MOLECULAR AND CELLULAR BIOLOGY, Feb. 2010, p. 626–639
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 30, No. 3
Expanded Roles of the Origin Recognition Complex in the
Architecture and Function of Silenced Chromatin in
Bilge O ¨zaydın and Jasper Rine*
Department of Molecular and Cell Biology, California Institute of Quantitative Biosciences, 392 Stanley Hall,
University of California, Berkeley, California 94720-3220
Received 11 May 2009/Returned for modification 21 June 2009/Accepted 19 November 2009
The silenced chromatin at the cryptic mating-type loci (HML and HMR) of Saccharomyces cerevisiae requires
a cell cycle event between early S phase and G2/M phase to achieve repression. Although DNA replication per
se is not essential for silencing, mutations in many of the proteins involved in DNA replication affect silencing.
Each of the four silencers, which flank the silenced loci, includes an origin recognition complex (ORC) binding
site (ACS). ORC directly interacted with Sir1 and recruits Sir1 to the silencers. This study describes additional
roles for ORC in the architecture of silenced chromatin. Using chromatin immunoprecipitation (ChIP)
analysis, we found that ORC physically interacts throughout the internal regions of HMR as well as with
silencers. This interaction depended on the presence of Sir proteins and, in part, on the HMR-I silencer. ORC
remained associated with the internal regions of HMR even when these regions were recombinationally
separated from the silencers. Moreover, ORC could be recruited to the silencers lacking an ACS through its
One mechanism for silencing in eukaryotes involves the for-
mation of heterochromatin, which blocks transcription in a
region-specific and non-gene-specific fashion. Once estab-
lished, silencing in such regions is stably maintained and in-
herited through multiple cell divisions despite the potentially
disruptive effects of DNA replication, recombination, and re-
pair. In Saccharomyces cerevisiae, heterochromatin is found in
three locations: telomeres (24), ribosomal DNA (rDNA) (54),
and the silent mating-type loci HML and HMR (51), which
contain functional copies of MAT? and MATa genes, respec-
tively. Silencing of HML and HMR is crucial for proper haploid
cell identity (26).
Transcriptional silencing of HML? and HMRa is controlled
by the E and I silencers that flank HML and HMR loci (7).
Silencers contain binding sites for the origin recognition com-
plex (ORC), Rap1p, and/or Abf1p, which together recruit Sir1,
Sir2, Sir3, and Sir4 proteins, which are essential for initiating
and spreading heterochromatin (8, 42, 48, 51). Deletion of
SIR2, SIR3, or SIR4 completely abolishes silencing; however,
deletion of SIR1 results in a population of cells in which HML
and HMR are silenced in some cells, but not in others. Both
states of HML and HMR are heritable in sir1 mutants. This and
other observations led to the view that Sir1 is required primar-
ily for the establishment of transcriptional silencing (44),
whereas Sir2 to Sir4 are required for both the establishment
and maintenance of silencing (4, 41). At HML and HMR, Sir1
associates mostly with chromatin at the silencers, whereas Sir2,
Sir3, and Sir4 associate with chromatin throughout the silenced
region (51). Sir2 is the only protein among the Sir proteins with
both structural and enzymatic roles in silencing (30, 31, 35).
Once recruited to the silencers via multiple interactions be-
tween the silencer binding proteins and other Sir proteins, Sir2
deacetylates the N-terminal tails of histones H3 and H4 of the
nearby nucleosomes (9). These hypoacetylated N-terminal tails
of histones provide new high-affinity binding sites for Sir3 and
Sir4, which are in a complex with Sir2 (25). Histone mutants
that mimic the hypoacetylated state rescue the binding and
spreading of a catalytically inactive Sir2, Sir2-345, at HML and
HMR (62). Thus, hypoacetylated histones provide a foundation
for silent chromatin assembly.
A classic study (41) revealed an S-phase dependence for the
establishment of silencing. Several proteins that have roles in
DNA replication, such as ORC (16, 39, 40), PCNA (64), Dna2,
Asf1, and Cac1, also contribute to HML and HMR silencing
(15, 32, 53, 55). These findings suggested that DNA replication
was required for establishment of transcriptional silencing.
However, multiple studies show that neither the initiation of
replication at origins that are part of silencers nor the passage
of a DNA replication fork through the HML and HMR loci is
required for establishment of silencing (30, 34, 37). Neverthe-
less, each silencer flanking the HML and HMR loci has an
ORC binding ARS consensus sequence, and mutations in dif-
ferent ORC subunits lead to decreased silencing (16, 39, 60).
ORC directly interacts with Sir1, but no such interaction was
detected between ORC and other Sir proteins (17, 63). In
addition, ORC’s role in silencing can be bypassed by tethering
Sir1 to the silencer through Gal4 binding sites (17). These
findings suggest that ORC’s only role in silencing is recruit-
ment of Sir1 to silencers. If that were the limit of ORC’s role
in silencing, then mutations in ORC should have no impact on
silencing if Sir1 were recruited to silencers by other means.
* Corresponding author. Mailing address: Department of Molecular
and Cell Biology, California Institute of Quantitative Biosciences, 392
Stanley Hall, University of California, Berkeley, CA 94720-3220.
Phone: (510) 642-7047. Fax: (510) 666-2768. E-mail: jrine@berkeley
?Published ahead of print on 30 November 2009.
To understand more fully the effects of ORC on the HML
and HMR chromatin and to characterize further the role of
ORC in silencing, we measured the silencing level in cells with
orc mutations and various configurations of silencers. These
experiments revealed unanticipated links between ORC, si-
lencing, and the architecture of the silenced chromatin.
MATERIALS AND METHODS
Yeast strains and genetic manipulations. Strains used in this study were
isogenic to S. cerevisiae W303 unless otherwise indicated (Table 1). Gene dele-
tions were done using one-step integration of PCR-amplified knockout cassettes
(22, 38) and confirmed by PCR and phenotypic validation. Epitope tagging for
immunoprecipitations was done similarly using tandem affinity purification
(TAP) tag cassettes (47) or constructs described previously (38). Oligonucle-
otide primer sets used for PCR, tagging, and knockout in this study are shown
in Table 2.
Yeast media and transformation. Rich medium (YPD) and minimal medium
(YM) are described previously (52). KanMX4 resistance marker was selected on
YPD containing 200 mg/liter of G418 (Geneticin). Modified lithium acetate
transformation was used as described previously (3).
Semiquantitative mating assay. After each culture was grown to late log
phase, it was diluted to a final optical density at 600 nm (OD600) of 1. Threefold
serial dilutions of each culture of interest were spotted onto YPD or synthetic
complete medium lacking uracil (SC-Ura) plates for growth and on YM plates
sprayed with excess MAT? mating tester strain (JRY2726) and incubated at 24°C
for 3 days.
