated by Tel1^ATM-MRX, and Mec1^ATR-Ddc2^ATRIP fulfills a back-up role for maintaining telomeres.

There are intriguing similarities between our current findings and findings in mammalian cells, where ATR-ATRIP was found to act upstream of ATM. In response to replication stress or UV irradiation during S-phase, the Nbs1 C-terminal ATM interaction domain is dispensable, but the N-terminus of Nbs1 is essential, for ATR to activate ATM in mammalian cells [12]. However, it is currently unknown whether ATR-ATRIP might also contribute to the recruitment of ATM to sites of stalled or distressed DNA replication forks in a kinase-independent manner, besides the previously established kinase-dependent role of ATR in converting inactive ATM dimers into active monomers [12]. However, the similar requirement for the N-terminus, but not the C-terminus, of Nbs1 for the ATR-ATM crosstalk in response to DNA replication stress in mammalian cells and telomere maintenance in fission yeast might suggest that fission yeast telomeres may be recognized primarily as stressed and/or abnormal replication forks by ATM/ATR kinases.

Studies in mammalian cells have also established the existence of an ATM-ATR crosstalk in response to DNA DSBs, where ATM and the MRN complex contribute to the recruitment of ATR-ATRIP to DSBs by promoting the generation of RPA-coated ssDNA at DSB sites [9]–[11],[47],[48]. The Nbs1 C-terminal ATM interaction domain is required to recruit ATM to DSBs induced by ionizing radiation (IR) and to promote ATM-dependent phosphorylation events [5],[6],[49], and it is thus critical for the ATM-ATR crosstalk in response to DSBs. While the critical role of Tel1^ATM in preferentially extending short telomeres in budding yeast has been well established [14],[38], mouse ATM was found not to be critical for extending short telomeres [50]. This difference might indicate that mouse ATR-ATRIP and ATM-MRN pathways work redundantly in extending critically short telomeres. Alternatively, since recruitment of human ATR to telomeres was found to occur earlier in S-phase than recruitment of ATM to telomeres [16], mammalian ATR-ATRIP may function upstream of ATM in telomere length maintenance, and perhaps also possess kinase-independent functions in recruitment of ATM to telomeres. While further analyses are clearly needed to test these speculations, our current findings highlight a complex molecular crosstalk between ATM-MRN and ATR-ATRIP pathways in recognizing an “open” configuration of telomeres to allow their stable maintenance.

Materials and Methods

Reagents and general methods

Fission yeast strains used in this study were constructed by standard techniques [51] and are listed in Table S1. Primers listed in Table S2 were used to construct new strains. For nbs1-myc, nmt-HA-rad3, myc-rad26 and YFP-rad26, original strains were described previously [15],[24],[27],[52]. For myc-rad3, nbs1-c60Δ-myc, myc-tel1 and nmt-HA-tel1, PCR-based methods [53],[54] were used to generate tagged strains. For nbs1Δ::kanMX, nbs1-c60Δ::kanMX6, rad3-kdΔ::ura4⁺, rad3Δ::LEU2 and rad26Δ::ura4⁺, original strains were described previously [5], [25]–[27],[55]. For nbs1Δ::natMX, PCR-based methods [53],[54] were used to generate deletion strains. Budding yeast strains used in yeast two-hybrid assays are also listed in Table S1. Plasmids used in this study are listed in Table S3. Yeast two hybrid assays were performed by mating S. cerevisiae MATa strains harboring GAL4-DBD plasmids with MATα strains harboring GAL4-AD plasmids, as described in the MATCHMAKER system manual (Clonetech). Positive two-hybrid interactions were identified by spotting mated cells onto SD-HTL plates. Sensitivities of fission yeast cells to IR, UV, HU (hydroxyurea) and CPT (camptothecin) were assayed as previously described [24].

Co-immunoprecipitation and western blot analyses

For most strains, cell extracts were prepared in lysis buffer 1 [50mM Tris pH8.0, 150mM NaCl, 10% glycerol, 5mM EDTA, 0.5% NP40, 50mM NaF, 1mM DTT, 1mM PMSF, 1mM Na₃VO₄, ‘Complete’ protease inhibitor cocktail (Roche)], either by glass bead disruption using FastPrep homogenizer (MP Biomedical) or by cryogenic disruption using MM301 Ball Mill (Retsch). For strains expressing myc-Rad3, myc-Rad3-kdΔ or myc-Tel1, lysis buffer 2 [25mM Tris pH7.5, 100mM NaCl, 10% glycerol, 15mM EDTA, 0.1% NP40, 1% Triton, 15mM MgCl₂, 0.1mM NaF, 0.5mM DTT, 1mM PMSF, 1mM Na₃VO₄, ‘Complete’ protease inhibitor cocktail] was used. For co-immunoprecipitation analyses, proteins were immunoprecipitated using either monoclonal anti-myc antibody (9B11, Cell Signaling) or monoclonal anti-HA antibody (12CA5, Roche), and protein G Dynabeads (Invitrogen) or protein G sepharose beads (GE) respectively. Proteins in whole cell extract or from immunoprecipitations were analyzed by western blots using monoclonal anti-HA antibody (12CA5), monoclonal anti-myc antibody (9B11), monoclonal anti-FLAG antibody (M2, F1804, Sigma) or monoclonal anti-GFP antibody (7.1/B.1, Roche). Anti-Cdc2 antibody (y100.4, Abcam) was used for loading control.

Pulsed-field gel electrophoresis (PFGE)

Chromosomal DNA samples were prepared in agarose plugs, digested with NotI restriction enzyme, and fractionated in 1% agarose gels using the CHEF-DR III system (Bio-Rad) as previously described [25]. C, I, L, and M probes specific for telomeric NotI fragments were prepared as previously described [56]. Except for Tel1 or Rad3 over-expression experiments, cells were extensively restreaked on YES agar plates to achieve terminal telomere states prior to harvesting. For over-expression experiments, minimal media was used for nmt1⁺ promoter-controlled over-expression.

Southern blot analyses

S. pombe genomic DNA samples were digested with EcoRI or ApaI, separated on 1.2% (EcoRI) or 2% (ApaI) agarose, transferred to Hybond-XL membrane (GE), and hybridized to telomere probe [57] in Church Buffer [0.25M sodium phosphate buffer pH7.2, 1mM EDTA, 1% BSA, 7% SDS] at 65°C overnight to monitor telomere length.

ChIP assays

Cells were processed for ChIP and analyzed as previously described [15], using either monoclonal anti-myc (9B11; Cell Signaling), anti-FLAG (M2, F1804, Sigma), or anti-HA (12CA5) antibodies. Percent precipitated DNA values (% ppt DNA) were calculated based on ΔCt between Input and IP samples after performing several independent triplicate SYBR Green-based real-time PCR (Bio-Rad) using telomere primers jk380 and jk381 [15]. For genetic backgrounds that cause eventual circularization of chromosomes due to a telomere maintenance defect, a Rad3 plasmid (pREP41H-rad3) was utilized to maintain linear chromosomes during strain construction [18]. Prior to ChIP experiments, single colonies that had lost the Rad3 plasmid were selected based on lack of growth on media lacking histidine and sensitivity to HU, and immediately utilized in ChIP experiments. Based on Southern blot analysis, the early generation strains that have just lost the Rad3 plasmid carry comparable or slightly longer telomeres than rad3Δ cells [18] (data not shown).

Statistical analysis

In order to determine statistical significance of our data, two-tailed Student's t-tests were performed, and P values ≤0.05 were considered as statistically significant differences.

Supporting Information

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