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Mol Cell Biol. Mar 2006; 26(5): 1955–1966.
PMCID: PMC1430255

Tolerance of Sir1p/Origin Recognition Complex-Dependent Silencing for Enhanced Origin Firing at HMRa


The HMR-E silencer is a DNA element that directs the formation of silent chromatin at the HMRa locus in Saccharomyces cerevisiae. Sir1p is one of four Sir proteins required for silent chromatin formation at HMRa. Sir1p functions by binding the origin recognition complex (ORC), which binds to HMR-E, and recruiting the other Sir proteins (Sir2p to -4p). ORCs also bind to hundreds of nonsilencer positions distributed throughout the genome, marking them as replication origins, the sites for replication initiation. HMR-E also acts as a replication origin, but compared to many origins in the genome, it fires extremely inefficiently and late during S phase. One postulate to explain this observation is that ORC's role in origin firing is incompatible with its role in binding Sir1p and/or the formation of silent chromatin. Here we examined a mutant HMR-E silencer and fusions between robust replication origins and HMR-E for HMRa silencing, origin firing, and replication timing. Origin firing within HMRa and from the HMR-E silencer itself could be significantly enhanced, and the timing of HMRa replication during an otherwise normal S phase advanced, without a substantial reduction in SIR1-dependent silencing. However, although the robust origin/silencer fusions silenced HMRa quite well, they were measurably less effective than a comparable silencer containing HMR-E's native ORC binding site.

A replication origin is a position on the chromosome where DNA replication initiates. A single eukaryotic chromosome relies on initiation by multiple replication origins during a single S phase to complete its duplication. Studies of replication in the yeast Saccharomyces cerevisiae indicate that individual origins vary in terms of their efficiency, the fraction of cell cycles in which they initiate (activate; fire), and their timing, the point during S phase in which they initiate (30; also see the commentaries in references 25 and 38). Several studies demonstrate that chromatin structures associated with transcription repression can prevent or delay initiation by some origins, but different mechanisms may be operating for other origins (reviewed in reference 42).

Silencers are DNA elements that direct the formation of repressive chromatin domains at the yeast silent mating-type loci, HMLα and HMRa (10, 33). Each silent locus contains two silencers, and each silencer binds sequence-specific DNA binding proteins, including the origin recognition complex (ORC) and one or both of the abundant multifunctional nuclear proteins Rap1p and Abf1p. ORC is essential for chromosome replication, binds to origins, and actively recruits additional proteins essential for initiation (1, 4). Since the yeast genome contains ~400 replication origins (30, 43), ORCs bind to hundreds of positions distributed over the genome. The vast majority of these origins are not associated with silencers. However, all four transcriptional silencers, two at HMRa (HMR-E and HMR-I) and two at HMLα (HML-E and HML-I), are associated with replication origins (reviewed in reference 21). Silencer-associated origins can fire efficiently in a plasmid but fire extremely inefficiently and late during a normal S phase (HMRa) or not at all (HMLα) in their chromosomal contexts (7, 26, 27, 31, 36, 41). Therefore, silencers contain elements necessary to function as replication origins, most notably a binding site for ORC, yet fail to do so efficiently in their native context.

Silencers nucleate the assembly of transcriptionally repressive Sir (silent-information regulator)-dependent chromatin at HMLα and HMRa that represses (silences) transcription. The silencer binding proteins, ORC, Rap1p, and Abf1p recruit a collection of four Sir proteins through direct protein-protein interactions (10, 13). In particular, ORC binds the HM-specific Sir protein Sir1p that helps recruit the Sir2 to Sir4 proteins to the HM loci (10). Sir2p is a conserved protein deacetylase (6) that modifies nucleosomes within the HM domains and thereby promotes the binding of additional Sir2, Sir3, and Sir4 proteins to chromatin, leading to silent chromatin that emanates from silencers (24, 33).

Sir-dependent chromatin can inhibit or delay origin firing in several contexts (42). For example, chromatin dependent on Sir2p, -3p, and -4p also forms at telomeres and can suppress origin firing within the subtelomeric repeats (37). Sir2p-dependent chromatin that forms within rRNA genes suppresses firing by many of the origins within these repeats (29) and may also inhibit many other genomic origins to some extent (28). In addition, tandem copies of the HMR-E silencer or a Gal4-Sir4p fusion engineered near normally early firing origins can delay origin activation in a Sir-dependent mechanism (44). Thus, suppression of origin firing may be an unavoidable by-product of SIR-dependent chromatin formation.

Given these observations, it is somewhat surprising that the inefficient origin activity and late replication time of the HM loci are not Sir dependent. The silencer-associated origins at HMLα are not activated in strains containing deletions of SIR genes, and the inefficient HMR-E origin is only somewhat enhanced in such strains (7, 26, 27, 41). Moreover, both HM loci are replicated at the end of S phase even in sir mutants. Thus, Sir-independent factors must contribute to the inefficient origin activity and late replication time of the HM loci (27, 36). An open question is whether these same factors also contribute to the stability of Sir-dependent chromatin.

