Figure 3. Analysis of overall telomere length and telomeric recombination. (A) Histogram of telomere lengths from all chromosomes in G3 ATR+/+ and ATRS/S MEFs. Arrows indicate critically short telomeres in G3 Terc/* MEFs that are rescued in G3 Terc+/* MEFs. Data from three independent MEF cultures per genotype are shown. (B) Schematic of CO-FISH telomere labeling resulting from leading-strand (red) and lagging-strand (green) DNA synthesis. T-SCE results in combined green and red fluorescence. (C) Representative CO-FISH images of full metaphases hybridized with probes against the leading (red) and lagging (green) telomeres. Arrowheads depict chromosomes where signals are interchanged at both sister telomeres. (D) Quantification of T-SCE. Only T-SCE events with exchanges between both leading- and lagging-strand probes simultaneously were considered positive. The number of T-SCE events scored out of total number of chromosomes is indicated. Data represent at least two independent MEF cultures per genotype. Statistical comparison using Fisher’s exact test is shown. Error bars represent SEM. Bar, 1 µm. recombination typically associated with ALT is unlikely to con- tribute to the significant increase in telomere length in ATRS/S/G3 Terc/* cells over their ATR+/+/G3 Terc/* counterparts, as both cells have similarly elevated telomere recombination fre- quencies. Although similarities in T-SCE frequencies alone are not sufficient to exclude the use of ALT, the increase in telomere length is more likely the result of delayed cell cycle kinetics in utero caused by limiting amounts of ATR (Murga et al., 2009) or may be the result of the existence of alternative recombination- independent, telomerase-independent mechanisms of telo- mere elongation. Severely reduced ATR levels do not augment end-to-end fusions caused by short telomeres Because of its essential role in DNA replication, ATR-deficient cells exhibit massive genome instability, particularly at fragile sites, that are sensitive to the formation of breaks and gaps after replication stress and are hotspots for SCEs, translocations, and deletions (Glover and Stein, 1987, 1988; Casper et al., 2004; Murga et al., 2009). Short dysfunctional telomeres also provoke genome instability, most often in the form of chromosome end- to-end fusions. In mouse primary cells, these fusions mostly involve p:p Robertsonian-like fusions (RLFs) and, to a lesser extent, q:q dicentrics and p:q fusions (Blasco et al., 1997; Herrera et al., 1999). Therefore, we were interested in determining the effects of combined ATR and Terc depletion on chromosomal instability. Short telomeres caused significantly increased end- to-end chromosome fusions in ATR+/+/G3 Terc/* cells com- pared with ATR+/+/G3 Terc+/* (P = 0.02), a majority of which were RLFs (Fig. 5 A, black bars). Interestingly, ATR depletion in ATRS/S/G3 Terc+/* cells did not increase RLFs over ATR+/+/G3 Terc+/*, but other types of fusions were significantly in- creased (rings, complex aberrations, dicentrics, p:q arm fusions, etc.), likely reflecting stochastic joining of broken chromo- somes and chromatids resulting from genome-wide instability in ATRS/S cells (Murga et al., 2009). The fact that the frequency of RLFs is similarly low in ATR+/+/G3 Terc+/* and ATRS/S/G3 Terc+/* (Fig. 5 A) indicates that severe ATR dysfunction does not, per se, lead to loss of telomere capping. Instead, the higher variety of chromosomal fusions in ATRS/S/G3 Terc/* double deficient cells in comparison with either of the single mutations (P = 0.004 in both cases) suggests that ATR depletion augments the dysfunctionality of short telomeres, as has been previously reported for ATM (Qi et al., 2003; Wong et al., 2003), and likely reflects opportunistic fusion of uncapped or fragile telomeres to chromosome breaks and fragments rather than a direct role for ATR in telomere capping, as suggested by the absence of any 644 JCB • VOLUME 188 • NUMBER 5 • 2010