Enzymatic activities of Sir2 and chromatin silencing
Heritable domains of generalized repression are a common
feature of eukaryotic chromosomes and involve the assembly of
DNA into a silenced chromatin structure. Sir2, a conserved
protein required for silencing in yeast, has recently been shown
to couple histone deacetylation to cleavage of a high-energy
bond in nicotinamide adenine dinucleotide (NAD) and the
synthesis of a novel product, O-acetyl-ADP-ribose. The
deacetylase activity provides a direct link between Sir2 and the
hypoacetylated state of silent chromatin. However, the unusual
coupling of deacetylation to cleavage and synthesis of other
bonds raises the possibility that deacetylation is not the only
crucial function of Sir2.
Department of Cell Biology, Harvard Medical School, 240 Longwood
Avenue, Boston, Massachusetts 02115, USA;
Current Opinion in Cell Biology
0955-0674/01/$ — see front matter
© 2001 Elsevier Science Ltd. All rights reserved.
nicotinamide adenine dinucleotide
nicotinic acid mononucleotide
silent information regulator
Gene silencing is the inactivation of large domains of DNA
by packaging them intoa specialized inaccessible
matin structure. This type of inactivation is involved in the
regulation of gene expression and is also associated with
the chromosome structures required for chromosome
maintenance and inheritance. For example, in metazoan
chromosomes, vast regions of DNA adjacent to cen-
tromeres and telomeres are packaged into an inactive type
of chromatin called heterochromatin (reviewed in ).
Mutations that disrupt the formation of heterochromatin
compromise chromosome inheritance, very probably as a
result of perturbed centromere function . Silenced chro-
matin is also found in unicellular fungi. In fission yeast
(Schizosaccharomyces pombe), similar to metazoans, large
regions of DNA adjacent to centromeres are packaged into
silenced chromatin, and the formation of these domains
plays an important role in chromosome inheritance [3,4].
In both fission yeast and budding yeast (Saccharomyces cere-
visiae), haploid cell identity is regulated by transcriptional
silencing of DNA domains that contain mating-type infor-
mation; also in both yeasts, telomeric DNA regions are
packaged into silenced chromatin [5–8].
Genetic and biochemical studies have identified the main
regulatory sites and proteins that collaborate to assemble
silenced DNA in budding yeast (reviewed in ). A major
recent development in this field has been the discovery
that the silencing protein Sir2 and other members of the
Sir2 family are NAD-dependent enzymes [10••–14••]. The
enzymatic activities of this protein family are unusual and
are only now beginning to come into focus. Here, we dis-
cuss the enzymatic activities of Sir2 and the current
speculation about the role of these activities in silencing.
The silencing machinery in yeast
Sir2 is required for all known examples of silencing in
yeast [15–19]. As a component of the SIR complex, it acts
together with Sir3 and Sir4 to assemble silent chromatin at
telomeric DNA regions and at the silent mating type loci
(called HML and HMR, homothallic left and homothallic
right, respectively; Figure 1; [20–22]). Together with Net1,
Cdc14 and perhaps other proteins, in a complex called
RENT (regulator of nucleolar silencing and telophase
exit), Sir2 is involved in ribosomal DNA (rDNA) silencing
(Figure 1; [23–25]). rDNA silencing is required for sup-
pression of hyper-recombination within the highly
repetitive rDNA array and repression of transcription of
pol II reporter genes inserted in rDNA [18,19].
Suppression of hyper-recombination by Sir2 plays a prima-
ry role in extension of replicative life span in yeast [26,27].
The SIR and RENT complexes are recruited to their chro-
mosomal targets via interactions with DNA-binding
proteins, which bind to specific regulatory sites and initiate
silencing. The DNA-binding protein(s) that target the
RENT complex to rDNA have not been identified yet,
but the mechanism of recruitment of SIR to DNA is rela-
tively well understood (reviewed in ; see Figure 1). In
addition to the two silencing complexes and DNA-binding
proteins, the conserved and highly basic amino termini of
histones H3 and H4 are required for silencing .
