Edited by John M. Denu Gcn5 and sirtuins are highly conserved histone acetyltransferase (HAT) and histone deacetylase (HDAC) enzymes that were first characterized as regulators of gene expression. Although histone tails are important substrates of these enzymes, they also target many nonhistone proteins that function in diverse biological processes. However, the mechanisms used by these enzymes to choose their nonhistone substrates are unknown. Previously, we used SILAC-based MS to identify novel nonhistone substrates of Gcn5 and sirtuins in yeast and found a shared target consensus sequence. Here, we use a synthetic biology approach to demonstrate that this consensus sequence can direct acetylation and deacetylation targeting by these enzymes in vivo. Remarkably, fusion of the sequence to a nonsubstrate confers de novo acetylation that is regulated by both Gcn5 and sirtuins. We exploit this synthetic fusion substrate as a tool to define subunits of the Gcn5-containing SAGA and ADA complexes required for nonhistone protein acetylation. In particular, we find a key role for the Ada2 and Ada3 subunits in regulating acetylations on our fusion substrate. In contrast, other subunits tested were largely dispensable, including those required for SAGA stability. In an extended analysis, defects in proteome-wide acetylation observed in ada3⌬ mutants mirror those in ada2⌬ mutants. Altogether, our work argues that nonhistone protein acetylation by Gcn5 is determined in part by specific amino acids surrounding target lysines but that even optimal sequences require both Ada2 and Ada3 for robust acetylation. The synthetic fusion substrate we describe can serve as a tool to further dissect the regulation of both Gcn5 and sirtuin activities in vivo.

Nicotinamide is both a reaction product and an inhibitor of the conserved sirtuin family of deacetylases, which have been implicated in a broad range of cellular functions in eukaryotes from yeast to humans. Phenotypes observed following treatment with nicotinamide are most often assumed to stem from inhibition of one or more of these enzymes. Here, we used this small molecule to inhibit multiple sirtuins at once during treatment with DNA damaging agents in the Saccharomyces cerevisiae model system. Since sirtuins have been previously implicated in the DNA damage response, we were surprised to observe that nicotinamide actually increased the survival of yeast cells exposed to the DNA damage agent MMS. Remarkably, we found that enhanced resistance to MMS in the presence of nicotinamide was independent of all five yeast sirtuins. Enhanced resistance was also independent of the nicotinamide salvage pathway, which uses nicotinamide as a substrate to generate NAD+, and of a DNA damage-induced increase in the salvage enzyme Pnc1. Our data suggest a novel and unexpected function for nicotinamide that has broad implications for its use in the study of sirtuin biology across model systems.KEYWORDS nicotinamide; sirtuins; DNA damage; checkpoint; Pnc1; NAD+ T HE DNA damage checkpoint is a highly conserved signaling cascade initiated in response to DNA lesions. In the budding yeast Saccharomyces cerevisiae, checkpoint activation begins with the exposure of single-stranded DNA (ssDNA), either from exonuclease-resected DNA doublestrand breaks (DSBs), or from stalled replication forks during S phase. Resected DNA coated by the ssDNA binding protein RPA is thought to act as a landing pad for Mec1-Ddc2 complexes (Melo and Toczyski 2002;Gobbini et al. 2013;Edenberg et al. 2014a). Mec1 is a sensor kinase that, in concert with adaptor proteins such as Rad9 or Mrc1, phosphorylates downstream checkpoint targets, including the Rad53 and Chk1 transducing kinases (Melo and Toczyski 2002;Gobbini et al. 2013; Bastos de Oliveira et al. 2015). Following autophosphorylation and release from adaptors, Rad53 is thought to move throughout the cell to phosphorylate targets that promote cell cycle arrest, the inhibition of late-firing origins of replication, and a global transcriptional response (Melo and Toczyski 2002;Jaehnig et al. 2013;Edenberg et al. 2014a). While the DNA damage response is traditionally associated with phosphorylationbased signaling cascades, it has recently emerged that other post-translational modifications including ubiquitylation, sumoylation, and acetylation play prominent roles in the response in both yeast and other eukaryotes (Downey and Durocher 2006a;Psakhye and Jentsch 2012;Panier and Durocher 2013;Edenberg et al. 2014a;Elia et al. 2015).Acetylation of lysine residues is catalyzed by histone acetyltransferases (HATs) and reversed by histone deacetylases (HDACs). Despite their names, these enzymes also have nonhistone targets that play critical roles in maintaining cellular homeostasis in organisms from ba...

ABSTRACT:Gcn5 and sirtuins are highly conserved HAT and HDAC enzymes that were first characterised as regulators of gene expression. Although histone tails are important substrates of these enzymes, these proteins also target many non-histone substrates that participate in diverse biological processes. The mechanisms used by these enzymes to choose their non-histone substrates is unclear. In this work, we use a unique synthetic biology approach in S. cerevisiae to demonstrate that a shared target sequence can act as a determinant of substrate selection for Gcn5 and sirtuins. We also exploit this system to define specific subunits of the Gcn5-containing ADA complex as regulators of non-histone acetylations proteome-wide.

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