RNA preparation and analysis. Total RNA was prepared using an RNeasy
minikit from Qiagen. Genomic DNA was digested on the column using RNase-
free DNase (Qiagen). Oligo(dT) primer-directed cDNA was synthesized using
the SuperScript III first-strand synthesis system for reverse transcriptase PCR
(RT-PCR) kit (Invitrogen). Quantitative PCR (QPCR) analysis was done at least
in triplicate on 3 or more independent RNA preparations of each strain. QPCR
was performed on a MX3000P machine (Stratagene) using SYBR GreenER
QPCR super mix (Invitrogen).
ChIP analysis. Chromatin immunoprecipitation (ChIP) analysis was done as
previously described (12) with minor modifications. Two hundred fifty milliliters
of mid-log-phase cultures was used to prepare whole-cell extracts, and 400 ?l of
this extract used for immunoprecipitation (IP) and 40 ?l was used for the input
sample. A 50-?l slurry of immunoglobulin G-Sepharose (Amersham Bio-
sciences) per IP was used for TAP-tagged proteins. Agarose-conjugated mouse
monoclonal anti-hemagglutinin (anti-HA) (Sigma) was used to IP HA-tagged
proteins. For immunoprecipitations with rabbit polyclonal ORC antibody (gen-
erous gift from Stephen Bell), 2 ?l of antibody was coupled to 50 ?l of protein
A slurry (Upstate) for 2 h. All immunoprecipitations were done overnight at 4°C.
Each ChIP experiment was done in triplicate or more for independently pre-
pared whole-cell extracts. For experiments where no enrichment was detected
(see Fig. 8B), ChIP was done in duplicate.
Genomic DNA analysis. The whole-cell extracts prepared for ChIP analysis
were used for DNA blot hybridization analysis. The whole-cell extracts were first
digested with proteinase K for 2 h at 37°C and then extracted with phenol-
chloroform. After isopropanol precipitation and a 70% ethanol wash, each pellet
was resuspended in 50 ?l of water. About 15 ?g of each sample was electro-
phoretically separated in a 2% agarose gel and then transferred to a Hybond N
membrane. Probes of interest were prepared by PCR and then radiolabeled
using [?-32P]dCTP with Amersham RediPrime random prime labeling system
(GE Healthcare). DNA blot analysis was done as previously described (56). Blots
were analyzed with a Typhoon scanner and ImageQuant software. The most
frequently occurring signal value, the mode, was determined for each lane. To
evaluate the extent of shearing among different DNA samples, the signal average
was determined for each 0.2 mm of each lane on the gel by multiplying the
number of signal counts for each fragment by the corresponding size of the
fragment and then dividing this value by the total number of counts. The final
averages were compared to determine the average shearing difference.
Protein analysis and immunoprecipitation. Yeast whole-cell extracts were
precipitated using 20% trichloroacetic acid (TCA) and solubilized in SDS load-
ing buffer. Anti-Flag antibody from rabbit (Sigma) was used to detect TAP-
tagged proteins, and anti-HA antibody from mouse (Sigma) was used to detect
HA-tagged Sir1 (Sir1-HA). The ORC was detected using polyclonal ORC anti-
body from rabbit. TAP-tagged Sir2 (Sir2-TAP) immunoprecipitations were as
previously described (2).
Previous work identified ORC’s role in silencing as recruit-
ment of Sir1 protein to the silencers. Indeed, the first 235
amino acids of Orc1, the largest subunit of the ORC complex,
contain a bromo-adjacent homology (BAH) domain that in-
teracts with a specific domain in Sir1, known as the ORC
TABLE 1. Yeast strains used in this study
JRY3009MAT? ade2-1 his3-11,15 trp1-1
leu2-3,112 ura3-1 can1-100
MATa ade2-1 his3-11,15 trp1-1
leu2-3,112 ura3-1 can1-100
MAT? ade2-1 his3-11,15 leu2-
3,112 ura3-1 orc5-1
MAT? ade2-1 his3-11,15 leu2-3
lys2? trp1-1 ura3-1 orc2-1
JRY3009 sir1::LEU2 sir2::HIS3
JRY2334 R. Rothstein
S. P. Bell
JRY3009 HMR-I? ORC5-HA-
JRY3009 HMR-SS (4?Gal4bs-
JRY4806 pJR1639 (GAL4 URA3)
JRY4806 pJR1815 (GAL4-SIR1
JRY8928 pJR1639 (GAL4 URA3)
JRY8928 pJR1815 (GAL4-SIR1
MATa ura3-52 lys2-801 ade2-101
his3-?200 trp1-?63 leu2-?1
JRY8934 ppr1::HIS3 ORC5-HA-
JRY8936 ppr1::HIS3 ORC5-HA-
W303-1A hmr::ra1a2 mat::URA3
YJS (YPH499)P. Hieter
MATa his4P. Schatz
aUnless noted otherwise, all strains were from the laboratory collection or
were constructed during the course of this study.
bbs, binding site.
VOL. 30, 2010ORIGIN RECOGNITION COMPLEX IN SACCHAROMYCES CEREVISIAE627
interaction region (OIR), but not with other Sir proteins (6, 27,
63). This study revealed additional roles for ORC in silencing
and in the architecture of the silenced chromatin.
ORC’s role in silencing beyond Sir1 recruitment. Two ORC
mutations, orc5-1 and orc2-1, cause loss of silencing at HML
and HMR (16, 18, 39, 58), particularly in strains in which
silencing has already been compromised or in strains with
synthetic silencers. The silencing defect of these orc mutations
can be bypassed by tethering a Gal4 DNA binding domain-Sir1
fusion protein (Gal4-Sir1) to the silencer through multiple
Gal4 binding sites in place of the ORC binding site (ACS) in
the HMR-E silencer (17).
If ORC’s role in silencing were exclusively to recruit Sir1,
then in the absence of Sir1, orc mutations should have no
further effect on silencing. The partial defect of sir1? mutants
allowed us to test whether orc5-1 and orc2-1 mutations had any
TABLE 2. Oligonucleotides used in this study
Primer set SequenceApplication(s)
PCR for blotting
PCR for blotting
SIR1 HA tagging
SIR3 TAP tagging
628O ¨ZAYDIN AND RINEMOL. CELL. BIOL.
effects on silencing in sir1? orc double mutants. Unexpectedly,
the orc5-1 mutation in combination with sir1? had a more
pronounced silencing defect as measured by mating efficiency
than did the sir1? mutant or individual orc mutants, indicating
that ORC had a role(s) in silencing HMR beyond Sir1 recruit-
ment (Fig. 1A).
