JCB: Article ATR suppresses telomere fragility and recombination but is dispensable for elongation of short telomeres by telomerase Carolyn J. McNees,1 Agueda M. Tejera,1 Paula Martínez,1 Matilde Murga,2 Francisca Mulero,3 Oscar Fernandez-Capetillo,2 and Maria A. Blasco1 1Telomeres and Telomerase Group and 2Genomic Instability Group, Molecular Oncology Programme and 3Molecular Imaging Core Unit, Biotechnology Programme, Spanish National Cancer Centre, Madrid 28029, Spain Y G O L O I B L L E C F O L A N R U O J E H T T lication is balanced by telomerase-mediated telo- elomere shortening caused by incomplete DNA rep- mere extension, with evidence indicating that the shortest telomeres are preferred substrates in primary cells. Critically short telomeres are detected by the cellular DNA damage response (DDR) system. In budding yeast, the important DDR kinase Tel1 (homologue of ATM [ataxia telangiectasia mutated]) is vital for telomerase recruitment to short telomeres, but mammalian ATM is dispensable for this function. We asked whether closely related ATR (ATM and Rad3 related) kinase, which is important for prevent- ing replicative stress and chromosomal breakage at com- mon fragile sites, might instead fulfill this role. The newly Introduction Telomeres are nucleoprotein structures that protect the ends of eukaryotic chromosomes from DNA repair and degradation activities. Telomeres comprise complexes of tandem DNA re- peats (TTAGGG in vertebrates) bound by a specialized multi- protein complex known as shelterin (Palm and de Lange, 2008). Shelterin contributes to the protective function of telomeres by cancelling DNA damage response (DDR) activation at telo- meres and preventing potential telomere fragility (Martínez et al., 2009b; Sfeir et al., 2009). In particular, recent studies demonstrate that the shelterin protein TRF1 is essential to Correspondence to Maria A. Blasco: mblasco@cnio.es Abbreviations used in this paper: ALT, alternative lengthening of telomeres; ATM, ataxia telangiectasia mutated; ATR, ATM and Rad3 related; CO-FISH, chromosome orientation FISH; CT, computed tomography; DDR, DNA damage response; DNA-PK, DNA-dependent protein kinase; DNA-PKcs, DNA-PK cata- lytic subunit; MEF, mouse embryonic fibroblast; MTS, multiple telomeric signal; PIKK, phosphoinositide-3-kinase–related kinase; Q-FISH, quantitative FISH; RLF, Robertsonian-like fusion; SCE, sister chromatid exchange; SFE, signal-free end; SKY, spectral karyotyping; TRAP, telomerase repeat amplification protocol; T-SCE, telomere SCE. The Rockefeller University Press $30.00 J. Cell Biol. Vol. 188 No. 5 639–652 www.jcb.org/cgi/doi/10.1083/jcb.200908136 created ATR-deficient Seckel mouse strain was used to ex- amine the function of ATR in telomerase recruitment and telomere function. Telomeres were recently found to re- semble fragile sites, and we show in this study that ATR has an important role in the suppression of telomere fra- gility and recombination. We also find that wild-type ATR levels are important to protect short telomeres from chromo- somal fusions but do not appear essential for telomerase recruitment to short telomeres in primary mouse embry- onic fibroblasts from the ATR-deficient Seckel mouse model. These results reveal a previously unnoticed role for mammalian ATR in telomere protection and stability. prevent telomere breakage associated with replication fork stalling at telomeres (Martínez et al., 2009b; Sfeir et al., 2009). TRF1 deletion leads to uncapping and fusion of telomeres and to increased numbers of telomeres with multiple telomeric sig- nals (MTSs), an aberrant structure that is enhanced by condi- tions known to cause replication fork stalling, such as aphidicolin treatments (Martínez et al., 2009b; Sfeir et al., 2009). Chromo- somal defects associated with TRF1 deficiency are also accom- panied by the activation of an ATM (ataxia telangiectasia mutated)/ATR (ATM and Rad3 related)-dependent DDR (Martínez et al., 2009b; Sfeir et al., 2009). Interestingly, the im- portant DDR kinase ATR functions protecting and stabilizing stalled replication forks during DNA synthesis and is essential for preventing chromosomal gaps and breaks at common © 2010 McNees et al. This article is distributed under the terms of an Attribution– Noncommercial–Share Alike–No Mirror Sites license for the first six months after the pub- lication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/). JCB 639