The overarching goal of this renewal application is to understand the mechanisms of age-related genomic instability and develop strategies to counteract it. In the past funding period we demonstrated that DNA double- strand break (DSB) repair becomes less efficient with age. Next, we showed that SIRT6 is an upstream regulator of DSB repair and overexpression of SIRT6 in senescent cells rescues the repair decline. Additionally, we found that SIRT6 maintains genome stability by silencing LINE1 transposons. Next, we set out to understand SIRT6 regulation. We discovered that JNK phosphorylates SIRT6 on S10 and this phosphorylation stimulates SIRT6 function in DSB repair. Recently, we generated knock-in mice with non- phosphorylatable SIRT6 S10A and phospho-mimetic S10E mutations. Our preliminary data suggest that S10E mice are more resistant to stress and have lower levels of DNA damage. Furthermore, we identified that SIRT6 is phosphorylated by AMPK on T294 and this modification enhances SIRT6 function in DSB repair. Intriguingly, AMPK phosphorylation consensus site is only found in long-lived mammals including human, but is absent in short-lived mammals such as mice. Furthermore, our preliminary data suggest that SIRT6 mono-ADP ribosylates AMPK? on K60, located very close to T172 activation site, possibly directly regulating AMPK activity. This application seeks to examine how activating SIRT6 affects aging and genome stability using SIRT6 S10 mutant mice and dissect the cross talk between SIRT6 and AMPK. This application will address the following questions: Do mice with constitutively active SIRT6 live longer? Does constitutive activation of SIRT6 have negative effect on fitness? How does activation of SIRT6 affect silencing of LINE1s? What is the function of SIRT6 phosphorylaton by AMPK? What is the function of AMPK mono-ADP ribosylation by SIRT6? Aim 1: Examine in vivo function of SIRT6 S10 phosphorylation in the context of aging and stress. Subaim 1a. We will test the hypothesis that mice with activated SIRT6 (S10E) are more resistant to oxidative stress and longer-lived than the WT or S10A mice. This aim will also test if there are fitness trade-offs associated with constitutively activated SIRT6 and upregulated DNA repair. Subaim 1b. We will examine activation of LINE1 transposons in the WT, SIRT6 S10A and SIRT6 S10E mice. Our data show that SIRT6 is required for silencing of L1 transposons, while S10 phosphorylation recruits SIRT6 to DNA breaks. Here we will test whether SIRT6 S10 variants lead to higher or lower transposon activity during stress and aging. Aim 2: Examine the SIRT6-AMPK signaling axis and the function of SIRT6 T294 phosphorylation. Our hypothesis is that SIRT6-AMPK axis regulates DNA repair in response to nutrition status. Subaim 2a. We will examine the effect of SIRT6 phosphorylation by AMPK on SIRT6-mediated DNA repair and control of glycolytic genes. We will compare the SIRT6-AMPK axis in mouse and in human. Subaim 2b. We will examine the effect of mono-ADP-ribosylation of AMPK by SIRT6 on AMPK activity in vitro and in vivo.