Two studies of the HMR-E silencer provide evidence that the ORC-silencer DNA interaction within HMRa contributes both to the formation of silent chromatin and the overall inefficiency of origin firing at this locus (26, 27). One study demonstrates that a mutation of the ORC binding site within the HMR-E silencer [to create the HMR-E(A-) mutant] reduces silencing, while it simultaneously enhances origin firing from HMRa (26). This apparently paradoxical result, given ORC's established positive role in origin firing, can be explained by the presence of several neighboring near matches to the A element surrounding the defined HMR-E silencer (21) that can bind ORC in vitro (M. A. Palacios DeBeer and C. A. Fox, unpublished) and provide for ORC-dependent origin firing in vivo (18, 26). We postulate that when an ORC normally binds its site within HMR-E, it somehow prevents additional ORCs from binding to the several near matches to the A element surrounding the silencer. However, when the silencer is mutated [as in HMR-E(A-)], ORCs now bind to these sites where they are better able to function in origin firing but considerably less able to function in silencing.

The second study exploited an engineered HMR-E silencer in which the neighboring sequences that can bind ORC described above have been deleted such that the silencer contains the only ORC binding site within HMRa (27). In this context and as expected, a mutation that substantially weakens the silencer's binding affinity for ORC in vitro reduces silencing in vivo. However, unexpectedly, this same mutation simultaneously enhances origin firing within HMRa. Thus, the silencer's native ORC binding site binds ORC in such a way that silencing is promoted while origin firing is inhibited. Together the data from these studies are consistent with the notion that HMR-E's ability to act as a silencer is intrinsically linked to its inherently weak activity as an origin.

In this report we addressed three related questions relevant to this idea by examining silencing and origin firing at HMRa. First, we examined yeast cells containing the HMR-E(A-) mutation to ask whether the more robust origin activity caused by this mutation was compatible with efficient SIR1-dependent silencing. Second, we engineered fusions between normally robust replication origins and the HMR-E silencer to determine whether it was possible to substantially enhance origin firing by the silencer and advance the replication time of HMRa during an otherwise normal S phase. Third, we asked whether more robust origin activity and earlier origin firing from the silencer itself was compatible with efficient SIR1-dependent silencing.


Yeast strains and plasmids.

Yeast strains and plasmids used in this study are described in Tables Tables11 and and2.2. All yeast strains are isogenic to W303-1A unless otherwise noted. Standard yeast genetic methods (15) and recombinant DNA methods (34) were used.

Yeast strains used in this study
Plasmids used in this study

Plasmid and strain construction.

ADH1pr→SIR1 was inserted into the multiple cloning site of pRS304 to create plasmid pCF968, which was cut within the TRP1 coding sequence and transformed into a strain containing HMR-E(A-)ΔI (CFY108). Single integration events were determined by screening TRP+ colonies by DNA blot hybridization. The control constructs, empty plasmid (pRS304) or pRS304 with ADH1pr→sir1-101 (pCF1470), were introduced into CFY108 similarly. Genetic crosses introduced these integration constructs into additional strains used in this study. For construction of ARS1/SS (synthetic silencer), the ARS1 region (Saccharomyces Genome Database coordinates 462484 to 462612) was amplified from pCF111 by PCR. Primers were designed to engineer BamHI and SphI sites flanking the origin. ARS1 was then introduced into the BamHI/SphI sites within EcoRI/HindIII fragment containing HMR-SS (an engineered version of the HMR-E silencer named the synthetic silencer) in pUC18 (pCF63). This replaced the A-element within HMR-SS with ARS1 (pCF1532). A similar strategy was used to replace the A-element of HMR-SS with ARS307 (pCF1537). The EcoRI-HindIII fragments containing these ARS/SS fusions were introduced into the HMRa locus by transformation into a strain carrying hmrΔ::URA3 (CFY31) followed by selection for one-step integration on medium containing 5-fluoroorotic acid.

2-D origin mapping.

Two-dimensional (2-D) origin mapping was performed as described previously (11, 19). For the timing experiments, yeast strains were MATa and contained bar1Δ::HIS3 to aid α-factor arrest and release. Cells were grown at 23°C in 4 liters of yeast rich medium (yeast extract-peptone-dextrose [YPD]) to an optical density at 600 nm of 0.7, concentrated for arrest in G1 phase with α-factor, and then released from arrest in 4.8 liters YPD plus 0.025 mg/ml pronase (CalBiochem). Aliquots (400 ml) were harvested every 10 min, and genomic DNA was isolated by CsCl gradients. DNA from each sample was analyzed for replication intermediates (RIs) by digestion with HindIII for HMRa, NcoI for ARS1 and ARS306, and EcoRI for R11. Digests were then separated in two dimensions and examined by DNA blot hybridization using primers specific for each region. Probes were created using Megaprime DNA labeling system (Amersham Pharmacia). For 2-D origin mapping of asynchronous cultures, DNA was enriched for RIs with BND cellulose after restriction digestion.

Quantification of RIs from 2-D origin mapping experiments.

