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PubMed2006

Bypassing the catalytic activity of SIR2 for SIR protein spreading in Saccharomyces cerevisiae.

Sir protein spreading along chromosomes and silencing in Saccharomyces cerevisiae requires the NAD+-dependent histone deacetylase activity of Sir2p. We tested whether this requirement could be bypassed at the HM loci and telomeres in cells containing a stably expressed, but catalytically inactive mutant of Sir2p, sir2-345p, plus histone mutants that mimic the hypoacetylated state normally created by Sir2p. Sir protein spreading was rescued in sir2-345 mutants expressing histones in which key lysine residues in their N-termini had been mutated to arginine. Mating in these mutants was also partially restored upon overexpression of Sir3p. Together, these results indicate that histone hypoacetylation is sufficient for Sir protein spreading in the absence of production of 2'-O-acetyl-ADP ribose by sir2p and Sir2p's enzymatic function for silencing can be bypassed in a subset of cells in a given population. These results also provide genetic evidence for the existence of additional critical substrates of Sir2p for silencing in vivo.

Catalysis↗
PubMed2001

Chemistry of gene silencing: the mechanism of NAD+-dependent deacetylation reactions.

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.

Adenosine Diphosphate Ribose↗
PubMed2006

Calorie restriction increases life span: a molecular mechanism.

Calorie restriction increases the life span of many organisms, from yeast to mammals. In yeast, the life span gene affected by calorie restriction is Sir2 (silent information regulator 2). In mammals, Sirt1, an ortholog of Sir2, controls the metabolism of white adipose tissue. Calorie restriction activates Sirt1, and the expressed Sirt1 protein inhibits the action of peroxysome proliferator-activator receptor gamma (PPARgamma), the nuclear receptor that promotes adipogenesis. The effect is lipolysis and loss of fat. Lowering of adiposity appears to be one mechanism whereby calorie restriction affects life span.

Caloric Restriction↗
PubMed2003

Mechanism of nicotinamide inhibition and transglycosidation by Sir2 histone/protein deacetylases.

Silent information regulator 2 (Sir2) enzymes catalyze NAD+-dependent protein/histone deacetylation, where the acetyl group from the lysine epsilon-amino group is transferred to the ADP-ribose moiety of NAD+, producing nicotinamide and the novel metabolite O-acetyl-ADP-ribose. Sir2 proteins have been shown to regulate gene silencing, metabolic enzymes, and life span. Recently, nicotinamide has been implicated as a direct negative regulator of cellular Sir2 function; however, the mechanism of nicotinamide inhibition was not established. Sir2 enzymes are multifunctional in that the deacetylase reaction involves the cleavage of the nicotinamide-ribosyl, cleavage of an amide bond, and transfer of the acetyl group ultimately to the 2'-ribose hydroxyl of ADP-ribose. Here we demonstrate that nicotinamide inhibition is the result of nicotinamide intercepting an ADP-ribosyl-enzyme-acetyl peptide intermediate with regeneration of NAD+ (transglycosidation). The cellular implications are discussed. A variety of 3-substituted pyridines was found to be substrates for enzyme-catalyzed transglycosidation. A Brönsted plot of the data yielded a slope of +0.98, consistent with the development of a nearly full positive charge in the transition state, and with basicity of the attacking nucleophile as a strong predictor of reactivity. NAD+ analogues including beta-2'-deoxy-2'-fluororibo-NAD+ and a His-to-Ala mutant were used to probe the mechanism of nicotinamide-ribosyl cleavage and acetyl group transfer. We demonstrate that nicotinamide-ribosyl cleavage is distinct from acetyl group transfer to the 2'-OH ribose. The observed enzyme-catalyzed formation of a labile 1'-acetylated-ADP-fluororibose intermediate using beta-2'-deoxy-2'-fluororibo-NAD+ supports a mechanism where, after nicotinamide-ribosyl cleavage, the carbonyl oxygen of acetylated substrate attacks the C-1' ribose to form an initial iminium adduct.

