A Role for the Replication Proteins PCNA, RF-C, Polymerase and Cdc45 in Transcriptional Silencing in Saccharomyces cerevisiae

Corresponding author: Jasper Rine, Department of Molecular and Cell Biology, 401 Barker Hall, University of California, Berkeley, CA 94720., jrine{at}uclink4.berkeley.edu (E-mail)

Communicating editor: F. WINSTON

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Transcriptional silencing in the budding yeast Saccharomyces cerevisiae may be linked to DNA replication and cell cycle progression. In this study, we have surveyed the effect of 41 mutations in genes with a role in replication, the cell cycle, and DNA repair on silencing at HMR. Mutations in PCNA (POL30), RF-C (CDC44), polymerase (POL2, DPB2, DPB11), and CDC45 were found to restore silencing at a mutant HMR silencer allele that was still a chromosomal origin of replication. Replication timing experiments indicated that the mutant HMR locus was replicated late in S-phase, at the same time as wild-type HMR. Restoration of silencing by PCNA and CDC45 mutations required the origin recognition complex binding site of the HMR-E silencer. Several models for the precise role of these replication proteins in silencing are discussed.

THE mating phenotypes of haploid cell types in the yeast Saccharomyces cerevisiae require transcriptional repression of mating-type genes at HML and HMR (LOO and RINE 1995 ). These loci contain mating-type genes identical to those at the expressed MAT locus, but are silenced in a mechanism comparable to position effect variegation in larger eukaryotes (WEILER and WAKIMOTO 1995 ). The silenced loci are flanked by sequence elements termed silencers that contain binding sites for the proteins Rap1 and Abf1 as well as for the origin recognition complex (ORC). The silencer binding proteins likely serve to attract other silencing factors, the silent information regulator (Sir) proteins, to form a specialized chromatin structure in the repressed regions that renders them inaccessible to transcription, thus achieving silencing (GARDNER et al. 1999 ).

Silenced genomic regions remain silenced throughout the cell cycle, including during DNA replication (FOX and RINE 1996 ). For replication, the replication factor C (RF-C) complex loads the sliding clamp PCNA onto the DNA double helix, which then facilitates the loading of the DNA polymerases and onto the template. Replication complexes move along the DNA and partially disrupt chromatin structures, raising the question of how silenced chromatin is reformed after replication.

Several observations indicate a link between DNA replication and silencing in yeast. The establishment of silencing requires passage through the S-phase of the cell cycle, suggesting a role for DNA replication independent of replication initiation at the silencer (MILLER and NASMYTH 1984 ; FOX et al. 1997 ). Also, mutations in the gene encoding Cdc7, a cyclin-dependent kinase acting at the transition from G1 to S-phase, restore repression to a HMR locus derepressed by point mutations in the silencer (AXELROD and RINE 1991 ). Similarly, LAMAN et al. 1995 identified mutations in several cell cycle regulators as suppressors of a silencing defect at HMR achieved by the deletion of the ORC binding site and a mutation in RAP1. Mutations in SWI6, a transcriptional regulator of cyclins, as well as mutations in the cyclins CLN3, CLB2, and CLB5 were capable of suppressing the silencing defect. Since these mutations lengthen specific phases of the cell cycle, this led to the hypothesis that perturbing the cell cycle was sufficient to reestablish silencing, perhaps by increasing the probability for silenced chromatin to be established.

In this study, we have further explored the relationship between replication and transcriptional silencing in yeast and have identified mutations in several replication proteins as novel suppressors of the silencing defects. The range of mutations affecting silencing, and those that do not, presents serious challenges to the cell cycle slowing model for suppression of mutant silencer function.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The genotypes of the yeast strains used in this study are presented in Table 1. Standard yeast media and genetic methods were used as described (SHERMAN 1991 ). Yeast transformations were performed by standard lithium acetate procedures. Quantitative mating assays were performed as described (EHRENHOFER-MURRAY et al. 1997 ).

Screening mutations for suppression of HMRa-e**:
Strains containing the mutation of interest were mated to a HMRa-e** carrying strain of the opposite mating type (JRY2069, JRY2611, JRY5273, JRY5471, JRY5472, and JRY5473, depending on which markers were most convenient for diploid selection); diploids were selected and subjected to sporulation and tetrad dissection. Even though MAT HMRa-e** strains are poor maters, the selection for prototrophic diploids was strong enough to produce diploid colonies in each mating. The mating phenotype of MAT segregants from at least 20 tetrads from each cross was determined. For mutations without a conditional phenotype, the mating tests were performed at 30°. Strains with conditional phenotypes were assayed at the permissive temperature for the mutation. Since both the HMRa and HMRa-e** alleles were in these crosses, and assuming linkage of 60% between MAT and HMR, 60% or 40% of MAT segregants (depending on whether the HMRa-e** allele was coupled to MAT or MATa in the diploid) are expected to carry HMRa and hence will be able to mate efficiently as cells. If a particular mutation were able to restore silencing at HMRa-e**, about half of the MAT segregants with HMRa-e** would be expected to mate efficiently due to the segregation of the suppressor in the cross. Moreover, if none of the poorly mating segregants from the cross (presumably MAT HMRa-e**) contained the suppressor mutation, as judged by their conditional phenotype or, for marked deletions, by marker segregation, then we concluded that the mutation suppressed the mating defect of MAT HMRa-e** strains.