For the timing experiments, we quantified the level of RIs as a fraction of the linear (unreplicated or fully replicated) DNA recovered for each fragment to acquire information about the peak replication time and overall replication pattern for each region during S phase. Separate boxes were used to quantify replication bubbles, small replication forks, large replication forks, and linear fragments using Molecular Dynamics Storm 820 PhosphorImager and ImageQuant 5.2 software. Each RI value was expressed as a fraction of the linear DNA that constitutes the substantial majority of DNA at each time point to reduce bias caused by differences in DNA recovery and to facilitate comparison between fragments that radiolabeled with different efficiencies. For early replication (extremely efficient replication origins) or late replication (R11 replication forks) controls, all RIs are generated by the same molecular event (i.e., either a replication origin [ARS306] or a passive fork [R11]). Therefore, the sum of RIs was divided by the value of linear DNA. However, HMRa is replicated by a mixture of molecular events—origin firing from HMR-E and passive replication from distant origins. To emphasize the timing differences at HMRa that were due to differences in origin firing, the values for small-fork RIs were subtracted from total RIs before dividing by the value for linear DNA. Small forks arise from passive replication of the fragment, not origin firing within the fragment, and thus, this subtraction step should emphasize the contribution origin firing is making to the RI signal. However, since small forks eventually become large forks, as do replication bubbles, this method does not exclude the contribution made by passive replication to the overall timing profile. However, we note that quantification of bubble RIs alone gave similar replication timing profiles, confirming that this subtraction method emphasized the contribution of replication bubbles to the value of RIs. Since wild-type HMR-E forms so few bubble intermediates, the values obtained by this more direct method were extremely small and subject to large variation due to slight differences in box placement during quantification. Therefore, we chose to use the value obtained from the subtraction method. For examining the effects of the ARS1/SS fusion on the replication timing profile of HMRa, we directly quantified bubble intermediates because they were formed quite efficiently. We note however, that the small-fork subtraction method produced a similar replication timing profile.

To effectively view the timing profile of each fragment on the same normalized graph, the peak replication value for HMRa was given an arbitrary value of 1.0, whereas the peak replication value for the control samples was given an arbitrary value of 0.9, and all other values were expressed as a fraction of these peak values. These differences in peak value between the different fragments were assigned to avoid overlap between profiles that made it difficult to view all of the data on a single graph.

RNA blot hybridization.

Total RNA was isolated from yeast as described previously (9), and 10 μg were used per lane. Blots were probed with a1 and SCR1, as a loading control, as described previously (9).

Quantitative RT-PCR analysis of a1 mRNA levels.

To determine the levels of a1 mRNA transcribed from HMRa in cells containing the ARS/silencer fusions, total RNA was prepared from cells grown to an optical density of 0.5 to 0.8 as described previously (9). Total RNA concentration was determined by both spectroscopic analysis and gel electrophoresis. Samples were diluted to a concentration of 1 μg/ml, treated with DNase I at 37°C for 45 min. DNase was heat inactivated at 75°C for 10 min. For reverse transcriptase PCRs (RT-PCRs), 5 μg of total RNA was used in a 20-μl reaction mixture using oligo(dT) primers and the Superscript II protocol (Invitrogen). Each RT-PCR mixture was diluted 1:20 to a total volume of 200 μl. Twofold serial dilutions were used in PCRs as follows: for measuring ACT1expression, 0.25, 0.5, and 1.0 μl were used with ACT1-specific primers in a 20-cycle PCR. For a1 mRNA, 1, 2, and 4 μl were used with a1-specific primers in a 31-cycle PCR, except for the HMR-SS RT-PCR sample that was diluted 1:10 prior to the PCR because this sample contained so much more a1 mRNA than the others. The PCR mixtures were analyzed on a 1% agarose gel stained with GelRed dye (Biotium) at a 1:1,000 dilution. The bands were quantified using an Epi Chemi II darkroom (UVP Laboratory Products).


Yeast strains used in this study contain a deletion of the HMR-I silencer (HMRΔI), as described elsewhere (23). In this context, mutation of the site required for ORC binding (A-element) within HMR-E [HMR-E(A-)] causes a silencing defect (26). Since the HMR-E silencer is both necessary and sufficient for HMRa silencing, we have continued to focus on HMR-E function in strains lacking HMR-I (HMRΔI) (Fig. (Fig.1A).1A). Since all strains described in this report harbor the same deletion (HMRΔI), we will note this fact explicitly only here and in Table Table1,1, Fig. Fig.1A,1A, and Fig. Fig.4A4A.

FIG. 1.
The HMR-E(A-) mutant silencer enhanced origin firing with HMRa and advanced the peak replication time of this locus but did not create an early firing origin. (A) Cartoon of HMRa HindIII (H) fragment examined in these experiments. The HMR-E silencer is ...
FIG. 4.
Sir1p/ORC-dependent silencing tolerated enhanced origin firing from the silencer itself. (A) Cartoon of silencers examined at HMRa. HMR-SS does not contain the neighboring regions present at natural HMR-E that contribute to origin firing in HMR-E(A-) ...

The HMR-E(A-) mutant silencer advanced the peak replication time of HMRa but did not create an early firing origin.

The HMR-E silencer is an inefficient origin; HMRa is replicated late in S phase primarily by forks that emanate from distant origins (30). As discussed in the introduction, a mutation of the ORC binding site within the HMR-E silencer [creating HMR-E(A-)] actually enhances the origin bubble signal at HMRa in a 2-D origin mapping experiment, presumably because ORC molecules can bind to neighboring near matches to the A-element (contained within regions flanking the HMR-E silencer depicted by the gray rectangles in Fig. Fig.1A;1A; see reference 21) where they function more efficiently in origin firing (26) (Fig. (Fig.1B).1B). Fork migration analyses allowed us to estimate that origin firing occurs ~30 to 40% more often from HMR-E(A-) than from wild-type HMR-E. We postulate that a silencer-bound ORC may suppress origin firing at HMRa by preventing ORCs from binding the regions flanking the silencer (26), perhaps by positioning nucleosomes, an activity reported for ORC bound to origins (20).

