DNA double-strand breaks (DSB) are the most deleterious type of DNA damage. In this work, we show that SIRT6 directly recognizes DNA damage through a tunnel-like structure that has high affinity for DSB. SIRT6 relocates to sites of damage independently of signaling and known sensors. It activates downstream signaling for DSB repair by triggering ATM recruitment, H2AX phosphorylation and the recruitment of proteins of the homologous recombination and non-homologous end joining pathways. Our findings indicate that SIRT6 plays a previously uncharacterized role as a DNA damage sensor, a critical factor in initiating the DNA damage response (DDR). Moreover, other Sirtuins share some DSB-binding capacity and DDR activation. SIRT6 activates the DDR before the repair pathway is chosen, and prevents genomic instability. Our findings place SIRT6 as a sensor of DSB, and pave the road to dissecting the contributions of distinct DSB sensors in downstream signaling.

DNA double strand breaks are the most deleterious type of DNA damage. In this work, we show that SIRT6 directly recognizes DNA damage through a tunnel-like structure, with high affinity for double strand breaks. It relocates to sites of damage independently of signalling and known sensors and activates downstream signalling cascades for double strand break repair by triggering ATM recruitment, H2AX phosphorylation and the recruitment of proteins of the Homologous Recombination and Non-Homologous End Joining pathways. Our findings indicate that SIRT6 plays a previously uncharacterized role as DNA damage sensor, which is critical for initiating the DNA damage response (DDR). Moreover, other Sirtuins share some DSB binding capacity and DDR activation. SIRT6 activates the DDR, before the repair pathway is chosen, and prevents genomic instability. Our findings place SIRT6 at the top of the DDR and pave the road to dissect the contributions of distinct double strand break sensors in downstream signalling.

The pituitary adenylate cyclase-activating polypeptide receptor (PAC1, also known as ADCYAP1R1) is associated with post-traumatic stress disorder and modulation of stress response in general. Alternative splicing of PAC1 results in multiple gene products, which differ in their mode of signalling and tissue distribution. However, the roles of distinct splice variants in the regulation of stress behavior is poorly understood. Alternative splicing of a short exon, which is known as the "hop cassette", occurs during brain development and in response to stressful challenges. To examine the function of this variant, we generated a splice-specific zebrafish mutant lacking the hop cassette, which we designated 'hopless'. We show that hopless mutant larvae display increased anxiety-like behavior, including reduced dark exploration and impaired habituation to dark exposure. Conversely, adult hopless mutants displayed superior ability to rebound from an acute stressor, as they exhibited reduced anxiety-like responses to an ensuing novelty stress. We propose that the developmental loss of a specific PAC1 splice variant mimics prolonged mild stress exposure, which in the long term, predisposes the organism's stress response towards a resilient phenotype. Our study presents a unique genetic model demonstrating how early-life state of anxiety paradoxically correlates with reduced stress susceptibility in adulthood. PAC1 (a.k.a. Adcyap1r1) is a G-protein coupled receptor (GPCR) that serves as the high-affinity receptor for the pituitary adenylate cyclase-activating polypeptide (PACAP). PAC1 has pleiotropic functions and was demonstrated to be involved in the regulation of several homeostatic processes including metabolic rate and food consumption 1,2 , circadian rhythm 3 and, in particular, stress response 4,5. Intracerebroventricular injection of PACAP increased phosphorylated cyclic AMP response element binding protein (pCREB) and corticotropin-releasing hormone (CRH) immunoreactivity in the rat paraventricular nucleus 6. PACAP knockout mice display blunted hypothalamic CRH levels in response to restraint challenge 7. PACAP/PAC1 signaling was also associated with hypothalamo-pituitary-adrenal activity and stress-related behaviors in humans and rodents 5,8,9. Furthermore, this pathway was correlated with stress-related risky behaviors in human and rodents 10,11. Overall, these findings support positive stress regulation by PAC1; yet, some reports suggest that it may also act to suppress stress phenotypes 4,12. It has been suggested that genetic vulnerability to post-traumatic stress disorder (PTSD) may depend on PAC1 expression and single-nucleotide polymorphism (SNP) in the PAC1 gene. Ressler et al. demonstrated that a specific PAC1 genotype is strongly correlated with susceptibility to PTSD in women, probably due to perturbed expression of PAC1 resulting in impaired stress responses 13. The same PAC1 SNP was associated with PTSD in African-American females, emotional numbing in traumatized earthquake Chinese survivors, dark-e...

25The pituitary adenylate cyclase-activating polypeptide receptor (PAC1, also known as 26 ADCYAP1R1) is associated with post-traumatic stress disorder and modulation of 27 stress response in general. Alternative splicing of PAC1 results in multiple gene 28 products, which differ in their mode of signalling and tissue distribution. However, 29 the roles of distinct splice variants in the regulation of stress behavior is poorly 30 understood. Alternative splicing of a short exon, which is known as the "hop 31 cassette", occurs during brain development and in response to stressful challenges. To 32 examine the function of this variant, we generated a splice-specific zebrafish mutant 33 lacking the hop cassette, which we designated 'hopless'. We show that hopless mutant 34 larvae display increased anxiety-like behavior, including reduced dark exploration and 35 impaired habituation to dark exposure. Conversely, adult hopless mutants displayed 36 superior ability to rebound from an acute stressor, as they exhibited reduced anxiety-37 like responses to an ensuing novelty stress. We propose that the developmental loss of 38 a specific PAC1 splice variant mimics prolonged mild stress exposure, which in the 39 long term, predisposes the organism's stress response towards a resilient phenotype. 40Our study presents a unique genetic model demonstrating how early-life state of 41 anxiety paradoxically correlates with reduced stress susceptibility in adulthood. 42 43 44 45 46 47 48 49 50 51 52 53 PAC1 (a.k.a. Adcyap1r1) is a G-protein coupled receptor (GPCR) that serves as the 54 high-affinity receptor for pituitary adenylate cyclase-activating polypeptide (PACAP). 55 PAC1 has pleiotropic functions and was demonstrated to be involved in the regulation 56 of several homeostatic processes including metabolic rate and food consumption 1,2 , 57 circadian rhythm 3 and, in particular, stress response 4,5 . Intracerebroventricular injection 58 of PACAP increased phosphorylated cyclic AMP response element binding protein 59(pCREB) and CRH immunoreactivity in the rat paraventricular nucleus 6 . PACAP 60 knockout mice display blunted hypothalamic CRH levels in response to restraint 61 challenge 7 . PACAP/PAC1 signaling was also associated with hypothalamo-pituitary-62 adrenal activity and stress-related behaviors in humans and rodents 5,8,9 . Furthermore, 63 this pathway was correlated with stress-related risky behaviors in human and 64 rodents 10,11 . Overall, these findings support positive stress regulation by PAC1; yet, 65 some data suggest that it may also act to suppress stress phenotypes 4,12 . 66It has been suggested that genetic vulnerability to post-traumatic stress disorder 67 (PTSD) may depend on PAC1 expression and single-nucleotide polymorphism (SNP) 68 in the PAC1 gene. Ressler et al. demonstrated that a specific PAC1 genotype is strongly 69 correlated with susceptibility to PTSD in women, probably due to perturbed expression 70 of PAC1 resulting in impaired stress responses 13 . The same PAC1 SNP was associat...

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