To provide a molecular assessment of the effects of orc
mutations on residual silencing in sir1? mutant cells, we mea-
sured the a1 transcript level from HMRa1 by quantitative re-
verse transcription-PCR (QRT-PCR) in these mutants. As ex-
pected, orc2-1 and orc5-1 mutations by themselves had no
detectable effect on silencing in cells with wild-type silencers
grown at the permissive temperature (Fig. 1B). However, when
combined with sir1? in cells incubated at the semipermissive
temperature (30°C) for 3 h, a pronounced effect on silencing
was observed in both orc mutants, while not affecting the cell
viability (data not shown). In agreement with the mating assay,
the orc5-1 mutation enhanced the silencing defect of sir1?. On
the other hand, orc2-1 mutation did not show a significant
effect on silencing in the absence of sir1.
Effects of orc mutations on silencing proteins and ORC
association with HMR. To explore the mechanism by which orc
mutations affect silencing independently of Sir1, we measured
their effect on the association of Sir proteins at HMR and HML
using ChIP followed by QPCR analysis (Fig. 2). We evaluated
the levels of Sir1, Sir2, and Sir3 proteins at the HML-E and
HMR-E silencers and at positions internal to the silenced do-
main, corresponding to HMRa1 and HML?1, which were ap-
proximately 1.4 kb from silencers. As reported previously, Sir3
is enriched at both silencers and in internal regions of HML
and HMR. Neither the orc2-1 mutation nor the orc5-1 mutation
had any notable effect on Sir3 occupancy at these positions
Sir1 data offered a somewhat different perspective. As ex-
pected, Sir1 was much more enriched at the HML-E silencer
than at HML?1. Interestingly, the orc5-1 mutation resulted in
substantial reduction of Sir1 at the HML-E silencer, with a
similar though quantitatively smaller impact caused by orc2-1.
At HMR, the Sir1 enrichment at the HMR-E silencer was as
expected; however, there was still substantial enrichment of
Sir1 at the internal HMRa1. Enrichment of Sir1 at both HMR
positions was somewhat sensitive to orc5-1 and less so to orc2-1
The Sir2 localization at HML and HMR in wild-type cells
was similar to that of Sir3. However, in contrast to Sir1 and
Sir3, Sir2 exhibited a pronounced sensitivity to orc2-1 and
much less so to orc5-1 (Fig. 2C).
Sir2 interactions with the silenced domain in sir1? mutant
strains were reduced in the orc5-1 sir1? double mutant in
accordance with the further decreased silencing in this strain.
However, orc2-1 sir1? had very little effect on silencing relative
to sir1? itself, but the effect of orc2-1 sir1? on Sir2-TAP levels
at HMR was significant (about 3-fold less than in the sir1?
strain) (Fig. 2D). The synergistic effect of orc2-1 and orc5-1 in
combination with sir1? on Sir2 ChIP levels was unanticipated
by all earlier studies of ORC’s role in silencing. ChIP analysis
for ORC in these mutations showed that, when polyclonal
ORC antibody was used, there was more ORC interaction at
HMR-E in the orc2-1 sir1? double mutant than in either the
sir1? or orc5-1 sir1? mutant strain (Fig. 2E). This was surpris-
ing because the ORC complex in the orc2-1 strain is unstable,
and except for the Orc1 subunit, the levels of other subunits
were reduced (Fig. 2F). In fact, the level of Orc5 as reflected in
ChIP analysis was greatly reduced in the orc2-1 strain (data not
shown), suggesting that the enhanced ORC ChIP in the orc2-1
mutant was probably contributed by the Orc1 subunit. Taken
altogether, the data suggest that although both orc mutations
resulted in a drop of Sir2 recruitment at HMR, which would
negatively affect silencing, enhanced recruitment of Orc1 in the
orc2-1 strain may increase silencing through a Sir2-indepen-
To test whether orc mutations had any adverse effects on the
overall level of Sir proteins in cells, we evaluated the level of
epitope-tagged Sir proteins. Sir1-HA and Sir3-TAP levels were
similar in different wild-type and orc mutant strains (Fig. 2G
and H). Due to its low abundance, it was difficult to analyze the
levels of Sir2-TAP by immunoblotting whole-cell extracts (data
not shown). Therefore, to test whether Sir2-TAP was ex-
pressed and intact in all the strains, we immunoprecipitated
Sir2 and evaluated its integrity by immunoblotting. In all
strains, Sir2 was resolved into 3 species (Fig. 2I). The major
species was the expected size for Sir2-TAP (80 kDa). At least
one of the other bands was observed in previous Sir2 immu-
noprecipitations and was presumed to be a degradation prod-
uct (23). However, although Sir2 degradation was quite low in
wild-type and orc5-1 strains, it increased significantly in orc2-1
strains. Despite a considerable drop in the level of intact Sir2
and in the Sir2 ChIP levels at HMR (Fig. 2C), the silencing in
orc2-1 sir1? double mutants was comparable to that in sir1?
mutants (Fig. 1).
The synergistic effect of sir1? orc2-1 double mutants on Sir2
FIG. 1. Effects of orc mutations on HMR silencing in the absence of
Sir1. (A)Semiquantitative mating
(JRY3009), orc5-1 (JRY3961), orc2-1 (JRY4058), sir1? (JRY3010),
orc5-1 sir1? (JRY8891), and orc2-1 sir1? (JRY8892) strains. (B) QRT-
PCR analysis of HMRa1 transcript from the same strains used in panel
A. Quantities of a1 cDNA were normalized to ACT1 cDNA.
assay forwild-type (WT)
VOL. 30, 2010ORIGIN RECOGNITION COMPLEX IN SACCHAROMYCES CEREVISIAE 629
FIG. 2. Association of ORC and Sir proteins with HML and HMR in orc2-1 and orc5-1 mutants. (A) ChIP results for Sir3-TAP at HML and
HMR loci in wild-type (WT) (JRY8899), orc5-1 (JRY8900), and orc2-1 (JRY8901) strains. (B) ChIP analysis for Sir1-HA at HML and HMR loci
in wild-type (JRY8902), orc5-1 (JRY8903), and orc2-1 (JRY8904) strains. (C) ChIP results for Sir2-TAP at HML and HMR loci in wild-type
(JRY8893), orc5-1 (JRY8894), and orc2-1 (JRY8895) strains. (D) ChIP analysis for Sir2-TAP in the absence of Sir1 in sir1? (JRY8896),
630 O ¨ZAYDIN AND RINEMOL. CELL. BIOL.
levels at silencers was unexpected. orc5-1 alone or in combi-
nation with sir1? had little effect on the level of Sir2 protein in
cells (Fig. 2I), ruling out a trivial explanation for the reduced
Sir2 occupancy in Fig. 2D. The cause of reduced Sir2 levels in
orc2-1 mutants remains unexplained.
