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* Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3202
Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario M5G 1L6, Canada
Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada
1 Corresponding author: Department of Molecular and Cell Biology, University of California, 522 Barker Hall #3202, Berkeley, CA 94720-3202.
E-mail: jrine{at}uclink.berkeley.edu
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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The proper segregation of sister chromatids during anaphase depends on the establishment of sister chromatid cohesion during S phase and chromosome condensation before mitosis (NASMYTH 2002). An evolutionary conserved cohesin complex is required for cohesive linkage of sister chromatids (GUACCI et al. 1997; MICHAELIS et al. 1997). The cohesin complex binds to chromosomes at distinct cohesion sites from late G1 phase until the metaphase-anaphase transition when the Scc1p cohesin subunits are degraded (UHLMANN et al. 1999). Cohesin binding is enriched at centromeres where cohesion counteracts the pulling force of the mitotic spindle before the onset of anaphase. Physical and genetic evidence indicates that establishment of sister chromatid cohesion is also closely linked to DNA replication, most likely mediated by components of the replication fork (CARSON and CHRISTMAN 2001). CTF7/ECO1 is an essential gene that is required in S phase for establishment of sister chromatid cohesion. Ctf7/Eco1 has been linked genetically and physically to the replication apparatus (SKIBBENS et al. 1999; TOTH et al. 1999; KENNA and SKIBBENS 2003). Another link between sister chromatid cohesion and DNA replication came with the discovery of an alternative replication fork clamp loader (RFC) complex, mutations of which lead to loss of sister chromatid cohesion (MAYER et al. 2001). The Ctf4 protein was originally identified through its binding to DNA polymerase 
(MILES and FORMOSA 1992). Mutation of CTF4 also leads to a cohesion defect (HANNA et al. 2001). Likewise, a nucleotidyl-transferase activity (polymerase 
) that is encoded by TRF4 and TRF5 contributes to sister chromatid cohesion (WANG et al. 2000). These proteins may contribute to the passage of the replication fork through a cohesion site (see CARSON and CHRISTMAN 2001). Finally, the pol2-12 mutation in DNA polymerase 
also causes a cohesion defect, providing a direct connection between sister chromatid cohesion and a replicative polymerase (EDWARDS et al. 2003). Interesting links between cohesion and condensation of chromosomes to the epigenetic inheritance of transcriptional states have emerged in different organisms (reviewed in HAGSTROM and MEYER 2003). For example, in Schizosaccharomyces pombe, the high concentration of Scc1 protein at centromeres requires stable heterochromatin formation at centromeric repeats (BERNARD et al. 2001).
Synthetic lethal interactions have been used to establish functional relationships between genes (GUARENTE 1993; HARTMAN et al. 2001). In a previous synthetic lethal screen for genes that are required for viability of orc2-1 mutants, cdc7, cdc14, and orc3 mutants were isolated on the basis of the lethality of the double-mutant combinations (HARDY 1996). Strong genetic interactions between ORC and other replication genes have also been observed, but none of these studies approached genetic saturation of possible interactions (LIANG et al. 1995; LOO et al. 1995; KROLL et al. 1996; ZOU et al. 1997).
To provide a more comprehensive view of the processes linked to ORC and presumably therefore to origins of DNA replication, we used the synthetic genetic array (SGA) methodology to systematically evaluate double mutants between ORC genes and the deletion collection of nonessential genes. As expected, synthetic genetic interactions were seen between ORC mutants and mutations in genes involved in DNA replication. However, the combined network of interactions for ORC and other replication mutants reveals an extended link among replication initiation, sister chromatid cohesion, chromatin structure, checkpoint control, and DNA repair. Finally, new links were uncovered between the establishment of sister chromatid cohesion and transcriptional silencing.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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mfa1
::MFA1pr-HIS3 can1 his31 leu20 ura30 MET15+ lys20). The identity of the orc-ts allele was confirmed by its phenotype and correct integration was confirmed by PCR or DNA blot. SGA screening procedure with the marked orc2-1 and orc5-1 alleles was done essentially as described previously (TONG et al. 2001). Double-mutant selection was done at 23°, 26°, and 30° for the orc5-1 strain and at 22°–23° for orc2-1. Potential synthetic lethal/sick interactions were confirmed by tetrad analysis at different temperatures, 23° and 30° for orc2-1 and orc5-1, respectively. Positive results from the synthetic lethal screens were confirmed by tetrad dissection and growth at 23° and 30°, which are semipermissive temperatures for orc2-1 and orc5-1, respectively. For the orc5-1 screen, a MATa version of the orc5-1::natMX4 strain was crossed with the haploid collection strains for the confirmation of the results. A minimum of 10 tetrads were dissected from each cross. Some results from the orc2-1 screen were also confirmed by random spore analysis as described by TONG et al. (2004).
