Mammalian ATM plays a primary role in sensing and transducing the signals emanating from DSBs and also is required for the generation of ionizing-radiation-induced replication protein A foci that lead to ATR recruitment and subsequent ATR-dependent checkpoint activation (1
). On the contrary, the checkpoint functions of the budding yeast ATM ortholog, Tel1, appear more furtive than those of ATM, as Tel1-deficient cells do not suffer from obvious hypersensitivity to DNA-damaging agents and do not show major checkpoint defects (7
). On the other hand, the finding that the lack of Tel1 exacerbates both the sensitivity to DNA-damaging agents and the checkpoint defects of cells lacking the ATR ortholog Mec1 (42
) indicates that the Tel1 contribution to the checkpoint response is masked by the prevailing activity of Mec1. This suggests that Tel1 may differ from Mec1 and ATM in its intrinsic kinase activity, in its ability to interact with specific targets, and/or its ability to interact with damaged DNA. Indeed, high Tel1 levels suppress the hypersensitivity to DNA-damaging agents of mec1
), indicating that Tel1's ability to sense and transduce the DNA damage signals can be enhanced by increasing amounts of Tel1. This prompted us to ask whether it was possible to generate TEL1
mutations able to compensate for the lack of Mec1 checkpoint functions.
Here, we describe the new mutant alleles TEL1-hy184
, and TEL1-hy909
, which we identified as dominant suppressors of the hypersensitivity to DNA-damaging agents of mec1Δ sml1Δ
cells. Although to different extents, all of the TEL1-hy
alleles also are able to partially suppress the inability of Mec1-deficient cells to trigger checkpoint activation in response to UV or bleomycin treatment. Moreover, with the exception of the TEL1-hy544
allele, all of the other TEL1-hy
alleles cause telomere overelongation compared to that of wild-type TEL1
, with TEL1-hy909
showing the longest telomeres. Thus, they can both activate the checkpoint in response to different kinds of DNA lesions and promote telomere elongation more efficiently than wild-type Tel1. Because Tel1 is thought to mediate the recruitment at telomeres of the Est2 catalytic subunit of telomerase and of the Est1 telomerase accessory protein (13
), it is tempting to propose that Tel1-hy proteins promote the association of telomerase to telomeres more efficiently than wild-type Tel1, but further studies will be required to address this point. The MRX complex, which is known to recruit ATM/Tel1 to damaged sites (32
), still is necessary to allow the Tel1-hy-mediated suppression of mec1Δ
defects and telomere overelongation, indicating that both the checkpoint and telomeric functions of these variants still depend on MRX.
The differences between wild-type and Tel1-hy species may be ascribed to alterations in their amounts, in their intrinsic kinase activities, and in their abilities to interact with specific protein targets and/or with damaged DNA. While our data do not indicate significant changes in the amount of any Tel1-hy variant compared to that of the wild type, Tel1-hy385-HA, Tel1-hy394-HA, Tel1-hy680-HA, and Tel1-hy909-HA can phosphorylate in vitro the protein substrate PHAS-I more efficiently than wild-type Tel1, although they do so to different extents. Because Tel1 exerts all of its functions through phosphorylation events (25
), Tel1-hy variants with an increased intrinsic kinase activity may display an enhanced ability to phosphorylate their targets, thus activating the checkpoint and promoting telomere elongation more efficiently than wild-type Tel1. Whereas the Tel1-hy394 and Tel1-hy909 variants carried multiple amino acid substitutions, the specific roles of which remain to be determined, the TEL1-hy385
mutations result in the single-amino-acid changes N2692D and Q2764H, respectively, inside the approximately 330-amino-acid C-terminal domain that is shared by a number of phosphatidylinositol 3 (PI3) protein kinases (43
). It is tempting to speculate that these amino acid substitutions inside the conserved kinase domain facilitate the accessibility of target proteins, thus increasing the efficiency of their phosphorylation. On the other hand, human ATM is present as inert dimers or multimers that, after DNA damage, undergo a rapid intermolecular autophosphorylation, causing their dissociation in active monomers (3
). If this regulatory mechanism also applied to its ortholog Tel1, the enhanced intrinsic kinase activity of Tel1-hy variants also could increase the ability to promote their autophosphorylation and activation.