ORC interacted with internal regions of HMR. ORC is ex-
pected to bind at the silencers because each silencer includes at
least one ACS sequence, but ORC’s binding to both silencers
and silenced DNA has not been extensively evaluated before.
Prompted by the Sir1 association at HML?1 and HMRa1 (Fig.
2B), we speculated that ORC might have a similar distribution
due to its interaction with Sir1. To test the association of ORC
throughout the silenced domain at HMR, HA epitope-tagged
Orc5 (Orc5-HA) and Orc1 (Orc1-HA) subunits, as well as the
whole ORC complex, were immunoprecipitated using anti-HA
antibodies and polyclonal ORC antibodies, and the immuno-
precipitates were evaluated by ChIP analysis. All of these as-
says showed similar and unexpected results. The ORC was
found at HMR-E and HMR-I, as expected, both of which are
bona fide chromosomal origins of replication (29, 49). How-
ever, ORC was also associated throughout all 3 kb of the
silenced domain (Fig. 3B and C). Qualitatively similar results
were obtained at HML at which neither silencer is a bona fide
origin of replication (Fig. 3E).
Although DNase I footprinting studies show a discrete bind-
ing of ORC to the ACS of origins (36), the ORC ChIP signal
sir1? orc5-1 (JRY8897), and sir1? orc2-1 (JRY8898) strains. (E) ChIP analysis for the ORC complex in sir1? (JRY3010), orc5-1 sir1? (JRY8891),
and orc2-1 sir1? (JRY8892) strains using polyclonal ORC antibodies. (F) Immunoblotting for the ORC complex in wild-type, orc5-1 (JRY3961),
and orc2-1 (JRY4058) strains using polyclonal ORC antibodies. Immunoblotting for Pgk1 is included as a background control. (G to I)
Immunoblotting for Sir1-HA (G) and Sir3-TAP (H). An asterisk indicates a cross-reacting background protein. (I) Immunoprecipitation results
for Sir2-TAP in the same strains as well as sir1? combinations. N-terminal degradation products of Sir2-TAP are labeled.
FIG. 3. Physical associations of ORC throughout the HMR region. (A) Schematic of the HMR region and locations of primer sets A to H used
for QPCR analysis of the immunoprecipitated chromatin here and in Fig. 4. (B) ChIP analysis of Orc5-HA throughout HMR in the wild-type strain
(JRY8919). (C) ChIP analysis for Orc1-HA on HMR in strain JRY8976. (D) ChIP analysis for the ORC complex using polyclonal ORC antibodies
in the wild-type strain (JRY3009). QPCR analysis was done for HMR, ARS305, and 1 kb downstream of ARS305. (E) ChIP results for Orc5-HA
and Orc1-HA on HML and ARS305 and 1 kb downstream of ARS305.
VOL. 30, 2010 ORIGIN RECOGNITION COMPLEX IN SACCHAROMYCES CEREVISIAE631
throughout HML and HMR raised the possibility that ORC
might have unexpected binding properties as measured by
ChIP analysis at other sites, such as origins of replication not
related to silencing. Hence we evaluated the same immuno-
precipitates for ORC binding to regions flanking a bona fide
origin on the same chromosome as HML and HMR (ARS305).
As at the silencers, ORC bound strongly with ARS305. How-
ever, in contrast to HML and HMR, ORC did not detectably
interact with sequences only 1 kb from ARS305 (Fig. 3D and
E). Therefore, the chromatin association of ORC at HMR was
distinctly unlike that observed for ORC at a bona fide origin of
ORC’s ability to ChIP throughout HMR was dependent on
Sir proteins and HMR-I. The silenced chromatin domain ex-
tends beyond the silencers and ends at the boundary elements
on either side of HMR (13, 14). ORC association followed a
similar pattern (Fig. 3B and C), suggesting the possibility of an
expanded role for ORC in HMR silencing. We tested whether
Sir proteins and hence silencing were required for ORC to
ChIP internal regions of HMR in strains lacking SIR1, SIR2, or
both. Deletion of SIR2 completely abolishes silencing, whereas
deletion of SIR1 produces cells in one of two mitotically stable
phenotypes as described above. Individual deletions of SIR1 or
SIR2 decreased ChIP levels of ORC both at HMR-E and at the
internal HMRa1 site (Fig. 4A). Deletion of SIR3 and SIR4 gave
effects similar to that of SIR2 deletion and had modest effects
on ORC levels at HMR-E but resulted in a 2- to 3-fold drop at
HMRa1 (data not shown). Deletion of the Sir proteins did not
decrease ORC recruitment at ARS305 (Fig. 4B) or ARS1 (data
not shown). Thus, silencing and/or Sir proteins were required
for ORC association throughout HMR. Surprisingly, the com-
bination of sir1? and sir2? showed a synergistic effect and led
to further decrease in ORC ChIP levels relative to sir2? alone.
Hence, Sir1 contributed to ORC’s association with internal
regions of HMR even in cells completely defective in silencing
HMR. Due to the presence of ORC binding sites at the silenc-
ers, ORC could be immunoprecipitated at silencers even in
sir1? sir2? double mutant cells; however, no ORC was de-
tected at the internal regions of HMR in the double mutants.
Recent work suggests that an interaction between the
HMR-E and HMR-I silencers enable Rap1-bound HMR-E to
interact with the internal regions of HMR as well as HMR-I
(61). To test whether the ORC association throughout HMR
was dependent upon this silencer interaction as well, we ana-
lyzed ORC distribution throughout HMR in cells lacking the
HMR-I silencer (Fig. 4C). Although the ChIP levels of ORC
for the internal regions decreased approximately 2-fold (com-
pare HMRa1 levels in Fig. 4A to enrichment at locus F in Fig.
4C), there was still 4-fold more ORC at 2 kb from HMR-E than
there was at 1 kb from ARS305. Thus, although HMR-I con-
tributed to ORC interaction at HMRa1, significant association
remained in the absence of HMR-I.
Heterochromatin was partially refractory to shearing. The
unexpected interaction of ORC throughout silenced chromatin
led us to consider various alternative explanations other than
direct association of ORC with the heterochromatin at these
FIG. 4. Dependence of ORC on Sir proteins and HMR-I. (A) ChIP results for Orc5-HA at HMR in WT (JRY8919), sir2? (JRY8920), sir1?
(JRY8921), and sir1? sir2? (JRY8922) strains. (B) ChIP for ORC complex in wild-type (JRY3009), sir1? (JRY8921), sir2? (JRY8920), and sir1?
sir2? (JRY8922) strains at ARS305. (C) ChIP results for Orc5-HA in HMR-I? (JRY8923). QPCR-analyzed locations are described in the legend
to Fig. 2A.
