The MRX complex is required for the recruitment of Tel1 to DSB ends (39
). On the other hand, the lack of Tel1 causes a decrease of MRX binding at telomeres (26
), suggesting that Tel1 exerts a positive-feedback loop on MRX. However, the physiological relevance of this control was unknown. By studying the effects on telomere processing and elongation of the lack of Tel1 compared with the effects of the dominant Tel1-hy909 variant, we provide evidence that Tel1 is crucial for counteracting Rif2-dependent negative regulation of telomere resection and elongation.
We show that the lack of Tel1, which is known to cause telomere shortening (22
), decreases both MRX-dependent resection at a DSB end carrying telomeric repeats and the amount of ssDNA formation at native telomeres. Together with the notion that MRX association at telomeres is impaired by the lack of Tel1 (26
), these data indicate that Tel1, once loaded onto DNA ends by MRX, regulates 5′-3′ nucleolytic degradation of telomere ends by promoting MRX activity. This role of Tel1 is not restricted to telomere ends, as the lack of Tel1 was shown to slightly impair generation of ssDNA also at intrachromosomal DSBs (34
Consistent with a role of Tel1 in regulating telomere end resection, the TEL1-hy909
mutation, which causes telomerase-dependent telomere overelongation, increases the amount of ssDNA at telomeres. This increase becomes apparent even in the absence of telomerase, indicating that it is due to an enhanced resection rather than to telomerase action. Moreover, the TEL1-hy909
mutation accelerates the onset of senescence of telomerase-negative cells. As telomeric ssDNA has been proposed to trigger senescence (1
), this finding further supports the hypothesis that the Tel1-hy909 variant enhances nucleolytic degradation at telomeres. Accordingly, the lack of Tel1, which reduces the amount of telomeric ssDNA, attenuates the senescence phenotype of telomerase-negative cells (19
How does Tel1-hy909 enhance telomere processing and elongation? Both the enhanced resection and elongation of TEL1-hy909
telomeres require Tel1-hy909 kinase activity, further confirming that Tel1 acts as a kinase at telomeres (33
). Like most of the other Tel1-hy variants that we found by virtue of their ability to suppress the hypersensitivity to genotoxic agents of mec1
Δ cells, the Tel1-hy909 variant has enhanced kinase activity in vitro
compared to wild-type Tel1 (3
). However, only the Tel1-hy909 variant confers a striking telomere overelongation phenotype (3
), implying that this phenotype cannot be entirely ascribed to the high Tel1-hy909 kinase activity.
Tel1-hy909 is more robustly associated than wild-type Tel1 to DSB ends carrying telomeric repeats during both the G1
cell cycle phases, likely leading to increased MRX and Est1 persistence at telomeric ends. In fact, the amounts of Mre11 and Est1 bound to telomeric DNA ends are higher in TEL1-hy909
cells than wild-type cells. While enhanced MRX association could explain the increased efficiency of TEL1-hy909
telomere resection, stabilization of Est1 binding to telomeric ends may account for the overelongation of TEL1-hy909
telomeres. Because MRX is required to load Tel1 onto DNA ends (39
) and to support Tel1-hy909 activities, this robust Tel1-hy909 association to telomeres may be due to an increased ability of Tel1-hy909 to interact with MRX compared to wild-type Tel1, as also suggested by the dominant effects of the TEL1-hy909
allele. Unfortunately, we have so far been unable to coimmunoprecipitate Tel1 with MRX to assess this possibility.
MRX-dependent generation of ssDNA at telomeres is prevented by Rif2 during both G1
). We show that neither processing nor elongation of TEL1-hy909
telomeres is inhibited by Rif2, indicating a failure of Rif2 to counteract MRX activity in the presence of the Tel1-hy909 variant. As Rif2 has been proposed to compete with Tel1 for binding to MRX (26
), the dominant Tel1-hy909 variant might escape the Rif2-mediated negative control by binding to MRX more efficiently than wild-type Tel1. Because Tel1 exerts a positive-feedback loop on MRX, this robust Tel1-hy909 recruitment may in turn stabilize MRX association to telomeric ends, possibly through phosphorylation events.