Mutations in several conserved lysines in histones H3 and
H4 abolish silencing at the telomeres and the silent mat-
ing-type loci. These lysine residues are reversibly
acetylated, and mutational analysis suggests that their
deacetylated state is important for silencing [29–31].
Moreover, two of the proteins in the SIR complex, Sir3 and
Sir4, are histone tail binding proteins, and their interac-
tions with H3 and H4 are likely to play a key role in the
assembly of silent chromatin domains . Despite its
general requirement for silencing, Sir2 had not been iden-
tified with a biochemical function until recently.
Ribosyltransferase activity of Sir2 and Sir2-like
Sir2 is a member of an ancient family of proteins in organ-
isms ranging from bacteria to complex eukaryotes [33,34].
Members of this family contain a 250 amino acid core
domain that shares about 25%–60% sequence identity.
The eukaryotic Sir2-like proteins have been implicated in
a wide range of chromosome-associated phenomena,
Sir2-containing silencing complexes in budding yeast. (a) A model for
the recuitment of the SIR complex (Sir2/3/4) to a telomere. The
enzymatic activity of Sir2 is required for silencing and efficient
spreading of the SIR complex along the chromatin fiber. Ac denotes
acetylated histones in nucleosomes, which are shown as filled circles.
Within the silent chromatin domain (spanning ~3 kb from the telomere)
histones are hypoacetylated. See text and [9,52] for details and
additional references. (b) At the silent mating type loci (HML, HMR), a
different set of proteins, bound to regulatory sites called silencers,
recruits the SIR complex to DNA (reviewed in [9,53]). (c) The RENT
complex mediates rDNA silencing and suppression of rDNA hyper-
recombination. Protein(s) involved in its recruitment to DNA have not
been identified. See text for a discussion of the NAD-dependent
protein deactylase activity of Sir2. Nic, nicotinamide.
including gene silencing, cell cycle progression, chromo-
some segregation and DNA damage repair [33,35,36]. The
first clues regarding the biochemical function of a Sir2-like
protein came from studies of cobalamine (vitamin B12)
biosynthesis in the bacterium Salmonella typhimurium by
Escalante-Semerena and colleagues . A precursor in
the cobalamine pathway, DMB-5′-ribosyl-phosphate, is
synthesized by transfer of phosphoribose from nicotinic
acid mononucleotide (NaMN) to another small molecule,
dimethyl benzimidazole (DMB; Figure
Salmonella CobT protein catalyzes this phosphoribosyl
transfer reaction. However, Tsang et al.  discovered
that CobB, the Salmonella Sir2-like protein, could partially
compensate for the loss of CobT in the cobalamine path-
way, suggesting that CobB could perform a similar
The idea that Sir2-like proteins can perform phosphoribo-
syl transfer reactions, of the type described above, was
directly tested by Frye [10••]. In examining various pyri-
dine nucleotide derivatives as donors for the transferase
activity of Sir2, Frye made the key discovery that only
NAD, but not NaMN or other NAD derivatives, could act
as cofactors for E. coli and human Sir2-like proteins in vitro
(CobB and SirT2, respectively; Figure 2b; [10••]). Sir2-like
proteins were therefore suggested to be ADP-ribosyltrans-
ferases as they could transfer ADP-ribose from NAD to
The above studies prompted several groups to examine
whether the budding yeast Sir2 protein had a similar enzy-
matic activity and whether this activity was required for gene
silencing. Using NAD as a donor, Tanny et al. ([11••];
J Tanny, D Moazed, unpublished data) tested the ribosyl-
transferase activity of Sir2 on a wide range of substrates,
including histones and other silencing proteins. It was found
that Sir2 could perform a weak ribosylation reaction in which
it transferred the ADP-ribose moiety from NAD to itself and
histones (Figure 2c). The weak ribosyltransferase activity
was accompanied by an efficient histone-dependent NAD
breakdown activity. Furthermore, a point mutation in Sir2
that abolished these activities in vitro resulted in a complete
Phosphoribosyltransferase activity of Sir2-like proteins.