632O ¨ZAYDIN AND RINEMOL. CELL. BIOL.
positions. During ChIP analysis, before the immunoprecipita-
tion step, the formaldehyde cross-linked chromatin is sheared,
resulting in DNA fragments of 0.2 to 1 kb in length. It has been
widely assumed that all structures of chromatin are equally
vulnerable to physical shearing by sonication. If the condensed
nature of heterochromatic regions were sufficiently refractory
to physical shearing, resulting in larger DNA fragments being
recovered by immunoprecipitation, then proteins bound to
specific sites within heterochromatin might give the illusion of
binding across a wider region. Therefore, we tested whether
ORC’s apparent association with sequences far from its bind-
ing sites was due solely to variation in the ease with which
different structures of chromatin are sheared by sonication. To
address this possibility, whole-cell extracts of wild-type and
HMR-I?, and sir1? sir2? mutant cells were prepared as for
ChIP analysis. The resulting sheared genomic DNA was elec-
trophoretically separated, blotted onto a membrane, and hy-
bridized with radiolabeled probes from euchromatic (ACT1)
and heterochromatic (HMRa1) regions (Fig. 5). The overall
shearing of the DNA was similar in the wild-type strain and
sir1? sir2? double mutant and was slightly more extensive in
DNA from HMR-I?, as judged from the gel image (Fig. 5A).
ACT1 probe hybridization showed a pattern similar to the
overall shearing pattern, suggesting that shearing of chromatin
containing the ACT1 gene was not affected by these genotypes
(Fig. 5C). In contrast, the a1 probe hybridized to a slower-
migrating smear in DNA from wild-type cells compared to the
DNA from silencing-deficient cells (Fig. 5B). The data in this
image were quantified by analyzing the signal counts for each
fragment size (in 0.2-mm bins) in the autoradiograms (Fig. 5D
to F). These graphs clearly indicated the similarity in bulk
DNA shearing from these strains and some shearing resistance
at HMRa1 in wild-type versus silencing-defective strains. We
used the data in these graphs to calculate the modes of each
FIG. 5. Analysis of chromatin shearing at HMR. (A to C) Genomic DNA from wild-type (WT), sir1? sir2?, and HMR-I? strains were separated
on the gel (A), transferred to a membrane, and probed with labeled a1 (B) and ACT1 (C). (D to F) Plots for signal quantity versus the DNA size
for the agarose gel, as well as the a1 and ACT1 probed blots. (G) Mode and percentage of the DNA fragments in each strain that include both
HMRa1 and at least one of the silencers. N. A., not available.
VOL. 30, 2010 ORIGIN RECOGNITION COMPLEX IN SACCHAROMYCES CEREVISIAE633
sample. Although ACT1 showed some variation in the mode
due to sample-to-sample shearing variations, the wild-type
strain showed consistently higher modes for the HMRa1 locus
than the sir1? sir2? double mutant strain did (Fig. 5G).
To determine whether this extent of shearing resistance was
adequate to account for the data about ORC distributions, we
calculated the total number of fragments that were large
enough to include HMRa1 and one or both of the silencers
(those longer than 1,440 bp) after normalizing for shearing
differences as explained in Materials and Methods. Such frag-
ments would be immunoprecipitated by the ORC complex
bound exclusively at the silencers and would be detected by
QPCR analysis for HMRa1. The wild-type strain had only
2-fold-more (40%) of these fragments than the sir1? sir2?
double mutant did (22%), and the difference was much less for
HMR-I? (29%) (Fig. 5G). This modest difference in shearing
propensity was inadequate to explain the 8-fold ORC enrich-
ment at HMRa1 in wild-type strains. These data indicated that
ORC actually interacted with internal regions of HML and
HMR and that interaction could not be explained by reduced
shearing efficiency in heterochromatin. The data so far were
compatible with interaction resulting from either ORC binding
along the Sir proteins or with long-range interactions facili-
tated by higher-order structures of the heterochromatin as
described in the next section.
ORC binds internally to HMR in a Sir-dependent way. The
interactions of Sir proteins throughout silenced chromatin as
determined by ChIP analysis are commonly interpreted as
spreading these proteins across the chromatin. However, it is
also possible that some higher-order chromatin structure
brings other sequences close to proteins that bind only at their
recognition site, thereby enabling them to physically interact
and be cross-linked. Indeed, interactions between the two si-
lencers at HMR allow Rap1 bound to HMR-E to interact
through out the silenced region (61). The critical test of
whether ORC is actually associated with the internal se-
quences at HMR is to separate the internal silenced chromatin
from the silencers themselves prior to cross-linking and then
determine whether ORC is still bound to the silenced chroma-
tin at those internal sequences. For this experiment, we used
strains with two recombination sites (RS) that allow excision of
HMR as an episome, leaving HMR-E and HMR-I silencers in
the chromosome (Fig. 6A). Upon galactose induction of a
heterologous recombinase, site-specific recombination at the
recombination sites occurs in most cells within one cell cycle.
The silencing on this episome is unstable and lost within 2 h of
galactose induction (10; data not shown). Therefore, we ana-
lyzed ORC interaction at 60 min past induction, during which
time silencing is still robust, even though the “loop out” was
only 70% complete at this stage. To clearly distinguish HMR
sequences that had been looped out from those in the 30% of
chromosomes in which recombination had not yet occurred, we
used a primer set that specifically amplifies the excised circle
(the circle-specific primer set CSP) to differentiate ORC bind-
FIG. 6. Sir3 and ORC interaction with excised HMR. (A) Schematics of the HMR construct with recombination sites (RS) between HMR-E and
HMR-I silencers. Induction of the Gal promoter-controlled recombinase loops out the internal regions of HMR, leaving behind the silencers. CSP
is a circle-specific primer set that detects only excised molecules. (B) ChIP analysis of Sir3 in wild-type (WT) (JRY8865) and sir4? (JRY8953)
strains grown in raffinose (RAF) or galactose (GAL) for 1 h. (C) ChIP analysis of ORC in the same samples used for panel B. The no-antibody
(No Ab) control in panels B and C is for the wild-type strain grown in galactose (GAL). No Ct, no counts.
634 O ¨ZAYDIN AND RINEMOL. CELL. BIOL.
ing at the episome from the internal sequences left at the
chromatin. For a positive control, Sir3 bound both the silencers
and episome efficiently, and this interaction was lost in the
sir4? mutant strain (Fig. 6B). ORC enrichment at internal
regions of this construct while still resident in chromosome was
easily detected, though 2-fold lower than observed at wild-type
HMR (Fig. 2). The twofold reduction was perhaps due to the
RS sites, which introduce an additional 750-bp sequence be-
tween the silencers, potentially resulting in less efficient ORC
interaction at HMRa1 (Fig. 6C). Nevertheless, upon loop out,
the episome was still enriched in the ORC immunoprecipitate,
and this interaction was lost in the sir4? mutant strain. Despite
an overall decrease in the ChIP signal at HMRa1 of the
episome relative to the chromosome, ORC still showed signif-
icant interactions with internal regions of HMR on the epi-
some, suggesting that ORC was bound to internal regions in a
Sir protein-dependent manner.