Amino Acid Substitution↗
PubMed2003

Role for human SIRT2 NAD-dependent deacetylase activity in control of mitotic exit in the cell cycle.

Studies of yeast have shown that the SIR2 gene family is involved in chromatin structure, transcriptional silencing, DNA repair, and control of cellular life span. Our functional studies of human SIRT2, a homolog of the product of the yeast SIR2 gene, indicate that it plays a role in mitosis. The SIRT2 protein is a NAD-dependent deacetylase (NDAC), the abundance of which increases dramatically during mitosis and is multiply phosphorylated at the G(2)/M transition of the cell cycle. Cells stably overexpressing the wild-type SIRT2 but not missense mutants lacking NDAC activity show a marked prolongation of the mitotic phase of the cell cycle. Overexpression of the protein phosphatase CDC14B, but not its close homolog CDC14A, results in dephosphorylation of SIRT2 with a subsequent decrease in the abundance of SIRT2 protein. A CDC14B mutant defective in catalyzing dephosphorylation fails to change the phosphorylation status or abundance of SIRT2 protein. Addition of 26S proteasome inhibitors to human cells increases the abundance of SIRT2 protein, indicating that SIRT2 is targeted for degradation by the 26S proteasome. Our data suggest that human SIRT2 is part of a phosphorylation cascade in which SIRT2 is phosphorylated late in G(2), during M, and into the period of cytokinesis. CDC14B may provoke exit from mitosis coincident with the loss of SIRT2 via ubiquitination and subsequent degradation by the 26S proteasome.

Acetylcysteine↗
PubMed2004

Structural basis for nicotinamide cleavage and ADP-ribose transfer by NAD(+)-dependent Sir2 histone/protein deacetylases.

Sir2 enzymes are broadly conserved from bacteria to humans and have been implicated to play roles in gene silencing, DNA repair, genome stability, longevity, metabolism, and cell physiology. These enzymes bind NAD(+) and acetyllysine within protein targets and generate lysine, 2'-O-acetyl-ADP-ribose, and nicotinamide products. To provide structural insights into the chemistry catalyzed by Sir2 proteins we report the high-resolution ternary structure of yeast Hst2 (homologue of Sir two 2) with an acetyllysine histone H4 peptide and a nonhydrolyzable NAD(+) analogue, carba-NAD(+), as well as an analogous ternary complex with a reaction intermediate analog formed immediately after nicotinamide hydrolysis, ADP-ribose. The ternary complex with carba-NAD(+) reveals that the nicotinamide group makes stabilizing interactions within a binding pocket harboring conserved Sir2 residues. Moreover, an asparagine residue, N116, strictly conserved within Sir2 proteins and shown to be essential for nicotinamide exchange, is in position to stabilize the oxocarbenium intermediate that has been proposed to proceed the hydrolysis of nicotinamide. A comparison of this structure with the ADP-ribose ternary complex and a previously reported ternary complex with the 2'-O-acetyl-ADP-ribose reaction product reveals that the ribose ring of the cofactor and the highly conserved beta1-alpha2 loop of the protein undergo significant structural rearrangements to facilitate the ordered NAD(+) reactions of nicotinamide cleavage and ADP-ribose transfer to acetate. Together, these studies provide insights into the chemistry of NAD(+) cleavage and acetylation by Sir2 proteins and have implications for the design of Sir2-specific regulatory molecules.

Adenosine Diphosphate Ribose↗
PubMed2001

SIR functions are required for the toleration of an unrepaired double-strand break in a dispensable yeast chromosome.