The action of Tel1-hy909 is not restricted to telomeric ends, as Tel1-hy909 also enhances resection at intrachromosomal DSBs (3
). However, this Tel1-hy909 function is restricted to the G2
cell cycle phase, because Tel1-hy909 is not able to enhance resection in G1
(when Cdk1 activity is low) of a DSB end without telomeric sequences, while it does it when the DSB end carries TG repeats. Noteworthy, while Rif2 prevents MRX activity specifically at telomeres, MRX-dependent resection at DSBs devoid of telomeric repeats is counteracted by Yku, which exerts this inhibitory role only in G1
). These observations, together with our finding that Tel1 regulates telomere resection by promoting MRX function, suggest that Tel1-hy909 is unable to promote resection in G1
at DSB ends without telomeric repeats because it cannot overcome the inhibitory effect exerted by Yku on MRX. This inability does not prevent Tel1-hy909 from enhancing MRX-dependent resection at telomeres, because Yku was shown to protect them mainly from Exo1 and not from MRX (7
). Since Yku-mediated inhibition of MRX activity at intrachromosomal DSBs is relieved in G2
), Tel1-hy909 can promote resection of intrachromosomal DSBs only during this cell cycle stage. Consistent with different mechanisms inhibiting MRX activity at telomeres than at DSBs, MRX-dependent resection in G1
is completely abolished at a DSB end with TG repeats, whereas it takes place, although less efficiently than in G2
, when the DSB is devoid of telomeric sequences. Interestingly, the resection kinetics of DSB ends with no TG repeats in G1
-arrested wild-type cells is similar to that occurring in G1
cells when the DSB end carries TG tracts (compare and ). Furthermore, the amount of MRX bound to DSB ends with no telomeric sequences in wild-type cells is similar to the MRX amount that is recruited at DSB ends carrying TG repeats in TEL1-hy909
cells. Thus, the difference in terms of ssDNA generation at DSBs versus telomeric DNA ends appears to rely on Rif2-mediated inhibition of Tel1 and, hence, of MRX activity.
The molecular mechanism underlying Tel1-mediated regulation of telomere length has not yet been solved. The finding that the amount of telomeric G tails is reduced by the lack of Tel1, whereas it is increased in TEL1-hy909
cells, leads to a model, also suggested by Lundblad and colleagues (19
), where Tel1 function in telomere length maintenance can be linked to its role in generating telomeric G tails, which are the substrates of telomerase. In this model, the telomere length defect observed in tel1
Δ cells might be a consequence of the reduced ssDNA amount at the telomeric ends, whereas the increased ssDNA generation at TEL1-hy909
telomeres might improve telomerase-dependent elongation by increasing the substrates available for the telomerase enzyme. However, the finding that the single-stranded G-tail signal was lower in mre11
Δ cells than in tel1
Δ cells, which display a similar telomere length defect and are likely defective in the same telomere length maintenance pathway (41
), is inconsistent with the idea that Tel1 function in telomere length maintenance is limited to ssDNA generation.
The correlation between telomere overelongation and increased Est1 binding at TEL1-hy909
telomeres provides additional support for a model in which Tel1 is a positive activator of telomerase. Because the kinase activity of Tel1 is needed for its role in telomere maintenance (33
), phosphorylation of one or more telomere binding proteins by Tel1 could increase the frequency of elongation by making the telomeric chromatin more accessible to telomerase. In this scenario, the robust association of hyperactive Tel1-hy909 kinase at telomeres may improve the efficiency of this process.
In conclusion, regulation of telomere processing and elongation appears to rely on a balance between Tel1 and Rif2 activities. As Tel1 hyperactivation can improve both telomere resection and telomerase action, Rif2-dependent inhibition of Tel1/MRX function at telomeres is important to ensure the maintenance of telomere identity by limiting ssDNA generation and elongation. In contrast, such a control appears to be dispensable at intrachromosomal DSBs, where generation of ssDNA is necessary to repair the break.