ORC recruited by tethered Sir1 did not cross-link with in-
ternal regions. One argument for the ORC interaction with
the episome (Fig. 6) could be that the excised episome, though
covalently separated from silencers, might be restricted in its
mobility in the nucleus such that it could still cross-link with
the silencer-bound ORC. We tested this possibility by using a
synthetic silencer that was previously designed to bypass ORC
involvement in silencing. This synthetic silencer has four Gal4
binding sites replacing the ACS and has no HMR-I silencer
(HMR-GalSS). In cells with normal Sir1 protein, this silencer is
unable to silence HMRa1. However, expression of a Gal4 DNA
binding domain fused to Sir1 restores HMR silencing even in
cells with mutant ORC genes (17). Although the mechanism
for how this “supersilencer” bypassed the functional require-
ment for ORC is not well understood, it offered an opportunity
to address the “association by proximity” hypothesis, testing
whether ORC recruited to one position in silenced chromatin
could ChIP with another. We first determined whether the
synthetic silencer bound by Gal4-Sir1 actually recruited ORC
to the HMR-GalSS in the absence of an ARS consensus se-
quence (Fig. 7A). As expected, no ORC association was de-
tected in the absence of Gal4-Sir1. In contrast, ORC was
efficiently recruited to HMR-E in cells expressing Gal4-Sir1,
and the interaction was dependent on the BAH domain in the
N-terminal region of Orc1 known to interact with Sir1. This
result established the ability of tethered Sir1 to recruit ORC to
HMR in the absence of ORC’s binding site. However, in this
context, ChIP analysis revealed that ORC interacted only at
the silencer and not at the internal HMRa1 position.
A potential explanation for the absence of ORC’s internal
interaction in this synthetic construct could be the lack of
near-match ACS sequences present next to the core HMR-E
silencer. It was possible that, even though these sequences are
not sufficient for ORC’s long-range interactions at HMR in
cells lacking HMR-I (as suggested by the results shown in Fig.
4A), they may still contribute to binding of ORC at the internal
regions of HMR. Therefore, we analyzed ORC ChIP levels at
a silencer which includes only the 138-bp region of HMR-E
silencer that has the ARS and Rap1 and Abf1 binding sites but
lacks all the other near-match ACS sequences. Thus, this core
silencer was trimmed of all the flanking sequences and near
matches to the ARS consensus sequence and was directly com-
parable in structure to the synthetic silencer used in Fig. 7. This
trimmed core silencer showed similar ORC enrichment at
HMRa1 to that of its parent strain, which had the wild-type
silencer (data not shown). Thus, the ORC binding site in the
silencer itself was sufficient to mediate associations with inter-
nal sequences of HMR without benefit of near matches or
other ORC binding sites in its vicinity. Although the mecha-
nism behind the ORC independence of silencing in this context
(Fig. 7B) is unclear, the abundant ORC recruitment to the
silencer through tethered Sir1 did not lead to significant inter-
action between ORC and the internal sequence HMRa1.
Therefore, ORC enrichment with the episome in Fig. 6C was
most simply explained by ORC’s physical presence on the
episome rather than the physical proximity of the episome to
ORC bound at silencers.
ORC was not recruited to silenced telomeric tracts, and Sir1
was not recruited to other replication origins. Since ORC
enrichment at internal regions of HMR relied on silencing, we
wondered whether ORC could be recruited to silenced do-
mains per se, even in the absence of an ACS. Subtelomeric
sequences and polymerase II (PolII)-transcribed genes in
rDNA are silenced in yeast; however, both of these regions
FIG. 7. Associations of ORC with HMR in strains with tethered Sir1.
(A) ChIP analysis for ORC with polyclonal ORC antibodies in
HMRGalSS (JRY8925), HMRGalSS/Gal4-SIR1 (JRY8926), HMRGalSS
(JRY8930) strains. Similar results were observed when HA-tagged
Orc5 was immunoprecipitated (data not shown). (B) QRT-PCR re-
sults for HMRa1 transcript for the same strains. No Ab, no-antibody
VOL. 30, 2010ORIGIN RECOGNITION COMPLEX IN SACCHAROMYCES CEREVISIAE 635
harbor at least one ACS that can recruit ORC, precluding a
direct test of ACS-independent recruitment of ORC. However,
an array of a simple repetitive DNA, C1-3A, which lacks an
ACS, is apparently sufficient to assemble silent chromatin in
yeast (57). These internal tracts should allow a distinction
between ACS-dependent and Sir-dependent ORC recruit-
ment. Moreover, silencing of a URA3 reporter gene by internal
C1-3A repeats is Sir1 independent. We performed ChIP anal-
ysis for Orc5-HA enrichment at 2 different sequences (A and
B) that were proximal to the C1-3A repeats and the reporter
gene (URA3) that was silenced by these tracts (Fig. 8A). Nei-
ther sequence enriched in Orc5-HA immunoprecipitation.
Hence, at least as defined by this assay, it was possible to
assemble Sir-dependent silenced chromatin without the re-
cruitment of ORC. However, as discussed below, the strength
of silencing mediated by this C1-3A array is considerably
weaker than that evident at HML and HMR.
Previous work and the results in Fig. 7A suggested a strong
interaction between Sir1 and Orc1 (6, 17, 27, 63 ) and raised
the possibility that ORC would recruit Sir1 to chromosomal
origins of replication. However, ChIP analysis for Sir1-HA
showed no enrichment for any of the six ARS sequences tested
(Fig. 8B) in accordance with earlier data of Gardner and Fox
ORC had unexpected roles in the function and architecture
of the silenced chromatin. The work presented here offered an
expanded view of the interaction between ORC and silenced
chromatin. Previously, the only known role for ORC in silenc-
ing was by binding to ARS sequences in silencers, thereby
recruiting Sir1 to silencers through binding between Sir1 and
the BAH domain of Orc1 (27, 63). Indeed, previous studies
also concluded that the requirement for ORC was bypassed
when Gal4-Sir1 was tethered to a synthetic silencer because
under such conditions, the effects of orc mutations on silencing
are suppressed (17). These findings favored the idea that Sir1
may act only at the silencers and, in contrast to Sir2, Sir3, and
Sir4, not spread throughout the silenced domain. In fact, in
such strains with Gal4-Sir1 bound to the HMR-E silencer,
wild-type Sir1 protein in the same cells was not detectable at
the silencer or within the silenced domains (50). The work
reported here revealed that results with tethered Sir1 protein
obscured unexpected complexity regarding the interactions of
Sir1 and ORC in silenced chromatin.
