Telomeres differ from DSBs because they do not induce a DNA damage response. However, proteins required for the latter are intimately involved in the regulation of telomere function, suggesting a time window where functional telomeres can be recognized as DNA breaks. By using two different systems, here we provide evidence that telomeres elicit an MRX-dependent DNA damage response when they become competent for elongation. Specifically, ectopic Tel1 induction in cells with otherwise stable short telomeres results in telomere elongation and checkpoint activation. Similarly, conversion into a full-length telomere of a single Flp1-induced telomere with short TG tracts parallels DNA damage checkpoint activation. In both cases, checkpoint activation correlates with telomere elongation, strongly suggesting that telomeres are dealt with in a similar manner as DSBs during the time of their replication.
We also show that the MRX complex, which is required in both the systems above for checkpoint activation and telomere elongation, is recruited to short telomeric ends concomitantly with their elongation and checkpoint activation. Furthermore, both the lack of Sae2 and the rad50s mutation increase MRX persistence at short telomeres and prevent checkpoint switch off after Tel1 induction. On the contrary, Sae2 high levels reduce MRX telomere association and impair checkpoint activation in GAL-TEL1 cells. This strongly suggests that checkpoint activation during telomere elongation can be ascribed to MRX recruitment at telomeres.
Interestingly, the MRX complex is not required to activate the checkpoint when telomeres undergo extensive shortening due to the lack of telomerase or when they are exposed to exonuclease degradation leading to ssDNA accumulation due to the lack of Cdc13 (Ijpma and Greider, 2003
; Foster et al., 2006
). This indicates that elongating telomeres generate checkpoint signals that are different from those of uncapped telomeres. Consistent with this hypothesis, we found that accumulation of ssDNA at telomeres is not likely responsible for the MRX-dependent checkpoint activation at elongating telomeres.
The specific roles of Mec1 and Tel1 in activating the MRX-dependent checkpoint during elongation of multiple short telomeres is difficult to assess, because high Tel1 levels can bypass Mec1 requirement (Clerici et al., 2001
). On the other hand, we found that both Mec1 and Tel1 contribute to checkpoint activation in response to elongation of a single short telomere. This situation is reminiscent of the checkpoint activated by a single DSB, where MRX mediates the recruitment of Tel1 at the DSB ends (Nakada et al., 2003
) and is necessary to activate the Mec1-dependent pathway, possibly by allowing generation of RPA-coated ssDNA (Zou and Elledge, 2003
; Nakada et al., 2004
; Mantiero et al., 2007
). Once MRX is recruited at telomeres, checkpoint activation does not require telomere elongation. In fact, the sae2
Δ and rad50s
alleles, which cause MRX persistence at short telomeres and do not allow DNA damage checkpoint switch off in galactose-induced GAL-TEL1
cells, also prevent telomere elongation in the same cells, possibly by altering MRX nuclease activity (Clerici et al., 2005
). Furthermore, we found that inhibition of replication origin firing impairs telomere elongation in galactose-induced GAL-TEL1
cells without affecting either MRX association at short telomeres or checkpoint activation. These data indicate that telomere-bound MRX is sufficient to activate the checkpoint independently of telomere elongation, suggesting that MRX binding at short telomeres is the signaling event for checkpoint activation. They also imply that only telomeres that become susceptible to be bound by MRX and, therefore, suitable for elongation, can be recognized as DSBs by the checkpoint machinery. Indeed, MRX was shown to be enriched at telomeres during S phase (Zhu et al., 2000
; Takata et al., 2005
), and only telomeres with short TG tracts are avidly bound by MRX, as well as by the telomerase enzyme (Negrini et al., 2007
; this study).
In this context, our results indicate that, under unperturbed conditions, only S phase telomeres are potentially detectable as DSBs by the checkpoint machinery (). However, the yeast telomerase enzyme only acts on short telomeres within one cell cycle (Teixera et al., 2004
), and the rate of telomere elongation appears limited to a few base pairs per generation (Marcand et al., 1999
). This limitation may prevent unscheduled checkpoint activation during an unperturbed S phase, because the checkpoint signals do not persist long enough to be detected by the checkpoint machinery.
Figure 7. A model for DNA damage checkpoint activation by short telomeres. During the time of telomere replication (S phase), only short telomeres are suitable to MRX binding and elongation. Telomere-bound MRX, besides triggering telomere elongation, can activate (more ...)
MRX access, telomere elongation, and checkpoint activation are instead prevented at long or full-length telomeres, suggesting that the latter have acquired a structure that may physically hide the telomeric ends from MRX and telomerase. This protective cap can be formed immediately after DNA replication and maintained until the next S phase. Although the precise molecular nature of this capped structure is unknown, we have found that high levels of Rif2 inhibit both telomere elongation and checkpoint activation in galactose-induced GAL-TEL1
cells. Because it has been shown that Rap1 is necessary to suppress both Cdc13 and MRX recruitment at long TG tracts (Negrini et al., 2007
), this suggests that Rap1, Rif1, and Rif2 might favor the formation of a t-loop structure similar to the one observed in many other organisms (de Lange, 2005
In conclusion, we have shown that telomeres behave similarly to intrachromosomal DSBs when they are suitable for elongation. This biological response appears to be conserved throughout evolution, because functional human telomeres have been shown to undergo a structural change that elicits a DNA damage response during or after DNA replication (Verdun et al., 2005
; Verdun and Karlseder, 2006