(a) Phosphoribose transfer from NaMN to dimethyl benzimidazole
(DMB) is catalyzed by the Salmonella CobT. CobB, the Salmonella
Sir2-like protein, can compensate for loss of CobT function and
has been proposed to perform the same reaction in vivo [37••].
(b) E. coli CobB and human SirT2 proteins perform an
ADP-ribosyltransferase reaction, using NAD rather than NaMN as
a donor molecule, to transfer ADP-ribose to DMB or to an artificial
protein substrate in vitro [10••]. (c) The yeast Sir2 protein performs
an NAD cleavage and weak ribosylation reaction using acetylated
histones as a substrate [11••]. See text for further details.
Na, nicotinic acid; Nic, nicotinamide.
loss of silencing invivo, suggesting that the ribosyltransferase
(and/or NADase) activities of Sir2 were essential for silenc-
ing. However, as discussed below, the activities observed in
this study were partial reactions that stemmed from the abil-
ity of Sir2 to catalyze some novel chemistry with NAD and
NAD-dependent protein deacetylase activity
The reports of ribosyltransferase activity in Sir2 and Sir2-like
proteins were followed by studies showing that Sir2-like pro-
teins have an NAD-dependent histone deacetylase activity
([12••–14••]; Figure 3). In contrast to the ribosylation activity
mentioned above, the deacetylase activity of Sir2 is very effi-
cient, suggesting that it is the primary activity of the protein
in vitro. This discovery has been greeted with enthusiasm, in
part, because earlier evidence had suggested a role for histone
deacetylation in assembly of silent chromatin domains
[28,30]. In fact, Sir2 had earlier been suggested to be a histone
deacetylase because its overexpression results in general his-
tone hypoacetylation in yeast . Deacetylation by Sir2
therefore provides a direct link between the silencing
machinery and the observed hypoacetylated state of histones
within silent chromatin domains.
Nonetheless, from a mechanistic point of view the
requirement for NAD in a deacetylation reaction is diffi-
cult to explain. Deacetylation is an energetically favorable
amide hydrolysis reaction. Similar reactions are catalyzed
by numerous proteases without the need for a cofactor
. This reasoning, and a mutation in Sir2 that appeared
to separate its two activities, led to the suggestion that the
The NAD-dependent deacetylase activity of Sir2. Sir2 and Sir2-like
proteins from E. coli, yeast and mouse can deacetylate histones and
other proteins [12••–14••]. Acetyl-K-histone, histone containing acetyl-
lysine; K-histone, deacetylated histone.
ribosyltransferase/NAD breakdown and deacetylation
activities of Sir2 are separable and fundamentally differ-
ent [12••]. At this point, it was clear that Sir2 is a very
unusual enzyme, but in the absence of a coherent reaction
mechanism, its biologically significant activity remained
in question. However, more recent studies, discussed
below, provide evidence that the different enzymatic
activities of this remarkable family of proteins are
Acetyl transfer from substrate to ADP-ribose,
the generation of O-acetyl-ADP-ribose
Although early experiments by Imai and colleagues [12••]
had suggested that the ADP-ribosyltransferase and NAD
cleavage activities of Sir2 are fundamentally distinct from
its deacetylase activity, a number of observations suggest-
ed that these very different activities may be related. First,
the ribosyltransferase and NAD breakdown activities of
Sir2 required the presence of an acetylated substrate in the
reaction ([11••,12••]; J Tanny, D Moazed, unpublished
data), suggesting a relationship between these activities
and acetyl-lysine (the substrate for the deacetylation activ-
ity of Sir2). Second, Landry et al. [13••] showed that, in
addition to deacetylation, Sir2 could perform an exchange
reaction in which it generated labeled NAD from
14C-labeled nicotinamide and unlabeled NAD. Although
the efficiency of this exchange reaction is unknown, it
required the presence of an acetylated histone substrate.
Thus, acetyl-lysine was required for a second set of Sir2
activities, ribosylation, NAD breakdown and an exchange
reaction involving breakage of the same C-N bond in NAD
that is cleaved during ribosylation.
