Figure 6. Telomerase deficiency aggravates lifespan attenuation in Seckel mutant mice. (A) Kaplan-Meier survival curve of mice of the indicated geno- type. n, number of mice per genotype. Statistical comparisons using log- rank test are shown. (B) Reduced body weight of Seckel mice at 7 wk of age is not worsened by telomerase deficiency. Statistical comparisons using Student’s t test (unpaired, two sided) are shown. n, number of mice. (C) Severe osteoporosis associated with ATR Seckel mutation is not increased by telomerase deficiency (two to three mice per genotype). (B and C) Error bars represent SEM. et al., 2009). Similarly, the severe microcephaly of Seckel mice was not worsened by Terc deficiency, and there were no measur- able differences in the dimensions of the cranial cavity, the extent of kyphosis, the length and angle of the nasal bone, mandibular lengths, or the cross-sectional dimensions of the thorax (Fig. S2, A–H) between ATRS/S/G3 Terc+/* and ATRS/S/G3 Terc/* mice. Owing to the fact that Seckel mice have a greatly reduced life- span, these mice never develop tumors (Murga et al., 2009), Table I. Telomerase deficiency does not further reduce the low birth rates of ATRS/S mice Genotype ATR+/+/G3 Terc+/* ATR+/+/G3 Terc/* ATR+/S/G3 Terc+/* ATR+/S/G3 Terc/* ATRS/S/G3 Terc+/* ATRS/S/G3 Terc/* Number of mice (percentage) 18 (15.5) 23 (19.8) 30 (25.9) 32 (27.6) 7 (6.0) 6 (5.2) Table indicates the number of mice born for each genotype from a total of 19 ATR+/S/Terc+/ × ATR+/S/G2 Terc/ crosses. Statistical analysis using 2 test is shown for comparison of G3 Terc+/* versus G3 Terc/* for each ATR genotype. Parents were ATR+/S/Terc+/ × ATR+/S/G2 Terc/. Mean litter size was 4.8 pups. The total number of mice studied is 116. P = 0.435 compar- ing ATR+/+/G3 Terc+/* with ATR+/+/G3 Terc/*; P = 0.799 comparing ATR+/S/G3 Terc+/* with ATR+/S/G3 Terc/*; and P = 0.593 comparing ATRS/S/G3 Terc+/* with ATRS/S/G3 Terc/*. which prevented addressing the impact of combined Terc and ATR deficiencies in carcinogenesis. Collectively, these results suggest that combined telomerase and ATR deficiencies seem to have a negative impact of longevity but do not worsen charac- teristic phenotypes of ATRS/S mice, most likely because of the early onset of Seckel phenotypes. Discussion Given that the yeast Tel1 and Mec1/Rad3 checkpoint kinases are critical for telomere length maintenance, a similar role was expected for their mammalian counterparts, and because murine ATM was proven dispensable for the lengthening of short telo- meres, ATR was suspected to fulfill this role (Feldser et al., 2006; Sabourin and Zakian, 2008). We demonstrate in this study for the first time that a severe reduction in ATR levels does not impair telomerase-mediated elongation of short telomeres in mouse primary fibroblasts but has an important role in the pre- vention of telomere fragility and recombination. ATR primarily functions to prevent replicative stress and genomic instability. In particular, ATR depletion leads to increased chromosome breakage, especially at fragile sites, both in human and mouse ATR-deficient Seckel cells (Casper et al., 2004; Murga et al., 2009). Consistent with this, we observed an ATR- dependent, Terc-independent increase in MTSs at chromosome ends, an aberration diagnostic of telomere fragility resulting from stalled DNA replication (Martínez et al., 2009b; Sfeir et al., 2009), and we further confirmed these results in cells derived from human Seckel patients. Furthermore, we also observed increased T-SCE in both ATRS/S/G3 Terc+/* and ATRS/S/G3 Terc/* MEFs, indicating a role for ATR in the negative regulation of recombina- tion at telomeres. Common fragile sites are genomic regions prone to replication fork stalling that exhibit increased formation of chromosome gaps and breaks and are also hotspots for increased recombination by SCE (Durkin and Glover, 2007). Given that 70% of all gaps and breaks at a common fragile site also have SCEs at these sites, our observation of ATR-dependent increase in MTSs paralleled by ATR-dependent increase in T-SCE underscores the notion that telomeres are bona fide fragile sites Telomere defects in ATR-deficient mice • McNees et al. 647