As an independent evaluation of whether a mutation restored the mating ability of MAT HMRa-e**, MAT HMRa-e** strains containing the putative suppressor were backcrossed to a MATa HMRa-e** parent (JRY2611, JRY5471, or JRY5473), and the mating phenotype of the MAT segregants from each cross was determined at the permissive temperature of the mutations. Since all segregants contained HMRa-e**, the mating phenotype could be directly correlated with the segregation of the mutation. If a mutation suppressed HMRa-e**, all segregants able to mate efficiently as cells were expected to carry the mutation, and all weakly mating segregants were expected to be wild type. This second test confirmed that pol30-52, cdc44-5, cdc45-1, pol2-12, dpb2-1, and dpb11-1 were capable of suppressing the mating defect of MAT HMRa-e** strains.

Strain constructions:
Strains carrying suppressor mutations combined with hmrA::TRP1 rap1-12 were obtained by crossing the MAT HMRa-e** strains containing the suppressor to a MATa hmrA::TRP1 rap1-12 strain (JRY3372) and by identifying the desired segregants by their auxotrophies and conditional phenotypes. MAT HMRa-e** sir4::LEU2 strains carrying the suppressors were constructed by crossing the MAT HMRa-e** strains containing the suppressor mutation to a MATa sir4::LEU2 strain (JRY4581) carrying the SIR4-containing plasmid pJR368. The plasmid pJR368, which was present to allow mating, was lost by counterselection prior to sporulation and tetrad dissection. MAT sir4::LEU2 segregants were selected from tetrads in which the two loci cosegregated in two segregants. Of these, the segregants carrying the suppressor mutation of interest were subjected to DNA blot-hybridization to determine their HMR allele, and the mating phenotype of strains containing HMRa-e** was subsequently determined by patch mating assays.

Construction of a W303-isogenic cdc45-1 strain:
In a first step, the cdc45-1 allele was cloned onto a plasmid by gap repair. A CDC45 containing plasmid (ZHOU et al. 1989 ; pEM45-3, a pRS416 derivative, gift from Dr. B. Stillman) was digested with AflII and BglII and treated with Klenow polymerase and religated, thus recreating a BglII restriction site. This plasmid (pJR1741) contained a gap in the 1953-bp CDC45 open reading frame of 1860 bp. BglII-linearized pJR1741 was used to transform the cdc45-1 strain JRY4349 to uracil prototrophy. Ura⁺ transformants could arise only if the plasmid circularized, either by a nonhomologous joining at the plasmid ends or by repair of the plasmid by gap repair using the homologous sequence from the chromosome. To distinguish between these possibilities, plasmids rescued from Ura⁺ transformants were tested by restriction digest to determine whether they contained an insert the same size as the parent plasmid, pEM45-3. One such plasmid (pJR1747) was used for further experiments. pJR1747 and pJR1741 were unable to complement the cold sensitivity of the cdc45-1 strain (JRY4349), whereas pEM45-3 could, suggesting that pJR1747 carried the cdc45-1 allele. Subsequently, the 3.7-kb SalI fragment of pJR1747 was cloned into the SalI site of the integrating, URA3-marked plasmid pRS406 to generate pJR1755. The wild-type strain JRY3009 was transformed to uracil prototrophy with AflII-linearized pJR1755. The resulting strain was streaked onto 5-fluoro-orotic acid-containing medium to select for Ura^- recombinants, and candidates were tested for their cold sensitivity to assess whether they retained the cdc45-1 allele. Candidates were subjected to DNA blot-hybridization to determine whether they had regained an intact, though mutant, CDC45 locus.

