whose stability is protected by ATR (Glover and Stein, 1987). Telomeres present inherent difficulties for the replication machin- ery, including topological interference by the t-loop and the tendency for secondary structure formation (such as G-quadruplexes) within repetitive sequences (Gilson and Géli, 2007). Replication forks stall naturally at mammalian telomeres and require an ATR-dependent restart for replication to com- plete (Verdun and Karlseder, 2006) and depend on TRF1 for effi- ciency of completion (Sfeir et al., 2009). Similarly, the fission yeast homologue of TRF1, Taz1, is crucial for efficient replication of yeast telomeres (Miller et al., 2006). Thus, similarly to the case for TRF1, increased telomere fragility in the absence of ATR most likely results from replication fork stalling at telomeres. Interestingly, budding yeast Mec1 has been proposed to have roles facilitating fork progression at specific regions in the genome (Cha and Kleckner, 2002), which are characterized by slow repli- cation, as well as in regulating dNTP pools (Zhao et al., 1998), suggesting essential roles for Mec1 in replication. We know from a study in yeast that ATM and ATR homo- logues have crucial roles in telomerase recruitment and telomere length maintenance (Moser et al., 2009), but the same does not appear to be true for higher eukaryotes, suggesting a degree of evolutionary divergence and that other mechanisms exist to en- able telomerase recruitment to critically short telomeres in mice. Compared with yeast telomeres (300 bp), human (8–15 kb) and murine (30–50 kb) telomeres are extremely long, which may ac- count for the divergence in function of the checkpoint proteins in telomere length regulation, even though other elements of telomere biology are evolutionarily conserved. Factors regulat- ing the recruitment and action of telomerase may vary according to species and cell type or context and multiple factors may be required. For example, recent work by Zhao et al. (2009) sug- gests that in the case of some immortalized human cell lines under telomere equilibrium conditions, telomerase acts to lengthen most telomeres each cell cycle, whereas in yeasts, primary murine cells, and other human cell lines, telomerase has been shown by numerous groups to act preferentially on the shortest telomeres under conditions of telomere shortening similar to those used in the current study (Hemann et al., 2001; Perrem et al., 2001; Samper et al., 2001a; Liu et al., 2002; Teixeira et al., 2004; Zhao et al., 2009). Whether these very different findings reflect experimental artifacts caused by the usage of immortalized versus nonimmortalized cell lines, differences between higher eukaryote species, or differential regulation of telomerase under conditions of telomere disequilibrium remains undetermined, and careful future work is required to resolve this important discrepancy. Although we and others have shown that neither ATM or ATR alone are required for telomerase-mediated elongation of short telomeres (Feldser et al., 2006; Sabourin and Zakian, 2008; this study), it is possible that because of their functional redundancy, both kinases together might function in this capacity in mice, as recently reported for fission yeast (Moser et al., 2009). However, because of their combined essential func- tions in cell cycle checkpoints, codepletion of ATM and ATR re- sults in severe embryonic lethality when ATRS/S mice are crossed with ATM/ to generate doubly depleted offspring (Murga et al., 2009). The combined deficiency is severe enough to prevent 648 JCB • VOLUME 188 • NUMBER 5 • 2010 even the generation of viable MEFs (Murga et al., 2009), mean- ing that this hypothesis cannot be properly tested with the cur- rently available mouse models. ATM and ATR are both members of the PIKK family. Intriguingly, a third PIKK family member, DNA-dependent protein kinase (DNA-PK) catalytic subunit (DNA-PKcs), also has important functions at the telomere. DNA-PKcs (along with its heterodimeric regulatory subunit Ku) functions primarily in DNA double-strand break repair by non- homologous end joining but is also required for effective telo- mere capping (Bailey et al., 1999; Gilley et al., 2001; Goytisolo et al., 2001). The localization of DNA-PK at telomeres and the observation of progressive telomere shortening with increasing generations of DNA-PKcs deficiency raise the intriguing mech- anistic possibility that DNA-PKcs may function in telomerase recruitment in mammalian cells (Espejel et al., 2004). Further studies are required to determine whether this dramatic shorten- ing results from degradation of telomeres related to uncapping or the fascinating possibility of a telomerase recruitment defect. In murine cells, ATM functions to protect short telomeres (Qi et al., 2003; Wong et al., 2003). In late generation ATM// Terc/ mice, ATM loss results in a synergistic increase in telo- mere dysfunction and chromosome fusion. Similarly, in double mutant ATRS/S/G3 Terc/* cells, we also observe dramatic ge- nomic instability and increased chromosome end-to-end fusions compared with either mutant alone, suggesting that, like ATM, ATR also functions to protect the stability of telomeres in murine cells. We hypothesize that the synergistic increase in fusions in ATRS/S/G3 Terc/* cells likely results from a combination of fusion of short telomeres as a protective strategy with an in- creased availability of chromosome ends at breaks at fragile sites, including replicating telomeres, caused by ATR deficiency. A role for ATM and ATR in telomere length maintenance was recently studied in Arabidopsis thaliana (Vespa et al., 2005). In contrast to mammalian cells lacking ATR, A. thaliana lacking either ATM or ATR are viable, and the authors demon- strated that ATM- and ATR-null cells showed wild-type telomere length homeostasis for several generations, which is consistent with our findings in Seckel mice and previous data from ATM- mice (Feldser et al., 2006). Collectively, these results fur- ther demonstrate that ATM and ATR have distinct functions in the DDR and telomerase regulation in higher and lower eukary- otes. In both mice and A. thaliana, cells doubly for ATM and telomerase exhibit increased telomere dysfunction without gross telomere shortening (Qi et al., 2003; Vespa et al., 2005), but A. thaliana ATR/telomerase double mutants show profound genomic instability in concert with dramatic telomere loss. This differs to our observations of moderate telomere lengthening in ATR-deficient ATRS/S/G3 Terc/* MEFs and implies that in plants, ATR functions to protect telomeres from deletion or deg- radation but that in mammalian cells, the shelterin complex is sufficient to protect mammalian telomeres from drastic shorten- ing in the absence of ATR and Terc. Use of the Seckel mouse model has allowed us to monitor the effects of ATR depletion on telomere length maintenance. It is important to emphasize that ATR expression in MEFs is undetect- able by Western blotting and causes cell cycle arrest defects and extreme genomic instability (Murga et al., 2009). In particular,