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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.

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Poly(ADP-ribosyl)ation has been suggested to be involved in regulation of DNA repair, transcription, centrosome duplication, and chromosome stability. However, the regulation of degradation of poly(ADP-ribose) and its significance are not well understood. Here we report a loss-of-function mutant Drosophila with regard to poly(ADP-ribose) glycohydrolase, a major hydrolyzing enzyme of poly(ADP-ribose). The mutant lacks the conserved catalytic domain of poly(ADP-ribose) glycohydrolase, and exhibits lethality in the larval stages at the normal development temperature of 25°C. However, one-fourth of the mutants progress to the adult stage at 29°C but showed progressive neurodegeneration with reduced locomotor activity and a short lifespan. In association with this, extensive accumulation of poly(ADP-ribose) could be detected in the central nervous system. These results suggest that poly(ADPribose) metabolism is required for maintenance of the normal function of neuronal cells. The phenotypes observed in the parg mutant might be useful to understand neurodegenerative conditions such as the Alzheimer's and Parkinson's diseases that are caused by abnormal accumulation of substances in nervous tissue. P oly(ADP-ribosyl)ation process involves a posttranslational modification of target proteins catalyzed by the poly(ADPribose) polymerase (PARP) family of enzymes with NADϩ as the substrate, resulting in formation of long-branched polymers of ADP-ribose (1, 2). The covalently attached and negatively charged poly(ADP-ribose) units significantly affect several important biological functions, including DNA repair (3), transcription (4), regulation of telomere length, cell cycle progression, centrosome duplication (5, 6), and chromosome stability (7). One of the major members of the PARP family is PARP-1, which catalyzes poly(ADP-ribosyl)ation in response to DNA strand breaks. Recently, additional members of the PARP family of enzymes have been characterized; PARP-2, -3, tankyrase-1, -2, VPARP, and Ti-PARP (2). In Drosophila, only two PARP family members, corresponding to PARP-1 and tankyrase, have been reported (8, 9).Poly(ADP-ribose) attached to acceptor proteins is hydrolyzed rapidly to produce free ADP-ribose residues by poly(ADPribose) glycohydrolase (PARG) (10-12). In contrast to PARPs, only one gene for PARG has been detected in mammals and insects (13). Thus, it is likely that the regulation of PARG activity is required to complete protein modification cycles initiated by different PARPs. In fact, PARG has been proposed to shuttle between the nucleus and the cytoplasm and becomes localized to the centrosomes during mitosis (14). There is evidence for an alternative form in the cytoplasm (M. K. Jacobson, personal communication), indicating that regulation of poly(ADPribosyl)ation by PARG is very dynamic.Mutation of the parg gene, tej, in plants alters circadian rhythms (15), and increased sensitivity to DNA damage in parg knockout mouse embryonic stem cells has been reported (3). However, little is known about the effects...