An important clue to the mechanism of Sir2 came from
examination of the relationship between its deacetylation
and NAD breakdown activities. The first crucial observa-
tion was that Sir2 couples deacetylation to cleavage of the
high-energy glycosidic bond that links the ADP-ribose
moiety of NAD to nicotinamide [40••–42••]. A precise sto-
ichiometric relationship between deacetylation and NAD
breakdown was uncovered, indicating that for every
acetyl-lysine that is deacetylated one NAD molecule is
cleaved. A second crucial observation in these experi-
ments was the unexpected nature of the NAD cleavage
Enzymatic activities of Sir2 and chromatin silencing Moazed 235
Coupling of deacetylation to NAD cleavage
and acetyl transfer from substrate to ADP-
ribose by Sir2 and Sir2-like proteins
[41••,42••]. (a) The overall reaction scheme.
(b) Structures of NAD and O-acetyl-ADP-
ribose, the novel compound produced by Sir2
and Sir2-like proteins, are shown. This figure
shows the most likely position of the O-acetyl
group on the 1′ ribose carbon; an alternative
position for the O-acetyl group on the 2′
ribose carbon has not been ruled out. See text
and references [40••–42••] for details.
products. Straightforward cleavage of NAD at the glyco-
sidic bond that links its ADP-ribose moiety to
nicotinamide should result in the generation of ADP-
ribose and nicotinamide. However, in addition to
nicotinamide and small amounts of ADP-ribose, a third
major reaction product, related to but distinct from ADP-
ribose, was detected [41••,42••]. Label transfer
experiments showed that this novel reaction product
resulted from the transfer of the acetyl group from the
substrate to the ADP-ribose moiety of NAD [41••].
Furthermore, at high pH, this product was shown to decay
to ADP-ribose and acetate and was therefore concluded to
be O-acetyl-ADP-ribose [41••]. In an independent study,
Tanner et al. [42••] analyzed the reaction products pro-
duced by Sir2 and Hst2, a yeast Sir2-like protein, and
found that O-acetyl-ADP-ribose was a primary product of
deacetylation by these enzymes. This study also con-
firmed the identity of O-acetyl-ADP-ribose by mass
spectroscopy analysis [42••]. These observations provide
proof of the obligate coupling of deacetylation to NAD
cleavage and demonstrate a direct role for NAD in histone
deacetylation by Sir2. Consistent with these results,
Landry et al. [40••] find that a non-hydrolyzable NAD ana-
, is a competitive inhibitor of the
NAD-dependent deacetylase activity of Sir2. It is there-
fore abundantly clear that Sir2 has two coupled enzymatic
activities, deacetylation and NAD breakdown, and pro-
duces a novel compound, O-acetyl-ADP-ribose (Figure 4).
As mentioned earlier, Sir2-like proteins also have a weak
ribosyltransferase activity. Interestingly, this activity of Sir2
(as well as its NADase activity) requires the presence of an
acetyl-lysine-containing substrate in the reaction, suggest-
ing that it is linked to deacetylation. The above
observations now provide an explanation for the weak ribo-
syltransferase activity of Sir2-like proteins. Tanner et al.
[42••] suggest that, by analogy with NAD glycohydrolases,
Sir2 first generates an oxo-carbenium ADP-ribose interme-
diate, which then acts as an acceptor for the acetyl group.