The enhanced origin firing of HMR-E(A-) observed in a 2-D origin mapping experiment from asynchronously growing cells meant that a fraction of cells in the population used internal origins to replicate HMRa before forks emanating from distant origins arrived. However, these experiments could not reveal whether the enhanced origin signal was due to internal origins firing occurring very early in S phase, well before HMRa is normally replicated, or later in S phase but still prior to HMRa's normal replication time. Therefore, to distinguish between these possibilities and determine the extent to which the enhanced origin firing of HMR-E(A-) advanced the replication time of HMRa, we performed 2-D origin mapping experiments during cell cycle arrest and release (Fig. (Fig.1C).1C). Specifically, cells were released from arrest in G1 phase and harvested for analysis of replication intermediates at various points throughout S-phase progression.

These experiments revealed that the HMR-E(A-) mutation only slightly advanced the replication time of HMRa in a fraction of cells in the population. As expected, this effect was due to the contribution made by origin firing. For example, at 40 min the HMR-E(A-) cells produced RIs with a high ratio of bubble to small-fork intermediates (Fig. (Fig.1C).1C). In fact, small forks were barely detectable for HMR-E(A-) at 40 min (Fig. (Fig.1C),1C), indicating that the majority of RIs at the relatively early time point were generated by origin firing within HMRa. These data are consistent with enhanced origin firing in HMR-E(A-) cells causing replication of HMRa to occur before the arrival of replication forks from distant origins. Moreover, the HMR-E(A-) mutant cells consistently produced RIs, again with a high ratio of bubble intermediates to small forks, detectable in the following cell cycle whereas wild-type HMR-E cells did not (Fig. (Fig.1C,1C, 120 min). Thus, the HMR-E(A-) mutation advanced the replication time of HMRa within a fraction of the cells in the population.

To gain information about how substantially the HMR-E(A-) mutation advanced the replication time of HMRa, ARS306 (an early S-phase origin) and R11 (a late replicated region) were analyzed in addition to HMRa in an independent set of cell cycle arrest and release experiments. Quantification of these data were consistent with the HMR-E(A-) mutation advancing the peak replication time of HMRa by about ~10 min (Fig. (Fig.1D,1D, compare 50 and 60 min). For wild-type HMR-E, most cells replicated HMRa after they had replicated the late control locus, R11 (Fig. (Fig.1D).1D). In contrast, for the HMR-E(A-) mutant, most cells replicated HMRa slightly before or coincident with R11 replication. These data provided additional independent evidence that the enhanced origin firing within HMRa caused by the HMR-E(A-) mutation advanced HMRa's replication time to some degree. However, although the HMR-E(A-) mutant advanced the replication time of HMRa in some cells, it remained a relatively late replicating locus regardless, with a peak replication time occurring later than the peak replication time for the early firing origin ARS306 (Fig. (Fig.1D1D).

Silencing of HMR-E(A-) was restored by increased dosage of SIR1, and this silencing required the defined Sir1p-Orc1p interaction.

The behavior of the HMR-E(A-) mutant raised the possibility that the enhanced origin activity and/or perhaps the advanced replication time it caused was incompatible with silencing. Consistent with this notion, a mutation in ORC2 (orc2-1) that reduces origin firing by HMR-E(A-) enhances silencing by this mutant silencer (26). However, since mutations in many different replication genes can modulate silencing (8), to test this idea more definitively, we needed to identify conditions that, in contrast to the orc2-1 mutation, restored efficient silencing to the HMR-E(A-) mutant without causing obvious and/or global effects on chromosome replication.

Some silencing mutations can be suppressed by increased dosage of SIR genes, particularly SIR1. In one study increased SIR1 dosage suppressed silencing defects caused by a number of silencing-defective mutants, including HMR-E silencer mutants (39). Sir1p must bind the silencer to function in silencing (5), and this binding requires a direct and well-defined interaction between Sir1p and Orc1p as well as a less defined interaction between Sir1p and Sir4p (3, 12, 45). Residual silencing by HMR-E(A-) still requires SIR1 (Palacios DeBeer and Fox, unpublished), indicating that Sir1p must bind HMRa in this mutant by some mechanism. A reasonable idea was that the HMR-E(A-) mutation reduced the affinity of Sir1p for HMRa by reducing or abolishing its ability to interact with a silencer-bound ORC. Therefore, we tested whether increased dosage of SIR1 could restore silencing to HMR-E(A-) mutant cells (Fig. (Fig.22).

FIG. 2.
Silencing of HMR-E(A-) was restored by increased dosage of SIR1, and this silencing required the defined Sir1p-Orc1p interaction. (A) Cartoons of the chromosomal constructs used to overproduce SIR1 or a mutant version of SIR1, sir1-101, as stable integrants ...

To perform these experiments, we engineered SIR1 under the control of the constitutive ADH1 promoter and integrated this fusion at the TRP1 locus (TRP1::ADH1pr→SIR1) (Fig. (Fig.2A).2A). A stable integrant allowed us to grow cells in rich nonselective medium that enhanced chromosomal DNA recovery for 2-D origin mapping experiments (see below). TRP1::ADH1pr→SIR1 was introduced into MATα HMR-E(A-) cells, and silencing was measured by RNA blot hybridization of a1 mRNA. As expected, the MATα HMR-E(A-) cells exhibited reduced silencing of HMRa as indicated by expression of a1 mRNA (Fig. (Fig.2B,2B, lane 3). In contrast, the MATα HMR-E(A-) cells containing TRP1::ADH1pr→SIR1 produced no detectable a1 mRNA (Fig. (Fig.2B,2B, lane 4) and were indistinguishable from the wild-type MATα HMR-E cells (Fig. (Fig.2B,2B, lane 2). Therefore, ADH1pr→SIR1 fully rescued the silencing defect of HMR-E(A-).

