Figure 2. ATR proficiency is dispensable for elongation of short telomeres. (A) Telomere length histograms for each genotype (top, i–iv); mean lengths of p arm, q arm, and total telomeres (bottom left); and proportion of SFEs (bottom right) from Y chromosome as determined by Q-FISH are shown. Statistical comparison of proportion of chromosomes <1 kb using Fisher’s exact test is shown. (B) Representative image of combined Q-FISH and SKY. (C–E) Telomere length distribution histograms for each genotype (top, i–iv); mean lengths of p arm, q arm, and total telomeres (bottom left); and proportion of SFEs (bottom right) from chromosome 1 (C), chromosome 2 (D), and chromosome 11 (E), as determined by Q-FISH, are shown. Statistical comparison of proportion of chromosomes <1 kb (C) and <4 kb (D and E) using Fisher’s exact test is shown. (A and C–E) Error bars represent SEM. n, number of metaphases analyzed. Bar, 1 µm. Severely decreased ATR levels in primary Seckel MEFs do not significantly impair elongation of short telomeres by telomerase Next, we sought to directly test whether short telomeres inherited from the G2 Terc/ father are elongated properly in telomerase- reconstituted ATRS/S cells. For this, we determined the telomere length of the Y chromosome using quantitative FISH (Q-FISH; Poon et al., 1999) in male littermate MEFs of each genotype under study: ATR+/+/G3 Terc+/*, ATR+/+/G3 Terc/*, ATRS/S/G3 Terc+/*, and ATRS/S/G3 Terc/*. Fig. 2 A shows the telomere length distribution frequency for Y chromosomes from litter- mate MEFs. As expected, cells receiving the Terc-null allele had a large peak of chromosome ends with undetectable telomeres (bottom panel, signal-free ends [SFEs]), which were com- pletely rescued in cells inheriting the wild-type Terc allele (P < 0.0001; compare i with iii), which is in agreement with telomer- ase preferentially elongating critically short telomeres in MEFs (Samper et al., 2001a). Importantly, when a similar comparison is made for the ATR-deficient ATRS/S littermates (Fig. 2 A, compare ii with iv and see SFE in bottom panel), we found that telomerase-reconstituted ATRS/S/G3 Terc+/* were also able to properly elongate critically short telomeres in a similar manner to their ATR+/+ MEF siblings (P = 0.17 for frequency of SFEs; Fig. 2 A, compare i with ii), indicating that wild-type ATR pro- ficiency is dispensable for the rescue of critically short Y chromo- some telomeres and that Seckel cells are not compromised in telomerase recruitment to chromosome ends. Fig. 2 A (bottom left) also shows the mean Y chromosome telomere lengths for each genotype. As expected, ATR+/+/G3 Terc+/* cells show longer mean telomeres (17.87 kb) than their ATR+/+/G3 Terc/* littermates (13.45 kb), but this lengthening is observed most dramatically for Yp telomeres, which were 7.14 kb longer; Yq telomeres were only moderately shorter in Terc-null cells (1.69 kb shorter). A similar trend is observed when comparing ATRS/S/G3 Terc+/* and ATRS/S/G3 Terc/* cells, in which very short Yp telomeres were lengthened to a greater extent than Yq telomeres (2.74 kb longer and 0.42 kb longer, respectively). This pattern likely reflects preferential telomerase activity at short telo- meres in mouse primary cells, where it appears that only 642 JCB • VOLUME 188 • NUMBER 5 • 2010