TRF2 is a component of shelterin, the telomere-specific protein complex that prevents DNA damage signaling and inappropriate repair at the natural ends of mammalian chromosomes. We describe a temperature-sensitive (ts) mutation in the Myb/SANT DNA-binding domain of TRF2 that allows controlled and reversible telomere odeprotection. At 32 degrees C, TRF2ts was functional and rescued the lethality of TRF2 deletion from conditional TRF2(F/-) mouse embryonic fibroblasts (MEFs). When shifted to the nonpermissive temperature ( 37 degrees C), TRF2ts cells showed extensive telomere damage resulting in activation of the ATM kinase and nonhomologous end-joining (NHEJ) of chromosome ends. The inactivation of TRF2ts at 37 degrees C was rapid and reversible, permitting induction of short periods (3-6 h) of telomere dysfunction in the G0, G1, and S/G2 phases of the cell cycle. The results indicate that both the induction of telomere dysfunction and the re-establishment of the protected state can take place throughout interphase. In contrast, the processing of dysfunctional telomeres by NHEJ occurred primarily in G1, being repressed in S/G2 in a cyclin-dependent kinase (CDK)-dependent manner.