This study was initiated with the goal of defining the relationships between nucleolytic processing of DNA ends and checkpoint signaling in fission yeast. We found that IR-induced phosphorylation of Chk1 is strongly decreased in cells lacking Mre11 nuclease activity or Ctp1. These findings suggest a checkpoint mechanism in which Ctp1 and MRN complex initiate DSB resection, leading to RPA binding ssDNA, recruitment of Rad3 and other checkpoint proteins, and finally to phosphorylation of Chk1 by Rad3. These data are consistent with evidence that CtIP-dependent resection is rate limiting for ATR activation of Chk1 in mammalian cells treated with CPT (43
). In contrast, elimination of Sae2 does not impair Mec1-dependent activation of Rad53 in budding yeast cells suffering DSBs; in fact, Rad53 activation is enhanced in sae2Δ
cells and suppressed in cells that overexpress SAE2.
Moreover, HO-induced DSBs caused robust and sustained activation of Rad53 in a strain lacking Mre11 nuclease activity (6
Interestingly, we also found that deleting Ctp1 uncovers a second Chk1 activation pathway that uses Tel1 instead of Rad3. This pathway requires Mre11 nuclease activity, the 9-1-1 checkpoint clamp, and Crb2. In fact, the pathway of Crb2 recruitment mediated by its interaction with Cut5TOPBP1
via threonine-215 seems to be the dominant one, which suggests that this pathway is important in the checkpoint response prior to extended resection. Parallel studies in S. cerevisiae
have shown that deleting Sae2 enhances Tel1-mediated activation of Rad53, a Cds1/Chk2 ortholog. As for the Tel1-to-Chk1 pathway in fission yeast, this “TM” pathway requires Mre11 and the Crb2-related protein Rad9. However, the 9-1-1 complex seems to be dispensable, and the role of Mre11 nuclease activity is unknown (47
The requirement for Mre11 nuclease activity shows unequivocally that this activity can function independently of Ctp1 in vivo
and suggests that DNA end processing by Mre11 is required for Chk1 activation by Tel1. This activity is not required for Tel1 activity per se
, since we found that Tel1 efficiently phosphorylates histone H2A in both mre11-H134S
backgrounds. Instead, Mre11 nuclease activity likely generates a DNA structure that is essential for Chk1 activation, probably a short single-stranded overhang (SSO) that is used for loading the Rad9-Hus1-Rad1 checkpoint clamp. Such a nuclease activity has been described for Mre11-Rad50 complex from Pyrococcus furiosus
How does deleting Ctp1 uncover a Tel1-to-Chk1 pathway? Our data show that Mre11 residence at a DSB is fleeting in wild type, but sustained and enhanced in ctp1Δ
cells (Fig. ). Since Tel1 binds the Nbs1 subunit of MRN complex (52
), we propose that sustained binding of MRN complex allows Tel1 to remain at DSBs in ctp1Δ
cells, revealing a Tel1-to-Chk1 checkpoint pathway. It is uncertain whether this pathway is transiently activated in wild-type cells. Active Tel1 associates with DSBs in rad3Δ
cells; however, Chk1 phosphorylation has not been detected in rad3Δ
cells, nor is a checkpoint delay evident in rad3Δ
strains. On the other hand, weak IR-induced Chk1 phosphorylation was observed in cells arrested in G1
), a phase of the cell cycle when Ctp1 is not expressed (23
), leaving open the possibility that resection-independent signaling through Tel1 is a cell cycle-regulated event. It is important to note that in mammalian cells, Mre11 complex also associates with DSBs through chromatin-based interactions with Mdc1, which binds phospho-histone H2AX surrounding DSBs (45
), allowing for the possibility of sustained recruitment of ATM through its interaction with Mdc1.
Our data show that MRN complex persistently binds to DSBs in the absence of Ctp1. Ctp1 associates with DSBs via an interaction with the FHA domain at the N terminus of Nbs1, but Ctp1 does not a stably bind the MRN complex (23
). Thus, the duration of MRN binding at DSBs is potentially determined by the affinity of Ctp1 for the MRN complex. As Tel1 associates with DSBs by binding to the C terminus of Nbs1, the affinity of Ctp1 for Nbs1 may regulate signaling by Tel1. These relationships may explain why Ctp1 is not a stable subunit of MRN complex, because if it were, there would be little opportunity for Tel1 to associate with DSBs. Consistent with these observations, recent studies with Xenopus
egg extracts and mammalian cells show that the appearance of Nbs1 and ATM at DSBs precedes that of CtIP (53
From these data we can propose a biphasic or handoff mechanism of checkpoint signaling at DSBs that can operate whenever Ctp1/CtIP recruitment is significantly delayed relative to Tel1/ATM (Fig. ). As illustrated for fission yeast, the first step is MRN complex binding to DSBs, followed by Tel1 recruitment mediated by Nbs1. Tel1 phosphorylates histone H2A in chromatin flanking the DSB, activating one of two pathways that can recruit Crb2. Mre11 endonuclease creates a short 3′ overhang required for loading the checkpoint clamp and Cut5. With these factors in place, Crb2 mediates phosphorylation of Chk1 by Tel1.
