Acetyl-coenzyme A (CoA) synthetase (Acs) is an enzyme central to metabolism in prokaryotes and eukaryotes. Acs synthesizes acetyl CoA from acetate, adenosine triphosphate, and CoA through an acetyl-adenosine monophosphate (AMP) intermediate. Immunoblotting and mass spectrometry analysis showed that Salmonella enterica Acs enzyme activity is posttranslationally regulated by acetylation of lysine-609. Acetylation blocks synthesis of the adenylate intermediate but does not affect the thioester-forming activity of the enzyme. Activation of the acetylated enzyme requires the nicotinamide adenine dinucleotide-dependent protein deacetylase activity of the CobB Sir2 protein from S. enterica. We propose that acetylation modulates the activity of all the AMP-forming family of enzymes, including nonribosomal peptide synthetases, luciferase, and aryl- and acyl-CoA synthetases. These findings extend our knowledge of the roles of Sir2 proteins in gene silencing, chromosome stability, and cell aging and imply that lysine acetylation is a common regulatory mechanism in eukaryotes and prokaryotes.

The yeast Sir2 protein, required for transcriptional silencing, has an NAD ؉ -dependent histone deacetylase (HDA) activity. Yeast extracts contain a NAD ؉ -dependent HDA activity that is eliminated in a yeast strain from which SIR2 and its four homologs have been deleted. This HDA activity is also displayed by purified yeast Sir2p and homologous Archaeal, eubacterial, and human proteins, and depends completely on NAD ؉ in all species tested. The yeast NPT1 gene, encoding an important NAD ؉ synthesis enzyme, is required for rDNA and telomeric silencing and contributes to silencing of the HM loci. Null mutants in this gene have significantly reduced intracellular NAD ؉ concentrations and have phenotypes similar to sir2 null mutants. Surprisingly, yeast from which all five SIR2 homologs have been deleted have relatively normal bulk histone acetylation levels. The evolutionary conservation of this regulated activity suggests that the Sir2 protein family represents a set of effector proteins in an evolutionarily conserved signal transduction pathway that monitors cellular energy and redox states. T ranscriptional silencing is a regulatory mechanism that results in the inactivation of large blocks of chromosomes via an altered chromatin structure. In Saccharomyces cerevisiae, silencing is observed at the HM silent mating type loci (reviewed in ref. 1), telomeres (2), and at the rDNA locus (3, 4). Although a different subset of proteins is required for silencing at each of the three loci, all types of silencing require Sir2p (3, 5). The Sir2 family of proteins is highly conserved and found in Archaea, eubacteria, and metazoa (6-9). A recent study showed that yeast and mouse Sir2p have NAD ϩ -dependent HDA activity on histone peptides specific for Lys-16 of histone H4 (10), an important residue for silencing (11-13). Earlier work had suggested that Sir2p might have HDA activity. Acetylated histones were inefficiently immunoprecipitated from the silent mating type (HM) loci relative to the expressed mating type (MAT) locus, and overexpression of Sir2p led to changes in levels of bulk histone acetylation (14,15). Other recent papers demonstrated a phosphotransferase activity for Sir2p, with NAD ϩ as the source of phosphate and a variety of proteins implicated as targets of ADP ribosylation (9, 16). A sir2 missense mutation that destroys this in vitro activity also destroys silencing in vivo. These results suggest that the Sir2p family is a group of ADP-ribosyl transferases (ARTs).We show here that Archaeal, eubacterial, and human Sir2 proteins, like Sir2p, have potent NAD ϩ -dependent HDA activity in vitro. The importance of NAD ϩ to the in vivo activity of Sir2p is underscored by our finding that mutations in the S. cerevisiae NPT1 gene lead to severe silencing defects. NPT1 encodes a nicotinate phosphoribosyltransferase, required for NAD ϩ synthesis through a salvage pathway. Intracellular NAD ϩ levels are significantly lower in npt1 null mutants than in the wild type, providing independent evidence that NAD ϩ is critic...

Our results underscore the critical importance of Hst3/Hst4p in controlling histone H3 K56Ac and thereby maintaining chromosome integrity.