Replication timing:
Strains expressing Escherichia coli dam methylase were generated by transforming a MATa strain (JRY2334) and a MATa HMRa-e** strain (JRY5471) to leucine prototrophy with AflII-linearized pRS305-dam (a gift of Dr. B. Brewer and Dr. W. Fangman). To identify transformants expressing high levels of methylase, DNA of the transformants was cleaved with EcoRI and DpnI, analyzed by DNA blot-hybridization with an ARS1 adjacent probe, and transformants with strong DpnI digestion were chosen for further experiments. For the timing experiments, cell cultures were prearrested in nocodazole before synchronization in -factor-containing medium, which resulted in a more reproducible G1 arrest. For the release into S-phase, cells were harvested, washed with YPD, and suspended in YPD with 20 µg/ml pronase (Calbiochem, San Diego) to degrade residual -factor. Samples of 10⁸ cells were collected at 5-min intervals. DNA was isolated, digested with EcoRI, PstI, and DpnI for 2–3 hr at 37°, electrophoretically separated on 0.6% agarose gels, and transferred to Zetaprobe GT (Bio-Rad, Richmond, CA). Three probes were used sequentially: a 1.4-kb BglII fragment to the right of HMR-I; a 3.7-kb EcoRI fragment (fragment R11) from the right end of chromosome V; and a 2.85-kb EcoRI fragment immediately adjacent to ARS1 on chromosome IV. Band intensities were quantitated using a PhosphorImager (Molecular Dynamics). The lane containing the highest fraction of full-length fragment was assigned a value of 1, and the fragments of the other lanes were normalized to it.

Two-dimensional origin analysis:
The isolation and analysis of replication intermediates at HMR were performed as described (FOX et al. 1995 ), except that the DNA was digested with NheI and BglII.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

A silencing-deficient HMR-E silencer was a chromosomal origin of replication:
Repression at the silent mating-type loci in yeast is mediated by flanking sequence elements called silencers. Silencers have a modular arrangement of subdomains. The HMR-E silencer which is essential for silencing at HMR, contains three domains: (i) an ORC binding site; (ii) a Rap1 binding site; and (iii) an Abf1 binding site. In addition to being a silencer, HMR-E is also a chromosomal origin of replication. In a previous study, we constructed an allele of HMR-E (HMRa-e**) with mutations in two silencer domains, the Rap1 and Abf1 binding sites, thus leaving the ORC binding site as the only known functional silencer element (KIMMERLY et al. 1988 ). The HMR locus containing this double-site mutant silencer is derepressed. To characterize further the HMRa-e** silencer, the origin activity of this mutant silencer was tested. Replication initiation at the HMRa-e** silencer was compared to that at the wild-type HMR-E using two-dimensional origin-mapping gels (BREWER and FANGMAN 1987 ). The signal from bubble-shaped replication intermediates from the two silencers was comparable (Figure 1). Thus, the mutant HMR-E silencer was as strong a chromosomal origin of replication as the wild-type silencer. Therefore, the silencing defect of HMRa-e** was not due to a defect in replication initiation at the mutant silencer.

View larger version (110K): In this window In a new window Download PPT slide   Figure 1. The mutant HMR-E silencer of HMRa-e** was a chromosomal origin of replication. Replication initiation at HMR-E was monitored in a wild-type strain (JRY3009) and a HMRa-e** carrying strain (JRY5273) by two-dimensional origin-mapping gels. Arrows indicate bubble-shaped replication intermediates that result from replication initiation at HMR-E. The DNA was digested with NheI and BglII, and HMR was detected with two HMR-specific probes.

Identification of novel suppressors of silencing defects:
In previous studies, we characterized two very different suppressors of the silencing defect of HMRa-e**:(1) mutations in SAS2, which encodes a homolog of histone acetyltransferases, restore silencing at HMRa-e**, suggesting a role for acetylation in silencing (EHRENHOFER-MURRAY et al. 1997 ), and (2) mutations in CDC7, which encodes an S-phase-promoting protein kinase and restores silencing at HMRa-e**, establishing a link between silencing and cell cycle progression (AXELROD and RINE 1991 ). An earlier study found that null alleles of CLN3, CIN8, CLB5, and a mutation in SWI6, genes encoding cyclins and other cell cycle regulators, could restore silencing to HMR with a mutant silencer (LAMAN et al. 1995 ). These results lead to the hypothesis that slowing the cell cycle in any of a variety of ways restored silencing at mutant silencers and suggested that the principle defect in silencing by these mutant silencers was kinetic. According to this view, given enough time, the mutant silencers could still establish the silenced state. However, the results described below revealed an unanticipated specificity as to which cell cycle progression mutations restored silencing and which did not.

The involvement of CDC7 in silencing prompted a survey of the effect of mutations in 41 other genes that have a role in the cell division cycle, early S-phase, or DNA replication for whether they suppressed the mating defect caused by HMRa-e** in a MAT strain (expression of a information in a MAT strain causes a nonmating phenotype; see Table 2 and Table 3). Of the 41 genes, mutations in 4 different genes suppressed the mating defect (Figure 2, Table 2 and Table 4): pol30-52, a mutation in the proliferating cell nuclear antigen (PCNA; AYYAGARI et al. 1995 ); cdc44-5, a mutation in the large subunit of RF-C (MCALEAR et al. 1996 ); cdc45-1, which is genetically related to MCM genes (HENNESSY et al. 1991 ); and pol2-12, a mutation in the catalytic subunit of polymerase (Pol; BUDD and CAMPBELL 1993 ).