Since the silencer ORC binding site is defective in the HMR-E(A-) mutant, it was conceivable that the Sir1p-Orc1p interaction normally required for wild-type HMR-E silencing was not being used when silencing of HMRa was restored with a high dosage of SIR1. To test this possibility, we engineered a yeast strain that lacked the coding region for the N-terminal BAH (bromo-adjacent homology) domain of Orc1p (orc1NΔ). This region of ORC1 is dispensable for replication initiation but required for formation of a Sir1p-Orc1p complex and silencing (2, 16, 17). Increased levels of SIR1 (TRP1::ADH1pr→SIR1)failed to restore silencing to the MATα HMR-E(A-) orc1NΔ cells (Fig. (Fig.2B,2B, compare lanes 5 and 6). In addition, increased levels of a mutant form of SIR1 (sir1-101) that produces a Sir1psrd mutant protein (srd for silencer recognition defective) defective in interactions with ORC but not with Sir4p (3) also failed to restore silencing to HMR-E(A-) (Fig. (Fig.2B,2B, lane 7). Therefore, increased dosage of SIR1 restored wild-type levels of silencing to the defective HMR-E(A-) silencer through the established Sir1p-ORC interaction mechanism.

SIR1/ORC-dependent silencing did not reduce origin firing or delay replication time of HMR-E(A-).

As shown above, TRP1::ADH1pr→SIR1 restored silencing to HMR-E(A-). This effect provided a means to ask whether silencing was compatible with the enhanced level of origin firing caused by the HMR-E(A-) mutation. Specifically, if silencing were compatible, then the level of origin firing in the silent HMR-E(A-) cells should be the same as that from the nonsilent HMR-E(A-) cells. To test this idea, we compared origin firing in strains containing HMR-E(A-) that differed only in terms of their TRP1 locus (Fig. (Fig.3A):3A): one strain contained TRP1::pRS304 and expressed endogenous levels of SIR1 [SIR1 (+), Not silent; see also Fig. Fig.2A],2A], whereas the other contained TRP1::ADH1pr→SIR1 and overexpressed SIR1 [SIR1 (+++), Silent; see also Fig. Fig.2A2A].

FIG. 3.
SIR1/ORC-dependent silencing did not reduce origin firing or delay replication time of HMR-E(A-). (A) 2-D origin mapping of HMR-E(A-) from yeast cells that contain either endogenous levels of SIR1 (TRP1::pRS304, SIR1(+) Not silent, CFY1531) or ...

SIR1-dependent silencing of HMR-E(A-) did not reduce the efficiency of origin firing at HMRa; HMR-E(A-) produced an equally robust origin bubble signal regardless of whether HMRa was silenced (Fig. (Fig.3A).3A). In addition and as expected on the basis of the level of 2-D origin bubbles observed in Fig. Fig.3A,3A, SIR1-dependent silencing of HMR-E(A-) [SIR1 (+++)] did not delay timing of HMR-E(A-) origin firing within HMRa relative to nonsilent [SIR1 (+)] HMR-E(A-) cells (Fig. (Fig.3B).3B). Furthermore, new replication intermediates were detected in the following cell cycle during the course of these experiments for both the silent [SIR1 (+++)] and nonsilent [SIR1 (+)] HMR-E(A-) cells (Fig. (Fig.3B,3B, 110 min).

In an independent set of cell cycle arrest and release experiments that were quantified, the silent HMR-E(A-) mutant cells expressing high levels of SIR1 [SIR1 (+++]) produced a peak of replication for HMRa at 50 min that coincided with the replication peak of R11 (Fig. (Fig.3C).3C). This pattern was more similar to that observed for the nonsilent version of HMR-E(A-) than for wild-type HMR-E (Fig. (Fig.1D).1D). Therefore, the enhanced origin firing and associated advanced replication time caused by the HMR-E(A-) mutation was not inherently incompatible with or substantially inhibited by efficient Sir1p-dependent silencing of HMRa.

Sir1p/ORC-dependent silencing tolerated enhanced origin firing from the silencer itself.