The SIR2 (silent information regulator 2) gene family has diverse functions in yeast including gene silencing, DNA repair, cell-cycle progression, and chromosome fidelity in meiosis and aging. Human homologues, termed sirtuins, are highly conserved but are of unknown function. We previously identified a large imprinted gene domain on 11p15.5 and investigated the 11p15.5 sirtuin SIRT3. Although this gene was not imprinted, we found that it is localized to mitochondria, with a mitochondrial targeting signal within a unique N-terminal peptide sequence. The encoded protein was found also to possess NAD ؉ -dependent histone deacetylase activity. These results suggest a previously unrecognized organelle for sirtuin function and that the role of SIRT3 in mitochondria involves protein deacetylation.

The Sir2 enzyme family is responsible for a newly classified chemical reaction, NAD(+)-dependent protein deacetylation. New peptide substrates, the reaction mechanism, and the products of the acetyl transfer to NAD(+) are described for SIR2. The final products of SIR2 reactions are the deacetylated peptide and the 2' and 3' regioisomers of O-acetyl ADP ribose (AADPR), formed through an alpha-1'-acetyl ADP ribose intermediate and intramolecular transesterification reactions (2' --> 3'). The regioisomers, their anomeric forms, the interconversion rates, and the reaction equilibria were characterized by NMR, HPLC, 18O exchange, and MS methods. The mechanism of acetyl transfer to NAD(+) includes (1) ADP ribosylation of the peptide acyl oxygen to form a high-energy O-alkyl amidate intermediate, (2) attack of the 2'-OH group on the amidate to form a 1',2'-acyloxonium species, (3) hydrolysis to 2'-AADPR by the attack of water on the carbonyl carbon, and (4) an SIR2-independent transesterification equilibrating the 2'- and 3'-AADPRs. This mechanism is unprecedented in ADP-ribosyl transferase enzymology. The 2'- and 3'-AADPR products are candidate molecules for SIR2-initiated signaling pathways.

Sir2 proteins are NAD(+)-dependent protein deacetylases that play key roles in transcriptional regulation, DNA repair, and life span regulation. The structure of an archaeal Sir2 enzyme, Sir2-Af2, bound to an acetylated p53 peptide reveals that the substrate binds in a cleft in the enzyme, forming an enzyme-substrate beta sheet with two flanking strands in Sir2-Af2. The acetyl-lysine inserts into a conserved hydrophobic tunnel that contains the active site histidine. Comparison with other structures of Sir2 enzymes suggests that the apoenzyme undergoes a conformational change upon substrate binding. Based on the Sir2-Af2 substrate complex structure, mutations were made in the other A. fulgidus sirtuin, Sir2-Af1, that increased its affinity for the p53 peptide.

