SUMMARY Mammalian telomeres repress DNA damage activation at natural chromosome ends by recruiting specific inhibitors of the DNA damage machinery that form a protective complex termed shelterin. Within this complex, TRF2 plays a crucial role in end-protection as it is required to suppress ATM activation and the formation of end-to-end chromosome fusions1, 2. Here, we address the molecular properties of TRF2 that are both necessary and sufficient to protect chromosome ends. Our data support a two-step mechanism for TRF2-mediated end protection. First, the dimerization domain of TRF2 is required to inhibit ATM activation, the key initial step involved in activation of a DNA damage response. Next, TRF2 independently suppresses the propagation of DNA damage signaling downstream of ATM activation. This novel modulation of the DNA damage response at telomeres occurs at the level of the E3 ubiquitin ligase RNF168 3. Inhibition of RNF168 at telomeres involves the de-ubiquitinating enzyme BRCC3 and the ubiquitin ligase UBR5 and is sufficient to suppress chromosome end-to-end fusions. This two-step mechanism for TRF2-mediated end protection helps to explain the apparent paradox of frequent localization of DNA damage response proteins at functional telomeres without concurrent induction of detrimental DNA repair activities.

Summary When telomeres become critically short DNA damage response factors are recruited at chromosome ends initiating a cellular response to DNA damage. We performed Proteomic Isolation of Chromatin fragments (PICh) to define changes in chromatin composition that occur upon onset of acute telomere dysfunction triggered by depletion of the telomere-associated factor TRF2. This unbiased purification of telomere-associated proteins in functional or dysfunctional conditions revealed the dynamic changes in chromatin composition that take place at telomeres upon DNA damage induction. Based on our results, we describe a critical role for the polycomb group protein Ring1b in NHEJ-mediated end-to-end chromosome fusions. We show that cells with reduced levels of Ring1b have a reduced ability to repair uncapped telomeric chromatin. Our data represent the first unbiased isolation of chromatin undergoing DNA damage and are a valuable resource to map the changes in chromatin composition in response to DNA damage activation.