The experiments described above using HMR-E(A-) mutant cells indicated that Sir1p-dependent silencing could tolerate enhanced origin firing, or something about ORC that promoted this origin firing, at HMRa. However, since multiple regions within HMRa can bind ORC in vitro (M. A. Palacios DeBeer, unpublished) and function as origins in vivo (18, 26), these experiments could not address whether the same ORC molecule that bound Sir1p could also function in enhanced origin firing. To better address this issue, we used an engineered version of the HMR-E silencer named the synthetic silencer (HMR-SS) that contains a single ORC binding site required for both origin firing and silencing at HMRa (23, 32) (Fig. (Fig.4A).4A). Although HMR-SS can silence HMRa, it does so inefficiently, allowing the expression of some a1 mRNA (9, 27) (Fig. (Fig.4B,4B, lane 1). In addition, this silencer binds Sir1p inefficiently in vivo (27). Finally and importantly, these defects can be attributed to the engineered ORC binding site at HMR-SS that contains only an A-element but lacks a B1-element; substitution of this ORC binding site with the high-affinity ORC binding site (B1/A element) from native HMR-E (E/SS) fully rescues Sir1p binding and silencing (Fig. (Fig.4B,4B, compare lanes 1 and 2), while at the same time suppressing origin firing to the barely detectable level observed for native HMR-E (27) (Fig. (Fig.5B,5B, panels 2 and 3). These observations were consistent with the notion that the extremely inefficient origin activity intrinsic to the HMR-E silencer was linked inextricably to Sir1p-dependent silencing. Therefore, we tested whether an enhanced Sir1p-ORC interaction could reduce or abolish origin firing by HMR-SS by introducing TRP1::ADH1pr→SIR1 into the appropriate yeast cells and measuring silencer and origin activity at HMRa (Fig. 4B and C). Data from these experiments provided strong evidence that overexpression of SIR1 (TRP1::ADH1pr→SIR1) could restore silencing by HMR-SS (Fig. (Fig.4B,4B, lane 3) without suppressing origin firing by this silencer (Fig. (Fig.4C4C).

FIG. 5.
ARS1/SS and ARS307/SS silencer/origin fusions functioned more efficiently as both silencers and origins than HMR-SS did. (A) RNA blot hybridization of a1 mRNA expressed from HMRa in cells containing HMR-SS (lane 1, CFY35), ARS1/SS (lane 2, CFY1724), E/SS ...

Although HMR-SS fires more efficiently than either the native HMR-E silencer or the HMR-E/SS fusion silencer (27) (Fig. (Fig.5B,5B, compare panels 2 and 3), HMR-SS is still an inefficient origin, firing in less than half the cells in a population (9). Furthermore, since the level of SIR1 had to be increased to obtain full silencing by HMR-SS, these data were still consistent with the idea that something about ORC function at this silencer origin was responsible for a reduced ability to recruit Sir1p and silence in vivo. Therefore, the question remained as to whether normal Sir1p-dependent silencing could tolerate replication initiation from a robust replication origin that had evolved to fire extremely efficiently. To test this idea, we engineered fusions between ARS1 or ARS307, both well-defined, efficient replication origins (22, 40), and HMR-SS, such that a single ORC binding site (B1/A-element) was forced to function simultaneously in both a defined, robust replication origin and a silencer at HMRa (Fig. (Fig.4A4A).

In terms of silencing, the origin/silencer fusions silenced much more efficiently than HMR-SS, substantially reducing a1 mRNA expression from HMRa (Fig. (Fig.5A).5A). If this increase in silencing were dependent upon a reduction in origin firing, then replication bubble intermediates from these origin/silencer fusions should be reduced compared to those from HMR-SS. However, both the ARS1/SS and ARS307/SS fusion silencers fired more robustly than either the efficient E/SS silencer or the weaker HMR-SS silencer (Fig. (Fig.5B,5B, compare panels 1 to 3 for ARS1/SS, and Fig. Fig.5C,5C, compare panels 1 and 2 for ARS307/SS). Therefore, silencing controlled by native levels of Sir1p could tolerate enhanced origin firing from the silencer itself.

The enhanced origin firing of ARS1/SS meant that some cells must replicate HMRa from the internal ARS1/SS fusion origin before replication forks emanating from distant origins arrived. To determine to what extent the ARS1/SS fusion advanced the replication time of HMRa, we measured the replication timing pattern of HMRa by performing 2-D origin mapping during cell cycle arrest and release experiments (Fig. (Fig.5E).5E). These experiments revealed that a substantial fraction of cells containing ARS1/SS at HMRa replicated this locus relatively early in S phase, coincident with the replication of ARS1 at its endogenous location (Fig. 5E and F). However, although the peak replication times overlapped, a substantial number of ARS1 replication intermediates were detected at 30 min, whereas no HMRa intermediates were detected at this time point (Fig. (Fig.5E,5E, 30 min), indicating that ARS1 in its native location fired earlier than the ARS1/SS fusion at HMRa.

Quantification of the cell cycle arrest and release 2-D origin mapping experiment also indicated that the ARS1/SS silencer fusion advanced the replication time of HMRa in a fraction of cells examined (Fig. (Fig.5F).5F). In particular, many ARS1/SS cells replicated HMRa well before replication of the R11 late control, coincident with the peak replication times of the early firing origin controls, ARS306 and ARS1 (Fig. (Fig.5F).5F). We note that ARS306 fires prior to ARS1 as measured by higher resolution density-labeling experiments (30) but that in the lower-resolution experiments used here the two origins generated coincident replication timing profiles. Regardless, both ARS306 and ARS1 replicated prior to the late replication control R11, indicating that earlier and late replicated regions were separated in these experiments. Thus, the ARS1/SS silencer fusion advanced the replication time of HMRa to an even greater degree than the HMR-E(A-) mutation did (Fig. (Fig.1B)1B) yet silenced HMRa quite effectively even with endogenous levels of SIR1 (Fig. (Fig.5A5A).