The critical observation that led to this study was that the
orc5-1 mutation reduced silencing in sir1? cells (Fig. 1A and B)
and reduced recruitment of Sir2 at HMR and HML (Fig. 2C
and D). At face value, that result required that ORC have
additional contributions to silencing. Moreover, we found that
tethered Gal4-Sir1 fusion protein could recruit ORC to chro-
matin lacking an ACS (Fig. 7A). Our efforts to understand
these and other phenotypes led to several surprising conclu-
ORC and Sir1 physically interacted with the whole silenced
domain. ChIP assays of Sir1 in wild-type cells revealed that
Sir1 was associated with the silencers, as expected, but also
with internal regions of HML and HMR. Unlike Sir2, Sir3, and
Sir4, which interact as a complex, Sir1 interacts only with ORC
and Sir4 (6). Nevertheless, the Sir1 association we detected
throughout HML and HMR was similar to that of the other Sir
proteins. Sir1’s physical association throughout the HML and
HMR loci, combined with its ability to recruit ORC, led to our
discovery that epitope-tagged Orc5 and Orc1 subunits, and
presumably the entire ORC complex, associated with chroma-
tin throughout the HML and HMR loci (Fig. 3).
Because all previous studies of ORC revealed it to bind only
to ARS sequences, its association throughout the silenced do-
main far from the ARS sequences of silencers led us to explore
several explanations for how ORC could appear to interact
throughout silenced domains yet actually be bound only to
ARS sequences. In brief, we considered whether ORC had
peculiar properties in ChIP analyses when bound to ARS se-
quences, whether silenced chromatin is intrinsically resistant to
FIG. 8. Association of ORC with other heterochromatic regions
and of Sir1 with other replication origins. (A) ChIP analysis for
Orc5-HA at the internal telomeric tracts in STL (JRY8938), STL
ppr1? (JRY8940), and TTL ppr1? (JRY8941) strains. Enrichments for
2 different regions (A and B) in the vicinity of the telomeric repeats
were calculated. (B) ChIP analysis for Sir1-HA in strain JRY8902 at
HML-E and at origins ARS2, ARS305, ARS307, ARS309, and ARS315.
636O ¨ZAYDIN AND RINEMOL. CELL. BIOL.
shearing so as to confound ChIP assays, and whether higher-
order chromatin structures at HMR could poise internal se-
quences of HMR within “cross-linkable distance” of ORC
bound to silencers. Our data excluded all three possibilities as
an explanation for how ORC associated with sequences
throughout the silenced domain: ORC bound to ARS305 be-
haved in ChIP analyses in a manner expected for a site-specific
DNA binding protein, in contrast to the regional associations
detected at HML and HMR. We found evidence for a slight
shearing resistance of silenced chromatin, but our quantitative
analysis indicated that such resistance made, at best, only a
modest contribution to the interactions of ORC throughout
silenced chromatin (Fig. 5). Finally, we found that the inter-
action between the E and I silencers revealed by chromosome
configuration capture (3C) analysis could not account for the
interaction of ORC with HMR sequences recombinationally
severed from the silencers. (In principle, we cannot exclude the
possibility that long-range interactions accounted for a portion
of the signal in our studies due to small quantitative differences
in the signal produced from different experimental designs.
Indeed, the effect caused by the deletion of the HMR-I silencer
may well have affected the stability of the interactions of ORC
throughout the silenced domain independently of E-I interac-
In support of our results, an independent set of experiments
using the ChIP-Seq method to evaluate ORC distribution ge-
nome-wide with its higher resolution compared to traditional
ChIP methods has also detected ORC signal across HML and
HMR, including sequences not associated with the ARS ele-
ments at silencers (David MacAlpine and Stephen Bell, per-
sonal communication). Recent work from our lab establishes
that heterochromatic regions are prone to less enrichment in
ChIP-Seq analysis due to shearing resistance by sonication and
subsequent elimination at the size selection step (59). Despite
this bias against the heterochromatic regions, ORC interaction
was detected across the entire HML and HMR loci.
Associations of ORC with silenced chromatin lacking ORC
binding sites. Having exhausted alternative explanations for
ORC’s association throughout the silenced domain, we tested
directly whether ORC molecules associated with sequences
within the silenced domains when those sequences were no
longer physically linked to the silencers themselves. Using the
site-specific recombinase method to excise the internal se-
quences of HMR as silenced chromatin on an episome, we
found that, like the Sir proteins, ORC was clearly associated
with these episomal sequences (Fig. 6). Nevertheless, there was
a quantitative difference between the extent of association with
these excised sequences and with the same sequences left in
the chromosome. However, a similar difference was also ob-
served for Sir3 association. The reason for the reduced asso-
ciations in this experimental design could be because silencing
is somewhat labile on the excised episome, with some loss of
silencing evident within 60 min (data not shown), or due to an
influence on the RS sites added to allow for excision.
An alternative argument for the interaction between ORC
and the episome was that the silencer-bound ORC on the
chromatin could still interact with the excised episome because
of physical proximity. This argument was discounted with an
experiment with a synthetic silencer, whose silencing function
is independent of ORC. The ORC was efficiently recruited to
this silencer through Orc1 and Sir1 interactions, but ORC
recruited in this way did not show any interactions with the
internal regions (Fig. 7A). Therefore, the ORC interaction we
detected with the episome was most simply explained by
ORC’s physical presence on the episome.
ORC’s physical interactions throughout the HMR region
depended on Sir proteins. Formally, it was possible that, unlike
the DNA sequence flanking conventional ARS elements, there
was some special property of the sequences that make up HML
and HMR that allowed ORC to bind these sequences in the
absence of recognizable binding sites. Indeed, ORC from
larger eukaryotes typically lacks sequence-specific binding sites
as origins (21). However, ORC’s association with sequences
throughout HML and HMR was dependent upon silencing per
se (Fig. 4A) (data not shown) and not simply upon the DNA
sequence of the silenced domain. In fact, our analysis showed
that the deletion of an almost 700-bp sequence that flanks the
HMR-E core silencer results in an internal ORC enrichment
that is similar to the wild-type strain (data not shown). In
silencing-defective cells, ORC still bound the silencers, pre-
sumably through the ARS sequences of the silencers. Thus, it
seemed inescapable that ORC itself and Sir1 were structural
components of silenced chromatin. The data also suggested
that the combination of an ACS in the silencer and Sir1 con-
tributed to the extent of ORC association with the silencers
At present, we do not know how the interactions between
ORC and Sir1 are mediated throughout the silenced domain,
except that it was probably not through direct DNA binding.
