Abstract 1507 DNA stimulates SIRT6 to mono-ADP-ribosylate within histidine repeats

Genome stability is integral for organismal homeostasis and survival. Sirtuin6 (SIRT6) is a member of the sirtuin family and plays important roles as altering expression in both cellular and animal models leads to notable phenotypes. SIRT6 has diverse enzymatic functions acting as a class lll histone deacetylase which is associated with chromatin condensation and gene repression. SIRT6 is also an ADP-Ribosyltransferase and can modify itself as well as other targets notably during DNA damage conditions which is also a hallmark of ADP-Ribosylation. For this reason, we asked if the addition of DNA to in vitro SIRT6 ADP-Ribosylation assays would affect activity. Using Western Blot analysis, we show that dsDNA inhibits SIRT6 auto-modification. However, dsDNA promotes ADP-Ribosylation of BAF170(SMARCC2), a known SIRT6 target as well as novel targets MeCP2 and NLK1. Histidine has been identified to be ADP-Ribosylated through two proteomic datasets but there is no known enzyme responsible for this. We demonstrate that SIRT6 ADP-Ribosylates BAF170, MeCP2 and NLK1 within histidine repeats, identifying it as the first enzyme to ADP-ribosylate histidine. Our data also shows how SIRT6 binds DNA in specific molar ratios to inhibit auto modification and promote target modification. Cryo-EM structures have elucidated SIRT6 deacetylation function while bound to the nucleosome however SIRT6 does not ADP-ribosylate nucleosomes in our hands. Taken together, our results highlight the multifaceted role of SIRT6 by supporting the model that SIRT6 deacetylase activity is associated with nucleosome binding and SIRT6 ADP-Ribosylation is regulated by DNA binding.