View larger version (42K): In this window In a new window Download PPT slide   Figure 2. Mutations in genes encoding replication proteins suppressed the silencing defect of HMRa-e**. The strains were patched on complete medium, pregrown for 12 hr at 30° (left) or 23° (right) and replica plated onto minimal medium spread with a lawn of MATa mating tester. The plates were incubated for 2 days at 30° and 23°, respectively. The strains used were JRY3009, JRY5273, JRY5474, JRY5475, JRY5476, and JRY5477.

It has been suggested that cell cycle slowing enhances the establishment of transcriptional silencing (LAMAN et al. 1995 ). However, many cdc and orc mutations that cause cell cycle defects at their restrictive temperatures were unable to suppress the silencing defect of HMRa-e** at the permissive temperature (Table 3). A subset of mutations was also tested for their ability to suppress at semipermissive temperatures, where cell cycle defects are exacerbated. However, even under more stringent conditions, they were unable to suppress (Table 3, footnote a). Thus, slowing cell cycle progression alone was not sufficient to reestablish silencing.

Surprisingly, a mutation in DBF4, which encodes the regulatory subunit of the Cdc7 protein kinase (JACKSON et al. 1993 ), was also unable to suppress the silencing defect, whereas mutations in CDC7 do (AXELROD and RINE 1991 ). This result indicated that Cdc7 might, at least for its silencing function, associate with an as-yet-unidentified cyclin. It was also noteworthy that cdc45-1 could suppress HMRa-e**, but that mutations in the MCM genes MCM2, CDC46/MCM5, CDC47/MCM7, and CDC54/MCM4, which are genetically associated with CDC45, could not. Cdc45 interacts physically with Cdc46/Mcm5 (HOPWOOD and DALTON 1996 ) and has a role in initiation of DNA replication (ZOU et al. 1997 ). However, mutations in genes encoding initiator proteins (orc2-1, orc5-1) and other prereplication complex components (cdc6-1) were unable to suppress.

There were striking differences between mutations in the various DNA polymerases for their ability to suppress HMRa-e**. Mutations in the catalytic subunits of polymerase (pol1-17) and (cdc2-1, cdc2-7) did not suppress the silencer mutation, whereas a mutation in polymerase (pol2-12) did. Interestingly, as described above, mutations in subunits of the accessory proteins PCNA and RF-C shared the suppression phenotype with the Pol mutation.

In summary, our data supported the notion that mutations in some, but not other replication proteins, suppressed HMRa-e**, although we cannot exclude the formal possibility that special alleles of some nonsuppressors may exist that are capable of suppression.

We confirmed that null alleles of CLN3, CIN8, and CLB5 also restored silencing at HMRa-e** (Figure 3, Table 4). Similarly, a deletion of CLB6, which was unable to suppress in the earlier study (LAMAN et al. 1995 ), also did not restore repression in our assay. Hence, the different assays for suppression shared at least some suppressors.

View larger version (54K): In this window In a new window Download PPT slide   Figure 3. Mutations in genes altering cell cycle progression restored silencing at HMRa-e**. The experiment was performed as in Figure 2. The strains used were JRY3009, JRY5273, JRY5360, JRY5358, JRY5361, JRY5359, and JRY5357.

Mutations in other components of polymerase suppressed the silencing defect of HMRa-e**:
In the experiments described so far, mutations affecting Pol, PCNA, RF-C, and Cdc45 had a role in silencing HMR. Pol is a protein complex required for chromosomal replication and DNA repair, and PCNA and RF-C are accessory proteins to Pol and other polymerases. The catalytic subunit of Pol is encoded by POL2, and other subunits are encoded by DPB2 (ARAKI et al. 1991B ), DPB3 (ARAKI et al. 1991A ), and DPB11 (ARAKI et al. 1995 ). To test whether other components of this multiprotein complex contributed to silencing like POL2, we tested whether mutations in DPB2, DPB3, and DPB11 could restore silencing to HMRa-e** (as described above). Both dpb2-1 and dpb11-1 suppressed the mating defect of MAT HMRa-e** strains (Table 4), suggesting that they restored silencing at HMR. In contrast, the deletion of DPB3 (dpb3) did not suppress the mating defect of MAT HMRa-e** strains (data not shown). In contrast to Dpb2 and Dpb11, Dpb3 is a nonessential subunit of Pol, and the only known phenotype of dpb3 is a modest effect on the spontaneous reversion frequency of a missense mutation (ARAKI et al. 1991B ). Hence, dpb3 may not have suppressed HMRa-e** because it does not cause the same defect in Pol as dpb2-1 and dpb11-1. Nonetheless, these results suggested that suppression of silencing defects was a general characteristic of multiple, but not all, mutations in Pol subunits, including noncatalytic subunits.