An increased kinase activity cannot account for the enhanced DNA damage response mediated by Tel1-hy184, Tel1-hy544, and Tel1-hy628. In fact, neither Tel1-hy184 nor Tel1-hy628 show increased in vitro kinase activity compared to that of wild-type Tel1, whereas Tel1-hy544 kinase activity is even slightly lower than that of the wild type. Thus, the ability of Tel1-hy184, Tel1-hy544, and Tel1-hy628 to suppress mec1Δ
cells’ hypersensitivity to genotoxic agents and checkpoint defects might be due to changes in their ability to interact with specific proteins and/or with damaged DNA. Because TEL1-hy184
cells undergo both checkpoint activation and telomere elongation more efficiently than the wild type, altered interactions with proteins in common between checkpoint and telomere length controls may account for the effects of Tel1-hy184 and Tel1-hy628. On the other hand, TEL1-hy544
cells activate the DNA damage checkpoint more efficiently than the wild type, but they are defective in maintaining telomere length, indicating that checkpoint and telomeric functions of Tel1 can be separable, as previously suggested (5
). In this view, the reduced Tel1-hy544 kinase activity might explain the telomere length defects of TEL1-hy544
cells, whereas enhanced interactions with proteins specifically required to activate the checkpoint may account for their enhanced checkpoint response. Curiously, the Tel1-hy909 variant suppresses mec1Δ
defects and elongates telomeres more efficiently than any other Tel1-hy species, but it displays an in vitro kinase activity similar to that of Tel1-hy680 and Tel1-hy385 and lower than that of Tel1-hy394. This suggests that the increased kinase activity cannot, by itself, account for the Tel1-hy909 effects.
Only the TEL1-hy909
allele, which suppresses the hypersensitivity of mec1Δ sml1Δ
cells more efficiently than any other analyzed Tel1-hy variant, also is able to partially rescue the cell lethality caused by the absence of Mec1. Consistent with its ability to bypass all known Mec1 functions, this allele also suppresses the hypersensitivity to genotoxic agents of cells lacking Ddc1 or Ddc2, which are known to act in concert with Mec1 in the DNA damage response (24
). On the other hand, checkpoint activation induced by Tel1-hy909 has the same downstream requirements as wild-type Mec1 and Tel1. In fact, the Tel1-hy909-induced DNA damage response does not escape the requirement of Rad53 and of its mediators, Mrc1 and Rad9 (2
). Moreover, the Tel1-hy909 protein appears to sense and signal a single DSB, which cannot activate a Tel1-dependent checkpoint even in the absence of Mec1 (26
). Because the ability of wild-type Tel1 to sense and transduce the DSB signal becomes apparent by increasing the amount of either Tel1 or DSB (8
), the enhanced Tel1-hy909 kinase activity may account for its acquired ability to activate the checkpoint in response to a single DSB. Alternatively, because resection of DSB ends inhibits wild-type Tel1 signaling activity (26
), it also is possible that the Tel1-hy909 protein recognizes DSBs more efficiently than wild-type Tel1.
We also found that Tel1-hy909 increases the efficiency of 3′-ended ssDNA accumulation, which in turn leads to Mec1 recruitment to DSB ends and Mec1-dependent checkpoint activation (21
). Because wild-type Tel1 is known to contribute to DSB resection, and because this likely involves the activation of the MRX complex that is required to resect DSB ends (26
), the Tel1-hy909 variant might display an enhanced ability to activate MRX, thus promoting DSB resection more efficiently than wild-type Tel1.
The potential to generate Tel1 mutant variants that are more active than wild-type Tel1 suggests that Tel1 activity is tightly regulated in order to avoid unscheduled checkpoint activation. Consistent with this hypothesis, we found that TEL1-hy909 cells expressing functional Mec1 exhibit a slow-growth phenotype and a checkpoint-mediated delay of the metaphase-to-anaphase transition under unperturbed conditions. Moreover, they show an increased sensitivity to DNA-damaging agents, possibly due to an upregulation of the checkpoint response in the presence of small amounts of DNA lesions. Finally, they impair checkpoint switch off. These negative consequences of having Tel1-hyperactive variants indicates that Tel1 has evolved in order to have a specific amount of activity to minimize the risk of inappropriately launching the checkpoint response.
Taken together, our data provide evidence that it is possible to generate Tel1 variants that trigger checkpoint activation and telomere elongation more efficiently than wild-type Tel1 and can at least partially replace Mec1 in the response to genotoxic treatments. Both the differences and the similarities in the phenotypes of Tel1/ATM- or Mec1/ATR-defective cells can be explained in the light of the specific roles of these proteins in the DNA damage response and telomere homeostasis. In this scenario, the characterization of these Tel1-hy variants provides new insights into the dynamic interactions between Tel1/ATM and Mec1/ATR and may help in the development of new strategies in the diagnosis and treatment of human diseases related to ATM and ATR dysfunctions.