In this study, we employed two experimental systems to examine the functions of the Mre11 complex at mammalian telomeres. We present evidence that the Mre11 complex is required for the response to telomere dysfunction and further that the disposition of telomeric ends following DNA replication may be influenced by the Mre11 complex. First, the effect of Mre11 complex hypomorphism was examined during long-term serial passage of telomerase-proficient and -deficient (TertΔ/Δ
) cells. No telomere attrition was seen in immortalized Mre11ATLD1/ATLD1
cells over the course of over 400 population doublings, suggesting that the Mre11 complex does not influence telomere length regulation in murine cells. This is in apparent contrast to data from human cells showing that Nbs1, a component of the Mre11 complex, promotes telomerase-mediated telomere synthesis and, when defective, leads to shortened telomeres (36
). Similarly, the degree of telomere attrition in Mre11ATLD1/ATLD1 TertΔ/Δ
was indistinguishable from that in TertΔ/Δ
alone, further supporting the view that the Mre11 complex does not contribute to telomere length maintenance.
Whereas Mre11 complex hypomorphism did not overtly affect normal telomeres, several lines of evidence indicated that the complex governs the cellular response to dysfunctional telomeres. First, propagation of telomerase-deficient cells to the point of telomere dysfunction led to telomere fusions that were at least partially dependent on Mre11, as telomere fusions were less frequent in late-passage Mre11ATLD1/ATLD1 TertΔ/Δ cells than in TertΔ/Δ cells. This indicates that in WT cells, the Mre11 complex recognizes and promotes the “repair” (i.e., fusion) of dysfunctional telomeres. The reduction in fusions was not attributable to differences in telomere length or the disposition of the 3′ single-stranded DNA overhang. The same effect was observed, and was significantly more pronounced, upon the induction of acute telomere dysfunction in Mre11ATLD1/ATLD1 Trf2F/Δ and Nbs1ΔB/ΔB Trf2F/F cells, indicating that the Mre11 complex responds to both acute and nonacute telomere dysfunction.
The inhibition of telomere fusions seen here is reminiscent of the effect of Mre11 complex hypomorphism on chromosome fusions associated with DNA-protein kinase catalytic subunit deficiency. The rate of chromosome fusions is elevated in immortalized DNA-protein kinase catalytic subunit-deficient cells (3
), and this effect was significantly reduced in Nbs1ΔB/ΔB Prkdcscid/scid
). We propose that the reduction in telomere fusions observed here suggests a role for the Mre11 complex in NHEJ, consistent with recent studies of lymphoid cells from Nbs1ΔB/ΔB
). Because they retain extensive telomere sequence, acutely dysfunctional telomeres are more likely to fuse via the canonical DNA ligase IV-dependent pathway. Accordingly, chromatid fusions in cells depleted of both Trf2 and Nbs1 are DNA ligase IV dependent; hence, the same is likely to be true for chromatid fusions in Mre11ATLD1/ATLD1
. Given that the fusion of both acutely dysfunctional and eroded telomeres is inhibited by Mre11 complex hypomorphism, the most reasonable interpretation is that the underlying mechanisms of fusion are the same in both contexts. Nevertheless, we cannot rule out the formal possibility that Mre11 complex hypomorphism has similar effects on two distinct mechanisms of NHEJ at chromosome ends.
Given that telomere uncapping elicits a DNA damage checkpoint response (33
), we propose that the checkpoint defects associated with Mre11ATLD1/ATLD1
mutations may at least partially account for the preponderance of chromatid fusions among the residual joining events observed. The G1
/S checkpoint is inactive in all cells as a result of SV40 immortalization. Hence, WT G1
cells with uncapped telomeres will undergo fusions prior to S phase or enter S phase with uncapped telomeres. The latter will likely activate the intra-S and/or the G2
/M checkpoint. The detection of TIF in interphase cells supports this scenario. Subsequent fusion of those uncapped telomeres would relax the checkpoint and allow cells to progress to metaphase with both sisters fused (i.e., with chromosome fusions). Concurrently, cells with persistent uncapped telomeres will accumulate at the G2
/M boundary and simply not progress to metaphase (Fig. ). The intra-S and G2
/M checkpoints are both defective in Mre11ATLD1/ATLD1
). Therefore, the progression of Mre11 complex mutant cells entering S phase with uncapped telomeres will not be impeded to the same extent as WT cells, and so metaphase spreads with chromatid fusions are more likely to be obtained (Fig. ). Consistent with this view, the intra-S and G2
/M checkpoint defects are more severely impaired in Mre11ATLD1/ATLD1
than in Nbs1ΔB/ΔB
), and the yield of chromatid fusions is greater (48.5% versus 29.3%) (Fig. ) in those cells.