pol30-52, cdc44-5, cdc45-1, and pol2-12 suppressed silencing defects at HMR in a gene-independent, SIR-dependent manner:
In principle, the newly identified suppressors might restore the mating ability of a MAT HMRa-e** strain by means other than silencing HMR. To test whether the increased mating ability was the consequence of restored silencing at HMR or of a different mechanism, such as interfering with a1/2-mediated repression, we tested whether the presumptive silencing restored by the suppressor mutations displayed the expected characteristics of silencing. Silencing represses transcription of a variety of genes when placed near a silencer and depends upon the function of the four SIR genes. We used these two criteria to determine whether the novel suppressors restored silencing at HMR. All four mutations caused ~1000-fold repression of TRP1 inserted at HMR (hmrA::TRP1) in a strain carrying a mutation in the silencer binding protein Rap1 (rap1-12; SUSSEL et al. 1993 ; Figure 4), whereas they were unable to suppress TRP1 in its chromosomal location (data not shown). Thus, the suppressors of the HMRa-e** silencing defect could repress a gene unrelated to mating type when that gene was inserted at HMR. Furthermore, MAT HMRa-e** strains that carried one of the suppressors pol30-52, cdc44-5, cdc45-1, or pol2-12 and a null allele of SIR4 were constructed. All four strains were nonmaters (data not shown), indicating that the suppressor mutations were unable to silence HMR in the absence of SIR4. Taken together, these results suggested that the suppressor mutations established bona fide silencing at mutant HMR alleles.

View larger version (94K): In this window In a new window Download PPT slide   Figure 4. Mutations in genes encoding replication proteins restored silencing in a rap1-12 hmrA::TRP1 background. Expression of the hmrA::TRP1 reporter was assayed by growing the strains in rich liquid medium for 12 hr at 30° (pol30-52, cdc45-1, and cdc44-5 strains) or 23° (pol2-12), diluting the cultures to an optical density at 600 nm (OD600) of 0.3, and placing 10-fold serial dilutions thereof on minimal media that was either fully supplemented (+trp) or that lacked tryptophan (-trp). The plates were incubated at the appropriate temperature for 2 days. The strains used were JRY3372 (wt), JRY5369 and JRY5370 (pol30-52), JRY5373 and JRY5374 (cdc45-1), JRY5367 and JRY5368 (cdc44-5), and JRY5371 and JRY5372 (pol2-12).

pol30-52 caused a silencing defect at HML:
Other studies have identified mutations in SAS2 and SAS3 as suppressors of the silencing defect of HMRa-e**. Interestingly, mutations in SAS2 also decrease silencing at HML when combined with a deletion of SIR1 (REIFSNYDER et al. 1996 ; EHRENHOFER-MURRAY et al. 1997 ). To test whether the new suppressors of HMRa-e** had a SAS2-like effect at HML, strains containing pol30-52, cdc44-5, cdc45-1, or pol2-12 were crossed to sir1 strains, and the mating phenotype of the MATa progeny of the crosses was analyzed. Of the four mutations tested, only pol30-52 caused derepression at HML in a sir1 strain (Figure 5). Therefore, POL30 showed a second role in silencing other than suppression of HMRa-e**, namely that pol30-52 caused complete derepression of an HML locus that was already compromised for silencing by sir1. Interestingly, like sas2, pol30-52 did not enhance derepression by sir1 at HMR (data not shown). This showed that pol30-52 and sas2 have at least some similarities with respect to their effect on silencing. However, POL30 and SAS2 were not functionally redundant in HML silencing, since the combination of the two mutations in the absence of sir1 did not cause derepression at HML (data not shown).

View larger version (56K): In this window In a new window Download PPT slide   Figure 5. HML was derepressed in sir1 pol30-52 strains. The a mating ability of strains JRY2334, JRY5499, JRY5500, and two isolates of JRY5501 was assayed as in Figure 2 except for using a MAT tester strain.

cdc45-1 and pol30-52 required the ORC binding site of HMR-E to restore repression:
In theory, the HMRa-e** suppressors could increase repression at HMR by bypassing the need for the HMR-E silencer, perhaps by strengthening the HMR-I silencer. Alternatively, the suppressors might require particular silencer elements to restore repression, perhaps by enhancing interactions between some silencer binding proteins and other silencing factors. To distinguish between these possibilities, we tested whether two of the suppressor mutations, pol30-52 and cdc45-1, required particular silencer elements to restore repression. For this purpose, we used mutant versions of the synthetic HMR-E silencer (HMR-SS I; MCNALLY and RINE 1991 ). This silencer lacks much of the functional redundancy of the natural silencer and, in the absence of HMR-I, requires all three silencer elements for complete silencing. Strains were constructed that were MAT, either pol30-52 or cdc45-1, and contained alleles of the synthetic HMR-E silencer in which the ORC binding site (ars^-), the Rap1 binding site (rap1^-) or the Abf1 binding site (abf^-) was mutated (Figure 6). Both pol30-52 and cdc45-1 suppressed the loss of silencing in the absence of the Rap1 or the Abf1 binding site. In contrast, both mutations caused only a slight increase of silencing when the ORC binding site was absent. Because this suppression was less pronounced than that of the Rap1 and Abf1 binding sites, this indicated that both pol30-52 and cdc45-1 required the ORC binding site for repression, suggesting a direct link between PCNA, Cdc45, and ORC.