Although the ARS1/SS fusion fired more efficiently than HMR-SS, it failed to fire as efficiently as ARS1 in its native location (Fig. (Fig.5D,5D, panels 1 and 2, compare the ratio of bubbles to small forks). Furthermore, although some cells containing ARS1/SS replicated HMRa coincident with the replication peak of endogenous ARS1, it was clear that the replication peak for ARS1/SS was broader than that of endogenous ARS1 (Fig. 5E and F). These data provided evidence that some mechanism was suppressing origin firing by ARS1/SS effectively in at least some cells. As discussed earlier, Sir-dependent chromatin can suppress and/or delay origin firing (reviewed in reference 42). However, it is also clear that both HMRa and HMLα possess inefficient origins regardless of Sir genotype, indicating that components inherent to the DNA within these loci, and/or other non-Sir factors contribute to the inefficiency of their associated origins under normal conditions. Therefore, we tested whether it was even possible for the ARS1/SS fusion to fire as efficiently as native ARS1 in a normal cell cycle by introducing a sir2Δ mutation into a strain harboring ARS1/SS. Significantly, in the absence of SIR2 (sir2Δ) the ARS1/SS fusion fired as efficiently as ARS1 at its native location (Fig. (Fig.5D,5D, compare panels 1 and 3), indicating clearly that origin firing by ARS1/SS was suppressed to some degree by the assembly of silent chromatin. Therefore, in the context examined here, which differs from the native HMRa context as described above but which nevertheless allows for virtually full silencing, there are no Sir-independent factors or DNA elements at HMRa that suppress the firing of an otherwise efficient origin.

The origin/silencer fusions were less effective than the HMR-E silencer's native ORC binding site at silencing HMRa.

The data discussed above indicated clearly that Sir1p-dependent silencing must be capable of tolerating a significant level of origin firing within HMRa, since the ARS/SS fusions substantially improved silencing over HMR-SS (Fig. (Fig.5A)5A) yet fired more robustly (Fig. 5B and C). However, we noted that ARS1/SS and ARS307/SS allowed small but detectable levels of a1 mRNA transcription from HMRa, whereas the E/SS silencer, which contains the HMR-E silencer's ORC binding site did not (Fig. (Fig.5A).5A). To better quantify the differences in silencing efficiencies among these silencers, we performed quantitative RT-PCR with mRNA isolated from cells that differed only in terms of their HMRa silencer, as indicated (Fig. (Fig.66 and see Fig. Fig.4A4A for silencer descriptions). These experiments revealed that both the ARS1/SS and ARS307/SS fusions allowed for measurable levels of a1 transcription from HMRa, at least 10-fold and 20-fold greater, respectively, than the level of a1 transcription allowed from the efficient E/SS silencer (Fig. 6A and B). Thus, although both origin/silencer fusions silenced efficiently, each was slightly less effective at silencing HMRa than the E/SS silencer that contains the native HMR-E silencer's high-affinity ORC binding site (Fig. (Fig.4A4A).

FIG. 6.
The origin/silencer fusions were less effective than the HMR-E silencer's native ORC binding site at silencing HMRa. (A) Raw data from RT-PCR analysis of total RNA isolated from a series of isogenic strains that differed only in terms of the silencer ...


The silent loci must assemble into domains of SIR-dependent chromatin, and to achieve this goal, they depend on silencers, small DNA elements that nucleate the assembly of SIR-chromatin through specific protein-protein interactions (10). Silencers and their associated replication origins both rely on ORC for their functions. The silencer-associated origins are extremely inefficient and have late firing origins regardless of SIR genotype. In this study, we focused on HMRa to determine whether more robust origin firing within this locus caused by certain silencer mutations could coexist with efficient silencing. We also asked whether the inherent inefficiency of the HMR-E silencer-associated origin was important for Sir1p-dependent silencing.

Sir1p-dependent silencing tolerated enhanced origin firing within HMRa.

Although the simplest hypothesis to explain the silencing defect of HMR-E(A-) is a defect in Sir1p recruitment, our analysis of HMR-E(A-) indicated that other possibilities must be considered. First, the 2-D origin mapping data reveal that ORC(s) must still bind to some extent in the near vicinity of the HMR-E(A-) silencer in vivo, indicating that ORC(s) remains very near the silencer (26). In theory, these ORC(s) could bind Sir1p. Second, in addition to reducing HMRa silencing, the HMR-E(A-) mutant produced two additional phenotypes: enhanced origin firing and a slight advance in HMRa's replication time. Furthermore, silencing by HMR-E(A-) is partially restored by additional mutations that reduce or abolish the origin activity associated with this mutant, including the orc2-1 mutation (26). Thus, it was possible that the HMR-E(A-) mutation, by some unknown mechanism, converted ORC from a silencing role to a replication role that was incompatible with efficient Sir1p-dependent silencing. The data presented in this report provide evidence against this possibility. Specifically, wild-type levels of silencing could be restored to HMR-E(A-) without restoring the replication pattern of wild-type HMR-E. In addition, this restoration required the same Sir1p-ORC interaction used in wild-type silencing, suggesting that this interaction itself was not incompatible with the enhanced origin firing of HMR-E(A).