This mechanism would involve the formation of an
enzyme-ADP-ribose intermediate, providing an explana-
tion for the detection of a ribosylated form of Sir2 [11••]. A
similar reaction mechanism involves the cleavage of NAD
by nucleophilic attack of the isoamide form of acteyl-lysine
on the 1′ ribose carbon position to release nicotinamide
[42••]. Both the above mechanisms would also involve the
generation of a transient substrate ADP-ribose intermedi-
ate, providing a possible explanation for the weak histone
ribosylation activity of Sir2. In either case, it is very likely
that, at least in vitro, the ribosylation activity of Sir2-like
proteins results from either the trapping of reaction inter-
mediates or side reactions during deacetylation and NAD
Significance for the mechanism of silencing
The coupling of deacetylation, itself an energetically favor-
able reaction, to cleavage of a high-energy bond is rather
unusual. The free energy of hydrolysis of the glycosidic
bond that links ribose and nicotinamide in NAD is
~8.2 kcal/mol, in the same range as the energy of hydrolysis
of ATP to ADP . One is forced to ask why does Sir2 do
it this way. Is it just an unusual deacetylase or is there a role
for the NADase activity and/or O-acetyl-ADP-ribose pro-
duction in silencing? There is a long-standing correlation
between gene inactivation and histone hypoacetylation
[44,45]. In budding yeast, silent chromatin domains are
hypoacetylated and mutational analysis of histones H3 and
H4 suggests that the deacetylated state of one or more
lysine residues in the amino termini of these histones is crit-
ical for silencing [29,30,38]. It is therefore easy to suppose
that deacetylation is the important function of Sir2 in silenc-
ing. However, there is currently no direct evidence
supporting this attractive hypothesis, and the possibility that
one of the other two significant activities of Sir2, which are
associated with deacetylation, NAD cleavage and O-acetyl-
ADP-ribose synthesis, play a critical role in gene silencing
must be considered.
Although histones associated with silent chromatin are gen-
erally in a hypoacetylated state, there is also evidence
favoring a role for histone acetylation (rather than deacetyla-
tion) in transcriptional silencing in yeast and Drosophila. For
example, deletion of the yeast RPD3 gene, which encodes a
histone deacetylase, results in a dramatic increase in silenc-
ing at telomeric, mating-type and rDNA regions [46-48].
Furthermore, deletion of the yeast type B histone acetyl-
transferase gene, HAT1, in combination with histone H3
amino-terminal tail mutations, results in a significant defect
in telomeric silencing, and mutations in the MYST family of
acetyl transferases have both positive and negative effects
on silencing [49–51]. Contrary to general expectations, these
results suggest that histone acetylation, as well as deacetyla-
tion, plays an important role in silencing.
Rpd3, Hat1 and MYST family acetyltransferases may be
influencing the efficiency of silencing indirectly, for exam-
ple through global effects on gene expression or chromatin
assembly. However, an alternative possibility is that acety-
lation of a specific target protein(s) is required for Sir2
function and efficient silencing. In this model, acetyl-lysine
(in histones or other proteins) would be required to trigger
the NADase and O-acetyl-ADP-ribose synthesis activity of
Sir2. The ultimate function of Sir2 in silencing would then
be to use acetyl-lysine as a cofactor to breakdown NAD
and/or to produce O-acetyl-ADP-ribose. In this regard, sev-
eral intriguing possibilities have been proposed [41••,42••].
One possibility is that the energy of NAD breakdown
might promote a step in the assembly of silent chromatin,
such as the spreading of silencing complexes along the
chromatin fiber [41••]. Another possibility is that O-acetyl-
ADP-ribose could act as a local allosteric effector for other
silencing proteins or as an acetyl donor for a novel class of
protein acetyltransferases or as an ADP-ribose group donor
for a novel ribosyltransferase [41••,42••]. Distinguishing
between the above models requires examining the role of
Sir2 in silencing under conditions that can separate its
Over the past year or so, Sir2 has travelled from being a
protein of unknown biochemical function to being an
enzyme with remarkable activities that produces a novel
cellular metabolite. As is often the case, these new discov-
eries raise more questions than they answer. In the
simplest case, the deacetylase activity of Sir2 provides a
direct explanation for the hypoacetylated state of histones
within yeast silent chromatin domains (see Figure 1). But
whether this function of Sir2 is crucial for silencing or not
is far from clear. The possible role of the NADase activity
of Sir2 and the role of O-acetyl-ADP-ribose in silencing
and cellular physiology remain to be determined. Finally,
how any of the activities of Sir2 contribute to those unique
properties of gene silencing, such as the spreading of
repressors along the chromatin fiber or the stable inheri-
tance of the silent state during chromosome duplication,
remains the subject of future studies.
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