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.

Here, we describe a novel specific telomere-associated protein: TZAP (Telomeric Zinc finger-Associated Protein). TZAP binds preferentially to long telomeres that have a low concentration of shelterin complex, in virtue of competition with the telomeric repeat binding factors TRF1 and TRF2. When localized at telomeres, TZAP triggers “telomere trimming”, a process that results in the rapid deletion of telomeric repeats. Based on these results, we propose a novel model for telomere length regulation in mammalian cells. Binding of TZAP to telomeres is restricted to long telomeres and represents the switch that triggers telomere trimming, setting the upper limit of telomere length. In this model, the reduced concentration of the shelterin complex at long telomeres results in TZAP binding and initiation of telomere trimming.

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.

RING (Really Interesting New Gene) domain-containing E3 ubiquitin ligases comprise a large family of enzymes that in combination with an E2 ubiquitin-conjugating enzyme, modify target proteins by attaching ubiquitin moieties. A number of RING E3s play an essential role in the cellular response to DNA damage highlighting a crucial contribution for ubiquitin-mediated signaling to the genome surveillance pathway. Among the RING E3s, RNF8 and RNF168 play a critical role in the response to double stranded breaks, one of the most deleterious types of DNA damage. These proteins act as positive regulators of the signaling cascade that initiates at DNA lesions. Inactivation of these enzymes is sufficient to severely impair the ability of cells to respond to DNA damage. Given their central role in the pathway, several layers of regulation act at this nodal signaling point. Here we will summarize current knowledge on the roles of RNF8 and RNF168 in maintaining genome integrity with particular emphasis on recent insights into the multiple layers of regulation that act on these enzymes to fine-tune the cellular response to DNA lesions.

From crude protein extracts of Pseudomonas putida KT2440, we identified a small protein, TurA, able to bind to DNA fragments bearing the entire Pu promoter sequence of the TOL plasmid. The knock-out inactivation of the turA gene resulted in enhanced transcription initiation from the Pu promoter, initially suggesting a negative regulatory role of TurA on Pu expression. Ectopic expression of TurA both in P. putida and in Escherichia coli reporter strains and transcription in vitro of the Pu promoter in the presence of purified TurA confirmed the TurA repressor role on Pu activity. turA gene inactivation did not significantly alter two well characterized physiological regulations of the Pu expression in routine conditions of cultivation, exponential silencing, and carbon-mediated repression, respectively. However, the growth at suboptimal temperatures resulted in a TurA-dependent increase of Pu repression. These results strongly suggest that a physiological significance of the negative role of TurA on Pu activity could be limitation of the expression of the toluene-degrading enzymes at suboptimal growth temperatures. Therefore, the identification of TurA as Pu-binding protein revealed a novel physiological modulation of Pu promoter that is different from those strictly nutritional described previously.