genomic fragile sites (Casper et al., 2002, 2004; Murga et al., 2009). The recent description of telomeres resembling fragile sites (Sfeir et al., 2009) suggests that ATR may have an impor- tant function in the maintenance of telomere stability. A role for ATR at mammalian telomeres is further supported by evidence showing that ATR can be found at mammalian telomeres during late replication (Verdun and Karlseder, 2006) as well as by the known role of Drosophila melanogaster ATR in telomere func- tion (Bi et al., 2005). Telomere length can be modulated by telomerase, a telo- mere-specific reverse transcriptase that uses its intrinsic RNA subunit as a template to add telomere repeats to chromosome ends (Chan and Blackburn, 2002). Telomerase is expressed in stem cell compartments and cancer cells, but adult tissues do not express sufficient telomerase for continuous telomere main- tenance (Blasco, 2005). Because of incomplete replication of linear DNA ends, telomeres shorten with progressive rounds of cell division until critically short, leading to increased genomic instability and eventually to cell cycle arrest and apoptosis, which in turn contribute to organismal ageing (Blasco, 2005). Under conditions of telomere shortening, the shortest telomeres are thought to be the preferred substrate for telomere lengthen- ing by telomerase in yeast, primary mouse cells, and some human cell lines (Hemann et al., 2001; Perrem et al., 2001; Samper et al., 2001a; Liu et al., 2002; Teixeira et al., 2004; Zhao et al., 2009), although this is not the case for some human cancer cell lines (Zhao et al., 2009). However, the mechanism enabling telomerase to recognize short telomeres is not clearly defined. A paradigm exists in budding yeast Saccharomyces cerevisiae in which the Tel1 checkpoint kinase (homologue of mammalian ATM) is a critical telomere length regulator; Tel1 deletion re- sults in dramatic telomere shortening, telomere fusion, and ele- vated telomere recombination (Greenwell et al., 1995; DuBois et al., 2002). Telomere length in tel1 cells eventually stabilizes at a minimum length of 50 bp (about sevenfold shorter than wild-type telomeres; Lustig and Petes, 1986), with residual telomere length being maintained by the activity of the closely related Mec1 kinase (homologue of mammalian ATR). Deletion of Mec1 on its own has little effect on telomere length, but mec1 tel1 double mutants exhibit progressive telomere attri- tion and eventual complete loss of telomeres (Ritchie et al., 1999), a phenotype similar to cells lacking telomerase compo- nents (Singer and Gottschling, 1994; Lendvay et al., 1996). Telomerase activity is normal in these cells, and the short telo- mere phenotype of the tel1 and mec1 tel1 strains can be overcome by fusing telomerase to the telomere end–binding protein Cdc13 (Chan et al., 2001; Tsukamoto et al., 2001), sug- gesting that these cells are defective in the recruitment of func- tional telomerase to short telomeres (Pennock et al., 2001; Bianchi et al., 2004; Tseng et al., 2006). Furthermore, 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 replication, as well as in regulating dNTP (deoxyribonucleotide triphosphate) pools (Zhao et al., 1998), suggesting essential roles for Mec1 in repli- cation. Although Tel1 is the major regulator of telomerase re- cruitment in budding yeast, an opposite scenario exists in fission 640 JCB • VOLUME 188 • NUMBER 5 • 2010 yeast Schizosaccharomyces pombe, in which deletion of the ATR homologue Rad3 causes severe telomere shortening, but tel1 cells have normal telomeres (Dahlen et al., 1998; Nakamura et al., 2002). Simultaneous deletion of rad3 tel1 is synergistic and causes the complete loss of telomere signal, defective telomerase recruitment, elevated telomere recombina- tion, and reduced binding of the shelterin components Tpz1 and Ccq1 (Nakamura et al., 2002; Moser et al., 2009). Thus, in yeast cells, the phosphoinositide-3-kinase–related kinase (PIKK) family members ATM and ATR make important contributions to telomerase recruitment and telomere length maintenance. Given their resemblance to DNA double-strand breaks, it is not surprising that short dysfunctional telomeres activate DDRs mediated by the ATM and ATR checkpoint kinases (Guo et al., 2007; Longhese, 2008). In the presence of DNA damage, ATM and ATR phosphorylate a variety of downstream target proteins, variously affecting checkpoint-mediated cell cycle arrest, DNA repair, apoptosis, and senescence (Matsuoka et al., 2007). A short telomere–induced DDR is manifested through the accumulation at critically short telomeres of phosphorylated ATM, 53BP1, -H2AX, the MRN (Mre11–Rad50–Nbs1) com- plex, and Rif, forming so-called telomere dysfunction–induced foci (d’Adda di Fagagna et al., 2003; Takai et al., 2003; Verdun and Karlseder, 2006; Longhese, 2008). Paradoxically, several important DDR proteins also localize to telomeres in the ab- sence of DNA damage, some of which have a role in telomere protection from DDRs, telomere length maintenance, and telo- mere replication, including ATM, ATR, MRN, Ku86, ERCC1, PARP2, BLM (Bloom syndrome protein), and WRN (Werner syndrome protein; Blasco 2005; Verdun and Karlseder 2006), demonstrating that there is critical interplay between DDR pro- teins and telomere metabolism in eukaryotic cells. Because it was first observed that ATM-deficient human cells have shorter telomeres than normal cells (Metcalfe et al., 1996; Pandita, 2002) and this was later confirmed in ATM-null mice (Hande et al., 2001), a role for ATM in telomere length regulation in mammals has been sought. However, simultane- ous deletion of ATM and Terc in mice does not lead to acceler- ated telomere shortening with respect to the single Terc/ controls, although it causes increased genomic instability and increased apoptosis relative to mice lacking either gene alone, implying that ATM depletion increases dysfunction of short telomeres but is not required for inducing cell death or for telo- mere maintenance (Qi et al., 2003; Wong et al., 2003). In line with this, ATM was demonstrated to be dispensable for telomerase- mediated elongation of short telomeres in mammalian cells (Feldser et al., 2006). These findings prompted us to ask whether ATR might be a critical regulator of telomerase recruitment, as well as of the recently described resemblance between telo- meres and common fragile sites (Sfeir et al., 2009). Results Generation of mice and cells doubly deficient in ATR and Terc In contrast to ATM, analyzing the role of ATR in mammals has been hampered by the essential nature of the kinase