Although the origin activity of HMR-E(A-), as measured here, was unaffected by restoration of Sir1p-dependent silencing, full silencing by this mutant required an increased level of Sir1p. Thus, it is possible that enhanced origin firing and/or advanced HMRa replication time in this mutant contributed to a silencing defect by reducing the affinity of the silencer for Sir1p and/or other Sir proteins. Another issue is that there are at least three regions within HMRa (excluding HMR-I) that can bind ORC in vitro and provide some origin activity to HMRa (26) (Palacios DeBeer, unpublished). The activity of one or more of these origins may be reduced by Sir1p-dependent silencing, but since these origins are so close together, 2-D origin mapping cannot reveal a reduction in one origin, especially if another's firing increases as a result. Replication initiation point mapping might test this idea (14). Regardless of the limitations conferred by the complexity of this locus and the resolution of the 2-D origin mapping data, these data revealed that the level and timing of initiation in the HMR-E(A-) mutant were remarkably unaffected by silencing.

HMRa silencing coexisted with an enhanced level of origin firing from the silencer itself.

The complexity of origin organization in HMR-E(A-) prevented us from tackling a more definitive question about the silencer origin that was also relevant. Specifically, since the HMR-E silencer origin is so inefficient and because this inefficiency is attributable to the ORC binding site itself (27), could the silencer be “forced” to fire more robustly without affecting silencing? Such an experiment is not possible at HMLα, since origins placed at this locus become inactive regardless of SIR genotype, indicating clearly that this locus contains additional intrinsic features that suppress origin firing (36). In fact, origin activation at HMLα requires either the deletion of distant origins or the introduction of mutations that allow for the firing of very late origins under conditions of an elongated S phase, both situations that perturb chromosomal replication and/or maintain a similar replication timing pattern for HMLα (35, 41). In contrast, at HMRa, the synthetic silencer, HMR-SS, has more robust origin activity than HMR-E regardless of whether the locus is silent. Moreover, HMR-SS contains a single ORC binding site required for both origin and silencer function (27, 32). Thus, we knew that it was possible to enhance origin firing from the silencer at HMRa, at least to some extent. However, HMR-SS is also a weakened silencer, in part because of its ORC binding site (27). Thus, it was possible that the enhanced origin firing of HMR-SS was a cause of its inefficient silencing, and the question of whether an ORC that functioned at an optimized origin could both fire and silence efficiently within HMRa remained.

To address this question as directly as possible, we created the most optimal condition we could for silencer/origin firing by fusing two different characterized and robust origins (ARS1 and ARS307) in their entirety to HMR-SS such that a single ORC binding site was forced to function simultaneously in both replication initiation and silencing. Both origin/silencer fusions fired more efficiently than HMR-SS itself and, critically, both silenced HMRa efficiently and much more robustly than HMR-SS. The unequivocal conclusion was that an ORC could function efficiently and simultaneously in both initiation and silencing.

These data indicate that the inability of HMR-SS to silence efficiently is not caused by its ability to function as an origin. Rather, we propose that HMR-SS is a weak silencer because it is not fully occupied by ORC in vivo; whenever it is occupied by ORC, it functions well as both a silencer and an origin. In this view, HMR-SS is actually an efficient origin because whenever it is occupied by ORC, it fires. In contrast, the ORC binding site of native HMR-E binds ORC with a high affinity in vitro that should allow full occupancy by ORC in vivo. In addition, this ORC binding site is also sufficient to convert HMR-SS to a robust silencer, as good as native HMR-E, while virtually inactivating origin firing at HMRa (27). The HMR-E ORC binding site must therefore present a form of ORC or ORC-DNA complex that is ideal for silencing but repressive to origin firing.

Excess contacts for maximum stability: a possible explanation for why the HMR-E silencer origin is inefficient yet is an origin nonetheless.

If origin firing per se is not incompatible with efficient silencing, why is origin inactivity a common feature of silencers and, in the case of HMR-E at least, an intrinsic property of its ORC binding site? One speculative explanation is that even a small reduction in silencing efficiency as revealed by the more sensitive analyses of the ARS/silencer fusions presented here would be selected against under environmental growth conditions in which the ability to mate, and thus, the ability to silence, was critical. Although the levels of a1 mRNA expressed from the ARS/silencer fusions were unimpressive by laboratory standards, under conditions in which silencing offered even a slight competitive growth advantage, a cell lineage that expressed even such small amounts of mRNA would be diluted from a population over time. We note here that the silencing defect did not require that an entire ARS be fused with HMR-SS; substitution with the ORC binding sites (A/B1-elements) from ARS1 and other ARS elements produced similar effects (K. H. McConnell, P. Müller, and C. A. Fox, unpublished). Thus, we propose that HMR-E evolved an ORC binding site, perhaps originally part of an active origin, that is, in fact, perfect for silencing. The origin activity that remains at silencers may be a relic of some primitive origin for which there has been no positive selective pressure.

Although many mechanisms could explain how a silencer-ORC is optimized for silencing, a simple model is that the ORC-HMR-E-silencer-DNA complex presents a surface with maximal contact points for Sir1p binding. Other ORC binding sites reduce the number of contact points but not to a level below a threshold required for silencing. Alternatively, the ORC-silencer-DNA complex may be optimized for stabilizing any number of Sir1p-independent factors, either directly or indirectly, required for silent chromatin formation. In either scenario, an ORC binding site optimized for origin firing may not provide the number of protein-DNA and/or protein-protein contacts that are optimal for a stable silencer-protein nucleation complex.


We thank M. A. Palacios DeBeer for productive discussions and M. K. Raghuraman and B. Brewer for primer sequences to ARS306.

This work was supported in part by a predoctoral training grant award to K.H.M. (T32 GM07215) and by a grant from the NIH (GM056890) to C.A.F.


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