View larger version (66K): In this window In a new window Download PPT slide   Figure 6. cdc45-1 and pol30-52 required the ORC binding site of the HMR-E silencer to restore repression. The mating ability of MAT strains carrying versions of the synthetic HMR-E silencer with a mutation in the ORC binding site (ars-), the Rap1 binding site (rap-), or the Abf1 binding site (abf1-) was assayed as described in Figure 3. The strains used were JRY3009 (wt) and (top to bottom, left to right) JRY4531, JRY5278, JRY4889, JRY5478, JRY5479, JRY5480, JRY5363, JRY5364, and JRY5362.

The observation that both mutations required the ORC binding site was consistent with the fact that the silencer in HMRa-e** has an intact ORC binding site. However, it was surprising in light of the result that the mutations also suppressed hmrA::TRP1 rap1-12, in which the ORC binding site of HMR-E is removed (A). One possibility is that the multiple near matches to the ARS consensus sequence in this region have ORC bound.

HMRa-e** was replicated late in S-phase:
Several models have been put forward to explain how mutations enhance repression at the silent mating-type loci. One model invokes a critical role for replication timing in silencing. Inactive genes are typically replicated late in S-phase. In this model, replication late in S-phase may be required for repression, perhaps because of differences in the availability of chromatin components in early and late S-phase. Accordingly, HMRa-e** might be early replicating and hence silencing deficient, and the suppressor mutations might restore repression by restoring late replication to HMRa-e**.

To test this hypothesis, we measured the replication timing of HMR and HMRa-e** and compared it to the replication timing of two previously characterized genomic fragments: ARS1, which is replicated early in S-phase, and R11, that is replicated late. For this purpose, we used an assay that measures hemimethylation as an indicator of replication timing (FRIEDMAN et al. 1995 ). In this assay, yeast DNA is methylated in vivo by expressing the dam methylase of E. coli. When the DNA is replicated by a passing replication fork, it becomes temporarily hemimethylated. The hemimethylation is detected by the sensitivity of the DNA to the restriction enzyme DpnI, which can cleave only those restriction sites methylated on both strands. The in vivo DNA methylation did not interfere with silencing at HMR or HMRa-e**, since the mating phenotype of MAT strains carrying these HMR alleles and expressing dam methylase was indistinguishable from that of dam^- strains (data not shown).

To measure replication timing, cells constitutively expressing the dam methylase were synchronized in G1 using mating pheromone, released, and samples were collected in 5-min intervals throughout the ensuing S-phase. The DNA was cleaved with EcoRI, PstI, and DpnI and analyzed by DNA blot-hybridization. A 1.4-kb BglII fragment to the right of HMR-I, a 2.85-kb fragment adjacent to ARS1 (FERGUSON et al. 1991 ), and a 3.7-kb fragment containing R11 (FERGUSON et al. 1991 ) were used as probes to determine the replication time of the respective fragments. In both the HMR and the HMRa-e** strain, ARS1 DNA was maximally insensitive to DpnI digestion at 20 min after release into S-phase (Figure 7), indicating that the fragment was replicated at this time in the majority of the cells. In contrast to ARS1, the R11 fragment was replicated 40 min after release both in the wild-type and the mutant strain, consistent with earlier observations that R11 is a characteristically late replicating fragment.

View larger version (26K): In this window In a new window Download PPT slide   Figure 7. Replication late in S-phase was not sufficient for silencing. Replication timing at ARS1, R11, and HMR was assayed by measuring DpnI resistance of newly replicated DNA in strains expressing bacterial dam methylase. DNA harvested at the indicated times after release of the cells from the G1 arrest was digested with EcoRI, PstI, and DpnI, and probed with ARS1, R11, and HMR probes. Appearance of full length EcoRI/PstI fragments during the ensuing S-phase is indicative of hemimethylation resulting from passage of a replication fork. Full length fragments were quantitated using PhosphorImaging (Molecular Dynamics). The lane containing the highest fraction was assigned a value of 1 and used to normalize the fraction of full length fragment in the other lanes.

We next measured the replication timing of the fragment adjacent to HMR. The 6.3-kb EcoRI/PstI fragment lies on the telomere-proximal side of HMR-I, with 6.0 kb of this fragment being outside the region of HMR that is silenced (LOO and RINE 1994 ). Thus, this region should be equally accessible to the dam methylase in the repressed and the derepressed state of HMR. With a replication fork moving at an estimated 3.6 kb/min (RIVIN and FANGMAN 1980 ), this HMR-flanking fragment would be expected to be replicated within 1.75 min of the adjacent HMR region and thus be an accurate reflection of the replication timing of HMR itself.