Unrepaired DNA double-strand breaks (DSBs) typically result in G(2) arrest. Cell cycle progression can resume following repair of the DSBs or through adaptation to the checkpoint, even if the damage remains unrepaired. We developed a screen for factors in the yeast Saccharomyces cerevisiae that affect checkpoint control and/or viability in response to a single, unrepairable DSB that is induced by HO endonuclease in a dispensable yeast artificial chromosome containing human DNA. SIR2, -3, or -4 mutants exhibit a prolonged, RAD9-dependent G(2) arrest in response to the unrepairable DSB followed by a slow adaptation to the persistent break, leading to division and rearrest in the next G(2). There are a small number of additional cycles before permanent arrest as microcolonies. Thus, SIR genes, which repress silent mating type gene expression, are required for the adaptation and the prevention of indirect lethality resulting from an unrepairable DSB in nonessential DNA. Rapid adaptation to the G(2) checkpoint and high viability were restored in sir(-) strains containing additional deletions of the silent mating type loci HML and HMR, suggesting that genes under mating type control can reduce the toleration of a single DSB. However, coexpression of MATa1 and MATalpha2 in Sir(+) haploid cells did not lead to lethality from the HO-induced DSB, suggesting that toleration of an unrepaired DSB requires more than one Sir(+) function.

DNA Damage↗
PubMed2002

Yph1p, an ORC-interacting protein: potential links between cell proliferation control, DNA replication, and ribosome biogenesis.

Immunoprecipitation of the origin recognition complex (ORC) from yeast extracts identified Yph1p, an essential protein containing a BRCT domain. Two Yph1p complexes were characterized. Besides ORC, MCM proteins, cell-cycle regulatory proteins, checkpoint proteins, 60S ribosomal proteins, and preribosome particle proteins were found to be associated with Yph1p. Yph1p is predominantly nucleolar and is required for 60S ribosomal subunit biogenesis and possibly for translation on polysomes. Proliferating cells depleted of Yph1p arrest in G(1) or G(2), with no cells in S phase, or significantly delay S phase progression after release from a hydroxyurea arrest. Yph1p levels decline as cells commit to exit the cell cycle, and levels vary depending on energy source. Yph1p may link cell proliferation control to DNA replication, ribosome biogenesis, and translation on polysomes.

Blotting, Western↗
PubMed1997

Direct evidence for SIR2 modulation of chromatin structure in yeast rDNA.

The yeast SIR2 gene maintains inactive chromatin domains required for transcriptional repression at the silent mating-type loci and telomeres. We previously demonstrated that SIR2 also acts to repress mitotic and meiotic recombination between the tandem ribosomal RNA gene array (rDNA). Here we address whether rDNA chromatin structure is altered by loss of SIR2 function by in vitro and in vivo assays of sensitivity to micrococcal nuclease and dam methyltransferase, respectively, and present the first chromatin study that maps sites of SIR2 action within the rDNA locus. Control studies at the MAT alpha locus also revealed a previously undetected MNase-sensitive site at the a1-alpha 2 divergent promoter which is protected in sir2 mutant cells by the derepressed a1-alpha 2 regulator. In rDNA, SIR2 is required for a more closed chromatin structure in two regions: SRR1, the major SIR-Responsive Region in the non-transcribed spacer, and SRR2, in the 18S rRNA coding region. None of the changes in rDNA detected in sir2 mutants are due to the presence of the a1-alpha 2 repressor. Reduced recombination in the rDNA correlates with a small, reproducible transcriptional silencing position effect. Deletion and overexpression studies demonstrate that SIR2, but not SIR1, SIR3 or SIR4, is required for this rDNA position effect. Significantly, rDNA transcriptional silencing and rDNA chromatin accessibility respond to SIR2 dosage, indicating that SIR2 is a limiting component required for chromatin modeling in rDNA.

Chromatin↗
PubMed2006

High-level bacterial expression and purification of human SirT2 protein for NMR studies.

Silent information regulator 2 (Sir2) proteins are a class of protein deacetylase enzymes that play key roles in transcriptional gene silencing, DNA repair, and aging. Here, we describe the high-level bacterial expression and purification of a human SirT2 construct that yields high resolution NMR spectra. By removing the N-terminal helix alpha0 and using Thioredoxin as a fusion partner, greater than 10 mg/L of purified protein can be obtained from minimal media. The protein is fully functional and enables NMR-based screening and structural studies of this important protein.