Deacetylation of histone H3 K56, regulated by the sirtuins Hst3p and Hst4p, is critical for maintenance of genomic stability. However, the physiological consequences of a lack of H3 K56 deacetylation are poorly understood. Here we show that cells lacking Hst3p and Hst4p, in which H3 K56 is constitutively hyperacetylated, exhibit hallmarks of spontaneous DNA damage, such as activation of the checkpoint kinase Rad53p and upregulation of DNA-damage inducible genes. Consistently, hst3 hst4 cells display synthetic lethality interactions with mutations that cripple genes involved in DNA replication and DNA double-strand break (DSB) repair. In most cases, synthetic lethality depends upon hyperacetylation of H3 K56 because it can be suppressed by mutation of K56 to arginine, which mimics the nonacetylated state. We also show that hst3 hst4 phenotypes can be suppressed by overexpression of the PCNA clamp loader large subunit, Rfc1p, and by inactivation of the alternative clamp loaders CTF18, RAD24, and ELG1. Loss of CTF4, encoding a replisome component involved in sister chromatid cohesion, also suppresses hst3 hst4 phenotypes. Genetic analysis suggests that CTF4 is a part of the K56 acetylation pathway that converges on and modulates replisome function. This pathway represents an important mechanism for maintenance of genomic stability and depends upon proper regulation of H3 K56 acetylation by Hst3p and Hst4p. Our data also suggest the existence of a precarious balance between Rfc1p and the other RFC complexes and that the nonreplicative forms of RFC are strongly deleterious to cells that have genomewide and constitutive H3 K56 hyperacetylation. Hst3p and Hst4p belong to a highly conserved family of NAD 1 -dependent protein deacetylases, known as the Sir2 protein family or sirtuins (Brachmann et al. 1995;Imai et al. 2000;Landry et al. 2000;Smith et al. 2000). The importance of K56 deacetylation is evident from the high level of genomic instability observed in hst3 hst4 cells. Cells lacking HST3 and HST4 show a plethora of chromatin-associated phenotypes (Brachmann et al. 1995) resulting from hyperacetylation of K56 in H3; mutation of K56 to arginine (K56R) suppresses nearly all these hst3 hst4 phenotypes (Celic et al. 2006;Maas et al. 2006). hst3 hst4 cells also accumulate spontaneous suppressors at a high rate (Brachmann et al. 1995) and the majority of these suppressors appear to adapt to the high level of K56 acetylation rather than preventing acetylation . We show here that hst3 hst4 phenotypes are alleviated by overexpression of RFC1, encoding the large subunit of the clamp loader (Howell et al. 1994), supporting the notion that the inability to deacetylate K56 interferes with normal DNA replication. These phenotypes are also suppressed by 1

In Saccharomyces cerevisiae, histone H3 lysine 56 acetylation (H3K56Ac) is present in newly synthesized histones deposited throughout the genome during DNA replication. The sirtuins Hst3 and Hst4 deacetylate H3K56 after S phase, and virtually all histone H3 molecules are K56 acetylated throughout the cell cycle in hst3D hst4D mutants. Failure to deacetylate H3K56 causes thermosensitivity, spontaneous DNA damage, and sensitivity to replicative stress via molecular mechanisms that remain unclear. Here we demonstrate that unlike wild-type cells, hst3D hst4D cells are unable to complete genome duplication and accumulate persistent foci containing the homologous recombination protein Rad52 after exposure to genotoxic drugs during S phase. In response to replicative stress, cells lacking Hst3 and Hst4 also displayed intense foci containing the Rfa1 subunit of the single-stranded DNA binding protein complex RPA, as well as persistent activation of DNA damage-induced kinases. To investigate the basis of these phenotypes, we identified histone point mutations that modulate the temperature and genotoxic drug sensitivity of hst3D hst4D cells. We found that reducing the levels of histone H4 lysine 16 acetylation or H3 lysine 79 methylation partially suppresses these sensitivities and reduces spontaneous and genotoxin-induced activation of the DNA damage-response kinase Rad53 in hst3D hst4D cells. Our data further suggest that elevated DNA damage-induced signaling significantly contributes to the phenotypes of hst3D hst4D cells. Overall, these results outline a novel interplay between H3K56Ac, H3K79 methylation, and H4K16 acetylation in the cellular response to DNA damage.KEYWORDS DNA damage repair and checkpoint response; H3 lysine 56 acetylation; H3 lysine 79 methylation; H4 lysine 16 acetylation; chromatin structure C HROMATIN structure influences major DNA metabolic processes such as transcription, DNA replication, and DNA repair (Wurtele and Verreault 2006;Campos and Reinberg 2009). The basic building block of chromatin is the nucleosome core particle composed of 147 bp of DNA wrapped around the surface of a protein octamer consisting of two molecules each of histones H2A, H2B, H3, and H4. During DNA replication, preexisting (old) histones are segregated onto sister chromatids, while new histones are deposited onto replicated DNA in order to restore normal nucleosome density on nascent sister chromatids (Ransom et al. 2010;Li and Zhang 2012). In humans, newly synthesized histones H3 and H4 are acetylated on multiple residues within their N-terminal tails (Ruiz-Carrillo et al. 1975;Benson et al. 2006;Jasencakova et al. 2010) and then are deacetylated following their incorporation into chromatin (Jackson et al. 1976;