In the wild-type strain, the HMR adjacent fragment displayed maximal DpnI resistance at 40 min after release into S-phase, at the same time as the R11 fragment, confirming earlier observations that HMR was replicated late in S-phase (REYNOLDS et al. 1989 ). In the HMRa-e** strain, the HMR fragment was also replicated at 40 min after release into S-phase. Hence, the replication time of HMRa-e** did not shift measurably. HMRa-e** was still replicated late in S-phase, even though it was derepressed. In a second set of experiments, we measured replication times for HMRa and HMRa-e** to be at 40 min after release, whereas R11 and ARS1 were replicated at 40 and 20 min, respectively, showing that derepression of HMR by a silencer mutation did not alter the time during S-phase at which the fragment was replicated. This result suggested that late replication per se was not sufficient for silencing. Furthermore, this indicated that late replication of HMR was not caused by silencing, but was likely due to other factors influencing the replication timing of this chromosomal region. Also, the observation that the derepressed HMRa-e** locus was late replicating ruled out the model that the suppressor mutations were restoring silencing by restoring late replication to an early replicating locus.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

This study describes mutations in the genes encoding the replication proteins PCNA, RF-C, polymerase , and Cdc45 that restored repression at a HMR locus derepressed by point mutations in the HMR-E silencer. In addition, the PCNA mutation, but not the other mutations, strongly derepressed HML in sir1 strains. Thus, PCNA joins SAS2 as a gene that, when mutated, caused opposite effects on silencing at HMR and HML, whereas RF-C, Pol, and CDC45 resemble CDC7 and SAS3 in that mutant alleles suppressed silencing defects at HMR, but did not increase the silencing defect of sir1 at HML (REIFSNYDER et al. 1996 ; EHRENHOFER-MURRAY et al. 1997 ). Like SAS2, mutations in PCNA and CDC45 depended upon the ORC binding site of the HMR-E silencer for repression and hence did not completely bypass the silencer.

The ability of mutations in closely related replication proteins to suppress silencing defects strengthens the connection between replication and silencing. DNA polymerase is required for chromosomal replication (BUDD and CAMPBELL 1993 ). Cdc45 is required for replication initiation (ZOU et al. 1997 ); it interacts with Mcm5/Cdc46 (HOPWOOD and DALTON 1996 ) and migrates with the replication fork along the DNA (APARICIO et al. 1997 ). PCNA is a homotrimer that assembles as a "sliding clamp" around the DNA double helix, tethers DNA polymerases to the DNA template, and increases the processivity of DNA replication (AYYAGARI et al. 1995 ). RF-C is a five-subunit complex that interacts with primer-template junctions and loads PCNA onto the DNA (CULLMANN et al. 1995 ). Mutations in the gene encoding PCNA as well as overexpression of PCNA suppress the defects of mutations in the RF-C subunit CDC44 (MCALEAR et al. 1994 ; AYYAGARI et al. 1995 ).

How do mutations affecting replication proteins suppress silencing defects? One common feature of most of the suppressors is that the respective proteins function close to the moving replication fork. The suppression by some but not other mutations in proteins at the replication fork may suggest that these proteins are in some way specifically involved in the formation of silenced chromatin, perhaps by communicating to other protein complexes that establish silenced chromatin after the passage of replication forks. Because loss of function mutations in these genes led to silencing in a subset of cells in the population, one would infer that in wild-type cells, these proteins promote the assembly of newly replicated DNA into euchromatin.

Others have suggested that a decrease in the rate of replication through the genome might increase the likelihood of silencing to be established from a compromised silencer. Because only a subset of mutations slowing DNA replication restored silencing, we do not favor this model unless the suppressing mutations specifically slow down replication through the silent loci, but not through most of the genome.

Another feature that unites some of the suppressors is their dual role in replication and DNA repair. For instance, PCNA interacts physically with the Msh2/Msh3 heterodimer that recognizes insertion/deletion mismatches in the DNA, and certain mutations in POL30 cause repair defects (JOHNSON et al. 1996 ). Also, Pol may be responsible for repair-associated DNA synthesis. Furthermore, because RF-C loads PCNA onto the DNA template, reduced loading of PCNA may explain why mutations in the large RF-C subunit CDC44 also have a defect in DNA repair (MCALEAR et al. 1996 ). If the effect of the mutations in PCNA, RF-C, and Pol on silencing were due to their defects in repair, repair synthesis would have a negative effect on silencing. However, the deletion of several mismatch repair genes (MSH2, 3, and 6; MARSISCHKY et al. 1996 ) and of other DNA repair genes (RAD1, 6, 7, 14, and 52) did not restore repression at HMRa-e** (Table 3), suggesting that mismatch recognition and DNA repair did not impair silencing. Moreover, the suppressor mutations restored silencing in cells whose DNA was not changed by any extrinsic agents or conditions.