Presumably the BAH domain of Orc1 associates with Sir1
throughout HMR, just as it does at silencers. Because Orc1 and
Sir3 are paralogs (5), perhaps Orc1 enjoys interactions within
silenced chromatin similar to those used by Sir3. Indeed, per-
haps ORC binds H3 and H4 histone tails deacetylated at those
positions that are the targets of Sir2. It would be interesting to
evaluate whether ORC has a different distribution profile in
cells lacking the Sas2 acetyltransferase, whose acetylation of
histone tails blocks Sir3 binding (33).
How do ORC mutations affect silencing? The results pre-
sented here have forced a reevaluation of the previous idea
that ORC mutations affect silencing primarily through their
effect on recruiting Sir1 to the silencer. That view was inspired
by two independent studies (11, 17) showing that a Gal4-Sir1
fusion protein tethered to the silencer through Gal4 binding
sites could suppress the silencing defect of ORC mutations.
The fundamental question in such experiments is whether the
constructs used to reveal some of the functions of a regulatory
site have the capacity to obscure part of the story. Particularly
for this synthetic silencer, where Gal4-Sir1 is tethered through
four Gal4 binding sites, the silencing may be more robustly
established than with a wild-type silencer and hence mask any
silencing defect caused by the absence of ORC recruitment.
Our ChIP analysis showed that the whole ORC complex
could be recruited to regions lacking a DNA binding site for
ORC in a Gal4-Sir1-dependent way and that recruitment was
lost in strains lacking the Sir1 interaction domain of Orc1
(N235?orc1) (Fig. 7). Loss of the ORC recruitment in
N235?orc1 did not affect the silencing adversely as measured
by a1 transcript levels, arguing that in the context of this syn-
thetic construct, the absence of ORC had no demonstrable
VOL. 30, 2010ORIGIN RECOGNITION COMPLEX IN SACCHAROMYCES CEREVISIAE637
effect on silencing. Nevertheless, the additional loss of silenc-
ing caused by orc5-1 in sir1? strains (Fig. 1) was free of any
complications caused by use of a silencer with multiple Gal4-
Sir1 binding sites and is unequivocal in identifying a Sir1-
independent role of ORC in silencing. Although the precise
nature of this role was incompletely resolved, ORC’s absence
from internal regions of HMR in cells with Gal4-Sir1 tethered
to the silencer indicated that the occupancy of internal se-
quences by ORC depends upon ORC binding sites at the
Heterochromatin was resistant to shearing by sonication.
The control experiments in this study uncovered a 2-fold rel-
ative shearing resistance of silenced chromatin (Fig. 5). A
similar resistance is reported for nucleoli in human and mouse
cell lines, presumably due to its extensive decoration with pro-
teins and RNA (46). Although shearing resistance could not
account for the presence of ORC at HMRa1 in the ChIP
experiments, this resistance could nevertheless prove useful in
analyses of genomes. The power of deep sequencing enabled
by the recent generation of DNA sequencers suggests that
variation in the shearing of different chromatin structures will
be useful for detecting interesting domains of chromatin struc-
ture that will be recognizable by underrepresentation (and
perhaps overrepresentation) of regions of the genome in
whole-genome sequence reads. Indeed, we and others have
recently exploited such impacts on shearing differences to eval-
uate deep-sequencing strategies for detecting structural fea-
tures on the chromosomes of yeast and humans (1, 59).
On the specificity of ORC-Sir1 interaction. If the comple-
mentary surfaces on Sir1 and Orc1 responsible for the physical
interaction of these two proteins (4, 20, 63) were sufficient to
explain their interaction, then the presence of one protein
would predict the presence of the other. One half of this
prediction was confirmed. A tethered Gal4-Sir1 protein re-
cruited ORC to a silencer completely lacking an ACS site.
However, as previously reported elsewhere (19), we were un-
able to detect Sir1 at any of the five different bona fide origins
of replication (Fig. 8B). In principle, these data could be ex-
plained if something else occupies the BAH domain of Orc1 at
origins. If so, such an interaction would probably not be nec-
essary for origin function, since HMR-E and HMR-I are bona
fide origins in cells. Alternatively, perhaps something else at
silencers, such as Rap1, contributes to forming a complex be-
tween ORC and Sir1. At face value, the inability of the C1-3A
repeats, which are, in effect, a series of Rap1 binding sites to
recruit either ORC or Sir1 would challenge that model. How-
ever, the rather weak silencing achieved by these repeats leaves
room to consider this possibility. The bottom line is that the
source of specificity for ORC-Sir1 interactions remains an in-
teresting and unexplained problem.
Could ORC have roles at HML and HMR beyond silencing?
The loss of silencing caused by mutations in ORC subunits is
clear, but the magnitude of these effects has, in all cases, been
small except in strains sensitized to silencing defects. More-
over, a sufficient number of tethered Gal4-Sir1 fusion proteins
can recruit ORC to a silencer, but not to internal regions of
HMR, yet silencing seems robust in the absence of ORC under
these circumstances. So what is the role(s) of ORC at internal
regions of HML and HMR?
The most obvious possibility would be some role in mating-
type interconversion. Interconversion occurs by a gene conver-
sion mechanism, which requires DNA synthesis, but does not
require an origin of replication. Perhaps ORC’s presence at
HML and HMR and ORC’s interaction with other proteins,
such as the minichromosome maintenance (MCM) proteins,
contribute to the remarkable efficiency of interconversion. Al-
ternatively, perhaps ORC’s integration of cell cycle signals,
such as those from Cdc28, helps sharpen temporal control of
the HO endonuclease to the small temporal window between
start and replication of HML and HMR. The tethered Gal4-
Sir1 protein in cells lacking the N terminus of Orc1 would
provide a useful context for testing these ideas.
In other organisms, ORC also interacts with heterochro-
matic domains. For example, Drosophila melanogaster Orc2
colocalizes with the heterochromatin protein HP1 in hetero-
chromatin (43), and mutations of orc2 cause a defect in re-
cruiting HP1 to heterochromatin, establishing a role for ORC
in heterochromatin structure at a global scale (28). In human
cell lines, Orc2 is tightly bound to the heterochromatin and to
HP1? and HP1? (45), and this localization is specific to G1-
phase and early S-phase cells. Although ORC in S. cerevisiae
occupies origins at all stages of the cell cycle, posttranslational
regulation of its activity offers potential insight into the cell
cycle regulation of events at HML and HMR that have so far
We thank Marc Gartenberg, Virginia Zakian, and Stephen Bell for
strains and reagents critical to the execution of these experiments. We
thank the members of our lab and Michael Botchan for helpful dis-
cussions and comments on the manuscript. We also thank Stephen Bell
and David MacAlpine for sharing their unpublished data.
This work was supported by a grant from the National Institute of
Health (NIH GMS 31105).
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