those telomeres that fall below a critical threshold are length- ened and not all telomeres in the cell (Hemann et al., 2001; Samper et al., 2001a). To further confirm our observation of rescue of short telo- meres in ATR-deficient mouse cells, we extended this analysis to additional chromosomes. We used spectral karyotyping (SKY) analysis in combination with Q-FISH (Fig. 2 B) to identify and measure the telomere lengths of specific chromosomes. Fig. 2 C shows that, as we observed for telomeres from the Y chromo- some (Fig. 2 A), a population of critically short telomeres iden- tified in Terc-null MEFs on chromosome 1 was effectively rescued by telomerase reconstitution in ATR-deficient ATRS/S/G3 Terc+/* MEFs to a similar same extent as in ATR+/+/G3 Terc+/* MEFs (P = 1.00 for SFE; comparison of i with iii and of ii with iv shows rescue of critically short telomeres), again indicating that murine ATR-deficient cells are not compromised in their ability to rescue short telomeres. This pattern is also reflected in the mean telomere lengths observed for chromosome 1 for the different genotypes in Fig. 2 C (bottom left). Although the over- all mean telomere length did not change in each of the geno- types, the shorter chromosome 1p telomeres observed in Terc-null mice were increased to an equal length in both ATR+/+/G3 Terc+/* and ATRS/S/G3 Terc+/* cells (by 10.72 kb and 10.79 kb, respectively). Using combined Q-FISH and SKY, we also examined the specific lengths of telomeres from chromosomes 2 and 11 in the different littermate MEFs. Chromosome 2 was examined be- cause it has been found to have a shorter mean telomere length than other mouse chromosomes in several independent mouse strains (Zijlmans et al., 1997; Hande et al., 1999; Samper et al., 2001a), whereas chromosome 11 was chosen because it is ho- mologous to human chromosome 17, which was also reported to have relatively short telomeres (Martens et al., 1998). In the telomere length histograms depicted in Fig. 2 (D [chromosome 2] and E [chromosome 11]), we see that telomerase deficiency in the ATR+/+/G3 Terc/* MEFs causes the accumulation of a population of critically short telomeres that were rescued in their ATR+/+/G3 Terc+/* littermates. In the ATRS/S/G3 Terc/* MEFs, this critically short telomere population was less appar- ent than it was for ATR+/+/G3 Terc/*; however, the proportion of telomeres <4 kb was reduced by a statistically significant amount in ATRS/S/G3 Terc+/* MEFs (P = 0.014 for chromo- some 2; P = 0.025 for chromosome 11), indicating that telomerase is able to rescue short telomeres in MEFs devoid of detectable ATR, as previously demonstrated for telomeres on chromosome 1 and the Y chromosome (Fig. 2, A and C). In most cases, the absence of Terc in the ATR+/+/G3 Terc/* ATRS/S/G3 Terc/* caused increased frequency of SFEs in comparison with their Terc+/* counterparts, except for the telomeres of chromosome 2 in the ATRS/S MEFs. The reason for this anomaly is not clear but may be related to the presence of shorter telomeres in this chromosome and consequent telomerase-independent lengthen- ing by recombination in the ATRS/S/G3 Terc/* population or ATRS/S/G2 Terc/* parent, leading to fewer SFEs because Terc/ cells with short telomeres undergo more frequent telo- mere recombination than wild-type cells (Benetti et al., 2007b). Curiously, we noticed that the mean length of chromosome 11q telomeres was 50% shorter than that of 11p telomeres in this MEF population, which is in contrast to the usual trend for p-arm telomeres to be shorter than q-arm telomeres (Fig. 2 E, bottom left; Zijlmans et al., 1997). Further to our analysis of individual chromosomes, we also examined the telomere length distributions for all chromo- somes for each genotype, incorporating three independent MEFs for each. As shown in Fig. 3 A, comparison of ATR+/+/G3 Terc+/* with ATR+/+/G3 Terc/* and of ATRS/S/G3 Terc+/* with ATRS/S/G3 Terc/* shows significantly (P < 0.0001) longer telomeres in the respective G3 Terc+/* populations, demon- strating telomere elongation upon telomerase restoration in both ATR+/+ and ATRS/S lines. Note especially the dramatic re- duction in critically short undetectable telomeres in these cell populations, as shown by arrows in Fig. 3 A. We found that the median telomere length in telomerase-competent ATR+/+/G3 Terc+/* and ATRS/S/G3 Terc+/* cells was not significantly dif- ferent (P = 0.2173) but, surprisingly, the median telomere length of ATR-deficient ATRS/S/G3 Terc/* MEFs was significantly longer than ATR+/+/G3 Terc/* MEFs, even though the frequency of critically short telomeres was similar in both populations (P < 0.0001). Severely decreased ATR levels augment telomere recombination in Seckel MEFs The overall longer telomeres in ATR-deficient ATRS/S/G3 Terc/* MEFs are suggestive of either telomere lengthening caused by increased usage of recombination-based telomere lengthening mechanisms such as ALT (alternative lengthening of telomeres) in ATR-deficient MEFs in the absence of telomerase (Bailey et al., 2004; Gonzalo et al., 2006) or of reduced telo- mere shortening caused by compromised cell division of ATR- deficient cells (Brown and Baltimore, 2003; Murga et al., 2009). To explore the first possibility, that ATR might suppress telo- meric recombination in Terc-deficient cells, we used chromosome orientation FISH (CO-FISH; Fig. 3 B) to directly visualize telo- meric sister chromatid exchange (SCE [T-SCE]). A positive T-SCE was scored when chromatid exchange was observed be- tween both leading and lagging strand telomeres (Fig. 3, B and C). Similar to previous observations, we found that ATR+/+ cells lacking telomerase showed increased T-SCE (Fig. 3 D), likely reflecting increased recombination stimulated by short telomeres (Benetti et al., 2007a; Morrish and Greider, 2009). Interestingly, we also observed a significant increase in T-SCE in ATRS/S/G3 Terc+/* cells in comparison with ATR+/+/G3 Terc+/* cells, sug- gesting that ATR may negatively regulate telomeric recombina- tion (Fig. 3 D). In ATRS/S/G3 Terc/* cells, T-SCEs were not significantly different to ATRS/S/G3 Terc+/* or ATR+/+/G3 Terc/* cells, implying that the combined deficiency of ATR and Terc does not further increase telomeric recombination (Fig. 3 D). Importantly, global recombination frequencies (SCE) along whole chromosomes were not significantly influenced by either Terc or ATR deficiencies, suggesting a preferential impact of these deficiencies on telomeric repeats (Fig. 4, A and B). Collectively, these data indicate that ATR may negatively regulate telomeric recombination independently of the presence of functional telo- merase. These results further indicate that increased telomeric Telomere defects in ATR-deficient mice • McNees et al. 643