Another model to explain how mutations in replication proteins might suppress silencing defects concerns the time during S-phase at which HMR is replicated. The HM loci and other transcriptionally inactive genome regions are replicated late in S-phase (REYNOLDS et al. 1989 ), which has led to the hypothesis that late replication is required for silencing. Perhaps the suppressor mutations were restoring silencing by restoring late replication to an early replicating mutant HMR allele. However, the HMRa-e** locus was also replicated late in S-phase, suggesting that the suppressor mutations did not act by restoring late replication. Conceivably, the suppressing mutations may cause HMR to replicate even later in S-phase than HMR normally replicates, in some way favoring the formation of silenced chromatin. Although it would be interesting to measure replication timing of HMRa-e** in its repressed state, because the suppressor mutations restore silencing only to a fraction of cells within a cell population, analysis of replication timing at the repressed HMRa-e** locus is not currently feasible.

Because the derepressed HMR was replicated late in S-phase, late replication alone was not sufficient for silencing. Thus, replication timing and silencing may not be causally related in yeast. Also, this result indicated that late replication of this region was not a consequence of silencing, but rather an inherent property of the region, perhaps because of its vicinity to the telomere or to determinants of late replication similar to those identified on chromosome XIV (FRIEDMAN et al. 1996 ).

Previous studies have uncovered an apparent competition between telomeres and HML and HMR for limiting silencing components (BUCK and SHORE 1995 ). Thus, mutations that restore repression to a weakened silencer might cause decreases in telomere length, thus releasing silencer factors bound to the telomeres and increasing their concentration within the cell. However, pol30-52 did not affect telomere length (R. T. KAMAKAKA, unpublished results), and cdc44-5 caused telomere elongation rather than shortening (ADAMS and HOLM 1996 ). Thus, telomere length regulation did not correlate with repression at HMR for these suppressors.

Interestingly, CDC44/RFC1 has the opposite effect on silencing at the rDNA locus. A transposon insertion in the promoter of CDC44 caused a loss of rDNA silencing (SMITH et al. 1999 ). Also, a mutation in Pol caused a rDNA silencing defect. However, the mutation identified was a mutation in the nonessential DPB3 subunit, which had no effect in our HM silencing assay. Similarly, a mutation in Pol had an effect at the rDNA, but not at HMR. This supports the view that there are at least some similarities, but also profound differences, between silencing at the HM loci and at the rDNA.

At present, we have no adequate explanation for why cin8, clb5, and cln3 were able to restore silencing at the mutant HMR silencer. With respect to the two cyclin mutants, perhaps a silencing component is limited by the Cdc28 kinase combined with either of these two cyclins.

In summary, the data presented here established a connection between DNA replication and silencing and underscored the question of how silenced chromatin is reestablished after passage of the replication fork. Our data implicated the involvement of replication proteins themselves in this process, perhaps by communicating to factors that establish silenced chromatin. In this light, it is interesting to note that PCNA in human cells interacts with a DNA methyltransferase, which is associated with the establishment of epigenetic patterns of gene expression (CHUANG et al. 1997 ). Furthermore, mutations in Drosophila mus209, the gene encoding PCNA, suppress position effect variegation (HENDERSON et al. 1994 ), a form of silencing in Drosophila. Thus, whatever the mechanism, roles for replication proteins in silencing may be conserved among eukaryotes.

	FOOTNOTES

¹ Present address: Max-Planck-Institute of Molecular Genetics, Otto-Warburg Laboratories, Ihnestr. 73, D-14195 Berlin, Germany.
² Present address: Unit on Chromatin and Transcription, National Institutes of Child Health and Human Development, Bethesda, MD 20892.

	ACKNOWLEDGMENTS

We thank the following colleagues for providing yeast strains and plasmids: D. Shore, H. Araki, L. Hartwell, J. Thorner, D. Botstein, P. Burgers, C. Holmes, D. Koshland, S. Elledge, T. Weinert, J. Campbell, B. Tye, B. Stillman, R. Kolodner, and P. Kaufman. We thank K. Friedman and B. Brewer for plasmids and protocols for the timing experiments and the members of the Rine lab for many stimulating discussions. A.E.-M. thanks A. König and A. Barduhn for assistance with quantitative mating assays. This work was supported by the Swiss National Science Foundation (A.E.-M.) and National Institutes of Health grant GM-31105 (J.R.). Core support was provided by a National Institutes of Child Health and Human Development Mutagenesis Center Grant.

Manuscript received December 3, 1998; Accepted for publication July 23, 1999.

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