DNA stimulates the deacetylase SIRT6 to mono-ADP-ribosylate proteins with histidine repeats

Sirtuins are the NAD⁺-dependent class III lysine deacylases (KDACs). Members of this family have been linked to longevity and a wide array of different diseases, motivating the pursuit of sirtuin modulator compounds. Sirtuin 6 (SIRT6) is a primarily nuclear KDAC that deacetylates histones to facilitate gene repression. In addition to this canonical posttranslational modification "eraser" function, SIRT6 can use NAD⁺ instead to "write" mono-ADP-ribosylation (mARylation) on target proteins. This enzymatic function has been primarily associated with SIRT6's role in the DNA damage response. This modification has been challenging to study because it is not clear under what precise cellular contexts it occurs, only a few substrates are known, and potential interference from other ADP-ribosyltransferases in cells, among other reasons. In this work, we used commercially available ADP-ribosylation detection reagents to investigate the mARylation activity of SIRT6 in a reconstituted system. We observed that SIRT6 is activated in its mARylation activity by binding to dsDNA ends. We further identified a surprising target motif within biochemical substrates of SIRT6, polyhistidine repeat tracts, which are present in several previously identified SIRT6 mARylation substrates. This work provides important context for SIRT6 mARylation activity, in contrast to its KDAC activity, and generates a list of new potential SIRT6 mARylation substrates based on the polyhistidine motif.