Nicotinamide Suppresses the DNA Damage Sensitivity of Saccharomyces cerevisiae Independently of Sirtuin Deacetylases

Abstract

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...

A Screen for Candidate Targets of Lysine Polyphosphorylation Uncovers a Conserved Network Implicated in Ribosome Biogenesis

“…Details are contained in the Supplemental Experimental Procedures. Protein extracts were prepared using a trichloroacetic acid (TCA)-lysis method (Rö ssl et al, 2016) and analyzed on Bis-Tris NuPAGE gels (Thermo Fisher Scientific NP0336BOX). VTC4-dependent electrophoretic shifts of potential targets were considered a ''hit.''…”

Histone deacetylase SirE regulates development, DNA damage response and aflatoxin production in Aspergillus flavus

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Nicotinamide‐induced mouse embryo developmental defect rescued by resveratrol and I‐CBP112

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