Escherichia coli↗
PubMed2005

Mechanism of transcriptional silencing in yeast.

Transcriptional silencing is a phenomenon in which the transcription of a gene by RNA polymerase II or III is repressed or not, dependent only on the gene's chromosomal location. Two prevailing models exist for silencing: (1) steric hindrance in silenced chromatin inhibits the binding of upstream activator proteins or polymerase or (2) silencing primarily blocks steps downstream of transcription preinitiation complex formation. Here, we test these models quantitatively for the case of SIR2-dependent silencing in budding yeast, using foreign and endogenous reporter proteins, at transgenic and endogenous loci. Our results contradict both models and show instead that transcriptional silencing at several URA3 transgenes, and at the naturally silenced endogenous HMRa and HMLalpha mating type genes, acts downstream of gene activator protein binding to strongly reduce the occupancy of TFIIB, RNA polymerase II, and TFIIE at the silenced promoters.

Bacterial Proteins↗
PubMed2002

Structural basis for the NAD-dependent deacetylase mechanism of Sir2.

The NAD-dependent histone/protein deacetylase activity of Sir2 (silent information regulator 2) accounts for its diverse biological roles including gene silencing, DNA damage repair, cell cycle regulation, and life span extension. We provide crystallographic evidence that 2'-O-acetyl ADP-ribose is the reaction product that is formed at the active site of Sir2 from the 2.6-A co-crystal structure of 2'-O-acetyl-ADP-ribose and Sir2 from Archaeoglobus fulgidus. In addition, we show that His-116 and Phe-159 play critical roles in the catalysis and substrate recognition. The conserved Ser-24 and Asp-101 contribute to the stability for NAD binding rather than being directly involved in the catalysis. The crystal structures of wild type and mutant derivatives of Sir2, in conjunction with biochemical analyses of the mutants, provide novel insights into the reaction mechanism of Sir2-mediated deacetylation.

Adenosine Diphosphate Ribose↗
PubMed2003

Identification of selective inhibitors of NAD+-dependent deacetylases using phenotypic screens in yeast.

Sir2 and Hst1 are NAD+-dependent deacetylases involved in transcriptional repression in yeast. The two enzymes are highly homologous yet have different sensitivity to the small-molecule inhibitor splitomicin (compound 1) (Bedalov, A., Gatbonton, T., Irvine, W. P., Gottschling, D. E., and Simon, J. A. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 15113-15118). We have now defined a critical amino acid residue within a small helical module of Hst1 that confers relative resistance to splitomicin. Parallel cell-based screens of 100 splitomicin analogues led to the identification of compounds that exhibit a higher degree of selectivity toward Sir2 or Hst1. A series of compounds based on a splitomicin derivative, dehydrosplitomicin (compound 2), effectively phenocopied a yeast strain that lacked Hst1 deacetylase while having no effect on the silencing activities of Sir2. In addition, we identified a compound with improved selectivity for Sir2. Selectivity was affirmed using whole-genome DNA microarray analysis. This study underscores the power of phenotypic screens in the development and characterization of selective inhibitors of enzyme functions.

Amino Acid Sequence↗
PubMed2001

Sound silencing: the Sir2 protein and cellular senescence.

The model organism Saccharomyces cerevisiae is providing new insights into the molecular and cellular changes that are related to aging. The yeast protein Sir2p (Silent Information Regulator 2) is a histone deacetylase involved in transcriptional silencing and the control of genomic stability. Recent results have led to the identification of Sir2p as a crucial determinant of yeast life span. Dosage, intracellular localization, and activity of Sir2p all have important effects on yeast longevity. For instance, calorie restriction apparently increases yeast life span by increasing Sir2p activity. Since Sir2p-related proteins have been identified in many prokaryotic and eukaryotic organisms, the fundamental principles derived from the studies in yeast may prove valuable in directing our future research toward an understanding of the mechanisms of aging in higher eukaryotes. BioEssays 23:327-332, 2001.