FIG. 6. Biphasic DNA damage checkpoint. DNA damage checkpoint signaling at DSBs is depicted. The MRN complex recruits Tel1 to DSBs. Tel1 phosphorylates histone H2A. Tel1 can phosphorylate Chk1 in a pre-resection phase of the DNA damage checkpoint. Mre11 endonuclease (more ...)
The second phase of the checkpoint response commences with Ctp1 binding the N-terminal FHA domain of Nbs1 (25
). Ctp1-dependent resection disengages the MRN complex and presumably Ctp1 itself from DNA ends. It is unknown how this happens, but it is noteworthy that MRN disengagement appears to also require Mre11 endonuclease activity (49
). One possibility is that Mre11 complex has poor affinity for resected DNA ends. Alternatively, formation of an RPA-ssDNA filament may displace MRN complex from DNA ends. In vitro
studies support the latter possibility, since MRN complex in HeLa cell extracts preferentially binds blunt-ended duplex DNA substrates versus DNA substrates with single-strand overhangs, whereas purified MRN complex displayed no preference (44
). In either case, Ctp1-dependent resection of DNA terminates MRN complex association with DNA ends, and with it the DSB association of Tel1 and Ctp1 itself. Thus, both MRN and Ctp1 self-limit their association DNA ends, promoting a handoff to downstream elements of the HR repair pathway. Displacement of MRN, Ctp1, and Tel1 from DNA ends by an RPA-ssDNA filament would provide for a seamless transition to the second phase of the checkpoint response in which RPA recruits Rad3-Rad26 complex. The handoff between Tel1 and Rad3 can ensure that checkpoint responses are both fast and yet can be maintained while DSBs are processed for HR repair.
In support of this model, a recent study using DNA templates added to HeLa cell nuclear extracts provided strong evidence for an ATM-to-ATR switch coinciding with formation of single-stranded overhangs (SSOs) (44
). Double-stranded ends and single-stranded/double-stranded DNA junctions with 5- to 25-nucleotide SSOs activated ATM, but longer SSOs attenuated ATM activation and stimulated ATR. Using Chk2 (Cds1) and Chk1 as ATM- and ATR-specific substrates, respectively, we found that ATM and ATR are consecutively activated in HeLa cells treated with IR. A period of overlapping Chk2 and Chk1 phosphorylation occurring ca. 10 to 30 min after IR treatment was hypothesized to arise from asynchronous processing of DSBs, but our studies of fission yeast suggest an alternative explanation, namely, that ATM may contribute directly to the early period of Chk1 phosphorylation.
In mammalian cells, both ATR and ATM are critical for Chk1 activation in response to DSBs (16
). ATM was recently shown to be required for efficient recruitment of CtIP to DSBs, suggesting that a resection deficiency may underlie the IR-induced Chk1 activation defect in ATM-null cells (43
). However, regulating CtIP may not fully explain the role of ATM in Chk1 activation, since Chk1 is significantly activated in CtIP knockdown cells treated with CPT, which creates DSBs in S-phase (43
). By extrapolating from fission yeast, our data raise the possibility that ATM may be directly responsible for a portion of Chk1 activation observed in the absence of CtIP. This may explain the severe IR sensitivity, genetic instability and cancer predisposition of A-T patients. From the data currently available, the only crucial difference between fission yeast and mammalian cells with respect to resection and checkpoint signaling is that Tel1 is not required for efficient DNA end processing, hence is not required for Chk1 activation by Rad3 in fission yeast.
In the present study we found that the UV sensitivity of rad3Δ
cells is partially suppressed by ctp1Δ
, even though the ctp1Δ
mutation itself causes moderate UV sensitivity. Suppression of rad3Δ
requires Chk1, indicating that it occurs through the partial reestablishment of a Chk1-mediated checkpoint response that delays the onset of mitosis. However, we also found that rad3Δ
partially suppresses the IR and CPT sensitivity of ctp1Δ
cells, even though the rad3Δ
mutation causes substantial IR and CPT sensitivity. This suppression also requires Chk1. These genetic interactions are more challenging to explain. One possibility is that as both Rad3 and Tel1 activate Chk1 in ctp1Δ
cells, deleting Rad3 abbreviates but does not abolish the checkpoint response, which might confer some survival advantage to a subset of the ctp1Δ
cells that are unable to complete DNA repair. Indeed, cells lacking Dis2, a putative Chk1 phosphatase, were shown to accumulate phosphorylated Chk1, have a prolonged checkpoint arrest, and acute sensitivity to DNA damaging agents such as the alkylating agent, methyl methanesulfonate (8
). Another possibility is that Rad3 (but not Tel1) might negatively regulate an activity that can compensate for the absence of Ctp1. Elimination of the Ku protein complex required for nonhomologous end joining partially rescues ctp1Δ
). This suppression requires Exo1, a 5′-to-3′ exonuclease, suggesting that Ku prevents Exo1 from substituting for Ctp1 in catalyzing the resection of DNA ends required for homologous recombination repair of DSBs. Studies in both human cells and budding yeast have indicated that phosphorylation of Exo1 negatively regulates its activity. Exo1 phosphorylation appears to be regulated by the Mec1/ATR pathways in budding yeast and human cells (11
). Extrapolating to fission yeast, it is plausible that eliminating Rad3 enhances Exo1 activity and thereby allows Exo1 to substitute for Ctp1. Future experiments will address this possibility.