FIG. 5. Summary figure. (A) DNA damage-dependent checkpoints and telomeric end resection. Uncapped telomeres (asterisks; red represents the leading strand, and green represents the lagging strand) elicit checkpoint responses in WT cells. The smaller asterisk (more ...)
Although the significant increase in chromatid fusions seen in Mre11ATLD1/ATLD1
cells is likely to reflect checkpoint defects in those cells, it does not account for the strong bias toward fusion of leading-strand telomeres. We propose that the structure of the newly replicated telomeric ends in Mre11 complex mutants may influence this outcome. Replication of the leading-strand telomere presumably results in a blunt end, while semicontinuous lagging-strand replication creates a 3′ single-stranded overhang as a result of the terminal Okazaki fragment. This difference in the leading and lagging termini is transient, as resection of both ends to create the 3′ G strand overhang occurs (26
The bias for leading-strand fusions is consistent with two possibilities (Fig. ). First, resection of the leading-strand telomere to create the 3′ overhang may be delayed in Mre11ATLD1/ATLD1 and Nbs1ΔB/ΔB cells. The presence of the 3′ overhang is likely to impair NHEJ-mediated fusion; hence, delayed resection of the blunt end would favor NHEJ of the leading-strand telomere. The alternative, and nonexclusive, possibility is that lagging-strand fusions are inhibited in Mre11 complex mutants because the complex also promotes degradation the 3′ overhang of unprotected telomeres and thereby promotes NHEJ of lagging-strand telomeres. The predominance of chromosome fusions in all genotypes upon Trf2 deletion (Fig. ) suggests that ≥50% of 3′ overhangs are degraded to permit fusion. This could reflect the hypomorphic nature of the Mre11 complex mutants examined as well as redundancy with other nucleases.
It is unlikely that the Mre11 complex per se mediates telomeric resection. It is clear that bulk resection of DNA ends in S. cerevisiae
is not mediated by the Mre11 complex (22
), that the polarity of Mre11-dependent exonuclease activity is 3′ to 5′ but that 5′-to-3′ polarity is required (34
), and that telomeric end resection is unaffected in mre11Δ
yeast strains (15
). Nevertheless, in conjunction with Sae2 (or CtIP in mammals), the Mre11 nuclease appears to initiate the resection process via endonucleolytic removal of a terminal oligonucleotide prior to bulk resection by Exo1, Dna2, and the helicase Sgs1 (28
). This initiation step may be delayed in Mre11 complex mutants, or recruitment of the required activities to the telomere may be impaired in Mre11ATLD1/ATLD1
cells. In either scenario, the data support the view that the Mre11 complex influences resection of the leading-strand telomere. Whether this apparent effect on telomeric end processing can fully account for the overall reduction in fusions is not clear. In this regard, the complex's role in the bridging of DNA ends is also likely to at least partially underlie these effects (50
Finally, the data presented herein indicate that the Mre11 complex is required for activation of the response to dysfunctional telomeres. TIF formation in cre
-infected Mre11ATLD1/ATLD1 Trf2F/Δ
and Nbs1ΔB/ΔB Trf2F/Δ
cells was strongly impaired in response to Trf2
deletion. This result phenocopies the outcome seen in cre
-infected Atm−/− Trf2F/Δ
). Whereas many aspects of Atm-dependent responses to telomere uncapping, such as the regulation of NHEJ, are specific to the telomere (8
), the observation that the Mre11 complex is required for the response to telomere dysfunction suggests that the early events in this response overlap significantly with those during the response to interstitial DNA damage.