Figure 4. Determination of global recombination frequencies (SCE). (A) Quantification of global SCE frequencies. The number of SCE events scored out of the total number of chromosomes is indicated. Data represent at least two independent MEF cultures (n) per genotype. Statistical comparison using Student’s t test is shown. Error bars represent SEM. (B) Representative global SCE images of full metaphases. Arrowheads depict chromosomes where there are SCE events. Bars, 5 µm. telomere length anomalies (Fig. 2) and by similar frequencies of RLFs in ATR+/+ and ATRS/S cells (Fig. 5 A). Finally, as previ- ously reported by us, there was significant elevation in the number of chromatid breaks and fragments in the ATRS/S cells com- pared with their ATR+/+ littermates (Fig. 5 B; Murga et al., 2009), but these were not affected by the Terc status of the cells because these aberrations do not typically result from criti- cal telomere shortening. ATR prevents telomeres from becoming fragile sites Consistent with very recent findings, we observed a significant ATR-dependent increase in chromosome ends possessing MTSs, which is also increased in ATR+/+ cells upon aphidicolin treat- ment (Fig. 5 C; Martínez et al., 2009b). Previously, we have demonstrated that MTSs are increased under conditions of repli- cation stress, such as in ATR-deficient Seckel MEFs as well as in wild-type cells after treatment with the DNA polymerase  in- hibitor aphidicolin, and thus, telomeres exhibit hallmark charac- teristics of common genomic fragile sites (Martínez et al., 2009b). MTSs are also increased in cells lacking TRF1, which is required for efficient telomere replication, and therefore are a di- rect indicator of telomere fragility resulting from replication fork stalling (Martínez et al., 2009b; Sfeir et al., 2009). In this study, we further demonstrate that ATR-dependent MTSs occur inde- pendently of the Terc status of the cell, indicating that telomere fragility is unrelated to telomere length (Fig. 5 C), thus suggest- ing that short telomeres are not more prone to replication fork stalling. Importantly, we further extended these findings to cells derived from human Seckel patients (O’Driscoll et al., 2003), which also present significantly increased MTSs, as well as in- creased breaks and fragments, which are further aggravated upon aphidicolin treatment (Fig. S1 A). Of note, we also observed slightly shorter telomeres in human Seckel cells compared with ATR wild-type controls (Fig. S1 B), although this modest telo- mere shortening was not accompanied by detectable end-to-end chromosome fusions (Fig. S1 A) and therefore is unlikely to cause telomere uncapping. Notably, in Terc+/* cells with long telomeres, the ATR- dependent increase in MTSs was also paralleled by an ATR- dependent increase in T-SCE (Fig. 3 D). In addition to breaks and gaps, increased chromosomal instability means that fragile sites are also hotspots for increased recombination by SCE; 70% of all gaps and breaks at a common fragile site also have SCEs at the same site (Glover and Stein, 1987; Durkin and Glover, 2007). Therefore, we suggest that increased telomere recombination observed in ATRS/S MEFs is a direct consequence of increased telomere fragility and that our results indicate that we have revealed an important role for ATR in suppressing two outcomes of fragile telomeres: MTSs and recombination. Inter- estingly, we also observed a marked elevation in aneuploidy (primarily tetraploidy) in ATRS/S cells in comparison with ATR+/+ (Fig. 5 D), which was not caused by the presence of short telo- meres and may instead reflect defective G2/M checkpoint ac- tivation in ATR-deficient cells leading to an accumulation of cells with a 4N DNA content (Lin and Dutta, 2007), but the exact molecular mechanism of this remains to be confirmed. Severe developmental defects in Seckel mice simultaneously abrogated for Terc The ATR mutation linked to Seckel syndrome causes an array of characteristic phenotypes in both human patients and Seckel mice, including intrauterine and postnatal growth retardation, microcephaly, mental retardation, progeria, and facial and osteo- skeletal abnormalities (O’Driscoll et al., 2003; Murga et al., 2009). Because Terc deficiency has been shown to suppress the Telomere defects in ATR-deficient mice • McNees et al. 645