Animals↗
PubMed1996

A Leishmania major protein with extensive homology to silent information regulator 2 of Saccharomyces cerevisiae.

We have isolated a cDNA from the protozoan parasite Leishmania major (Lm) that encodes a protein homologous to the Saccharomyces cerevisiae and Kluyveromyces marxianus silent information regulator 2 (SIR2) proteins. The deduced Lm SIR2-related protein (termed LmSIR2rp) consists of 381 amino acids that share 40.5% identity with yeast SIR2, increasing to 60% when substitutions are included. Moreover, the LmSIR2rp aa sequence contains a single potential zinc-binding domain with a CysXaa2CysXaa20CysXaa2Cys motif, and its C-terminal part is rich in Ser (16 Ser residues over 40 aa) which constitute potential sites for phosphorylation. The characterization of a novel Lm gene product which shows considerable similarity to a yeast mating-type regulatory protein provides a new tool to investigate the parasite differentiation control mechanisms and gene expression regulation.

Amino Acid Sequence↗
PubMed2003

Structure of the yeast Hst2 protein deacetylase in ternary complex with 2'-O-acetyl ADP ribose and histone peptide.

Sir2 proteins are NAD(+)-dependant protein deactylases that have been implicated in playing roles in gene silencing, DNA repair, genome stability, longevity, metabolism, and cell physiology. To define the mechanism of Sir2 activity, we report the 1.5 A crystal structure of the yeast Hst2 (yHst2) Sir2 protein in ternary complex with 2'-O-acetyl ADP ribose and an acetylated histone H4 peptide. The structure captures both ligands meeting within an enclosed tunnel between the small and large domains of the catalytic protein core and permits the assignment of a detailed catalytic mechanism for the Sir2 proteins that is consistent with solution and enzymatic studies. Comparison of the ternary complex with the yHst2/NAD(+) complex, also reported here, and nascent yHst2 structure also reveals that NAD(+) binding accompanies intramolecular loop rearrangement for more stable NAD(+) and acetyl-lysine binding, and that acetyl-lysine peptide binding induces a trimer-monomer protein transition involving nonconserved Sir2 residues.

Amino Acid Sequence↗
PubMed2002

Human Sir2 and the 'silencing' of p53 activity.

Members of the evolutionarily conserved silent information regulator 2 (Sir2) protein family are nicotinamide adenine dinucleotide (NAD(+))-dependent histone deacetylases. In yeast, the founding Sir2 protein is known to function in transcriptional silencing processes through the deacetylation of histones H3 and H4, thus setting up a repressive chromatin structure. Yeast and Caenorhabditis elegans Sir2 are also involved in regulating the life span of these organisms. Until recently, the function of mammalian Sir2 family members was completely unknown. However, several recent studies have now determined a remarkable function for the human SIRT1 protein, which is the closest human homolog of yeast Sir2. SIRT1 specifically associates with the p53 tumor suppressor protein and deacetylates it, resulting in negative regulation of p53-mediated transcriptional activation. Importantly, p53 deacetylation by SIRT1 also prevents cellular senescence and apoptosis induced by DNA damage and stress.

Animals↗
PubMed2001

Promoter-specific binding of Rap1 revealed by genome-wide maps of protein-DNA association.

We determined the distribution of repressor-activator protein 1 (Rap1) and the accessory silencing proteins Sir2, Sir3 and Sir4 in vivo on the entire yeast genome, at a resolution of 2 kb. Rap1 is central to the cellular economy during rapid growth, targeting 294 loci, about 5% of yeast genes, and participating in the activation of 37% of all RNA polymerase II initiation events in exponentially growing cells. Although the DNA sequence recognized by Rap1 is found in both coding and intergenic sequences, the binding of Rap1 to the genome was highly specific to intergenic regions with the potential to act as promoters. This global phenomenon, which may be a general characteristic of sequence-specific transcriptional factors, indicates the existence of a genome-wide molecular mechanism for marking promoter regions.

Binding Sites↗