Activation of SIRT6 Deacetylation by DNA Strand Breaks

Abstract

SIRT6 is an emerging regulator of longevity. Overexpression of SIRT6 extends the lifespan of mice. Conversely, SIRT6 knockout mice demonstrate severe metabolic defects and a shortened lifespan. The discrepancy between SIRT6's weak in vitro activity and robust in vivo activity has led to the hypothesis that this enzyme can be activated in response to DNA damage in cells.Here, we demonstrate that the deacetylase activity of SIRT6 can be stimulated by DNA strand breaks for synthetic peptide and histone substrates. The mechanism of activation is further explored by using an integrative chemical biology approach. SIRT6 can be preferentially activated by DNA lesions harboring a 5′-phosphate. The N-and C-termini of SIRT6 are strictly required for DNA break-induced activation. Additionally, the defatty-acylase activity of SIRT6 is also sensitive to DNA breaks, although the physiological significance needs further investigation. Collectively, our study sheds important light on the cellular regulation of diverse SIRT6 activities and suggests possible strategies for effective SIRT6 activation.

DNA stimulates SIRT6 to mono-ADP-ribosylate proteins within histidine repeats

“…28,42 We compared the binding of SIRT6-WT and a SIRT6 C-terminal truncation (SIRT6-Δ298-355, abbreviated as "SIRT6-ΔC") to the ds601 DNA by gel shift assay. Our results recapitulated prior findings 27,28 in that both proteins bind the ds601 DNA, but the binding of the SIRT6-ΔC is weaker than the WT. The SIRT6-WT has a higher affinity for the DNA and exhibits a larger shift beginning at the 2.0 µM SIRT6 concentration that is not observed with the SIRT6-ΔC up to 7 µM protein (Figure S2C).…”

“…We next tested circular plasmid DNA and found that it did not activate mARylation by SIRT6 (Figure 2C), which is consistent with previous studies of SIRT6 DNA binding preferences. 27,29,30 Altogether, these data suggest that SIRT6 binding at DNA ends is important for the activation of its MARylase activity and are consistent with a model of SIRT6 dimerization at dsDNA ends. 29,30 All the mARylation assays were incubated at 37°C for 2 h unless otherwise noted.…”

“…[19][20][21][22] SIRT6's relatively low activity on acetylated peptides 21,[23][24][25] has led to an idea that SIRT6 alone possesses insufficient deacetylase activity. 26,27 However, biochemical and structural data show that SIRT6 binds to the nucleosome with high affinity, rendering it active toward this substrate, 19,20,1,28,24,22 providing a rationale for its low activity on histone peptides compared to the context of a nucleosome substrate. In addition, SIRT6 has been shown to bind non-nucleosomal DNA, or so-called "free DNA," and its ability to bind DNA has been linked to its role in the DNA damage response.…”

“…[29][30][31] Moreover, a recent paper reported that binding to free DNA increases SIRT6 activity on acylated peptides. 27 In addition to its deacetylase function, SIRT6 can deacylate, 32 defatty-acylate, 26,25 or mono-ADP-ribosylate ("mARylate") [33][34][35][36][37][38] various substrates. Much like PARP1/2-catalyzed poly-ADP-ribosylation (pARylation), SIRT6-catalyzed mARylation is associated primarily with oxidative stress and DNA damage conditions.…”