Yeast strains:
The genotypes of all yeast strains used are in Table 1
. PCR fragments containing ctf4::kanMX4, ctf18::kanMX4, dcc1::kanMX4, trf4::kanMX4, and trf5::kanMX4 were amplified by colony PCR from the knockout collection strains. PCR products were purified using the QIAEX buffer desalting protocol and transformed into a diploid strain derived from mating of JRY4012 and JRY3009. Gene disruption in haploid segregants was confirmed by PCR and characterization of phenotypes. The scc1-73::TRP1 allele was from strain ROY1063. Segregants from crosses with ROY1063 were tested for an intact HMR-I silencer by PCR.
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::kanMX4), and JRY7717 (ctf18::kanMX4). Plasmid-loss assays were done as described previously (LOO et al. 1995), except that growth in nonselective medium was for 9 or 10 generations at 30°.
Sister chromatid cohesion assay:
Tet repressor-green fluorescent protein (GFP)/Tet operator repeat constructs were used to visualize the URA3 region on chromosome V (MICHAELIS et al. 1997). TetR-GFP was expressed from K3524/yplac128tetR-GFP (URA3-5'NLStetR-SuperGlowGFP-ADH-T::LEU2::leu2-3,112). The K2524 construct was integrated into JRY4012 to generate JRY7468. JRY7469, JRY7470, and JRY7471 were generated from JRY7468 with JRY4285 (orc5-1) and ROY1063 (scc1-73). To mark the URA3 locus, these strains were transformed with plasmid pXH122 (p306tetO2X224 ChV-38; HE et al. 2000) that was linearized with EcoRV. Cells from overnight cultures were diluted to A600 
0.1 and grown for 3 hr at 31° in SC-Ura or YPD. To monitor sister chromatid cohesion in G2/M phase, cells were transferred to A600 0.2 in YPD + 15 µg/ml nocodazole and arrested for 2.5 hr at 31°. To control for aneuploidy, cells were arrested at A600 0.2 in YPD + 5 µg/ml -factor for 2.5 hr at 31°. Aliquots (0.9 ml) were fixed with 100 µl 37.5% formaldehyde for 10 min at 4°. Samples were washed twice with 1 ml ice-cold phosphate-buffered saline and sonicated for 10 sec. After the second wash, the pellets were resuspended in 200 µl phosphate-buffered saline and GFP dots were directly analyzed by fluorescence microscopy. The fraction of cells with two GFP dots was compared to the total number of arrested cells. Microscopy was done using a Nikon Eclipse E600 microscope, a Nikon x100 Plan Apo phase objective, and a Hamamatsu digital camera C4742-95. Slides were coded during scoring so that the scorer was blind to the genotype of the sample.
Silencing assays:
All silencing assays were done with ctf4::kanMX4, ctf18::kanMX4, and dcc1::kanMX4 in the W303 background. To assay silencing at HMR, strain JRY5329 with HMR::2EDA replacing HMRa was used (SUSSEL et al. 1993). Cultures were grown to A600 1 in liquid YPD medium and 100 µl from 1:104 dilutions was plated on YPD plates. The deletion strains containing ADE2 at the HMR locus were analyzed for development of red or pink color after 3 days at 30° and 3 additional days at 4°. To assay telomeric silencing for the URA3-TRP1 reporter at telomere VII-L, cultures were grown to A600 0.5 in liquid YPD medium and spotted in fivefold serial dilutions on SC, SC + 0.1% fluoroorotic acid (FOA), and SC-TRP plates. Strains containing the ADE2 reporter gene at telomere V-L were grown to A600 1 in YPD and plated on SC medium to obtain 100–200 colonies per plate. Photographs were taken after 3 additional days at 4°.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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(Figure 1B). More interacting genes were recovered in the orc2-1 screen than in the orc5-1 screen (Table 2) . However, all results from the orc5-1 screen were also found in the orc2-1 screen. This complete overlap suggests that both Orc2p and Orc5p function in only one essential process. Lower recovery of interactions with orc5-1 was in part due to minor differences in the conditions of the screens using the two mutants and, perhaps, due to orc2-1 being more defective than orc5-1 at some temperatures. The results of the ORC screens overlapped extensively with those from the synthetic lethal screens with cdc45-1 and cdc7-1 (Figure 1C), which are described elsewhere (TONG et al. 2004). Considering the occurrence of false-negative results from the robotic screen, the overlap could be even more extensive. Thus, the pattern of synthetic lethal interactions clearly supported the common view that the major role of ORC is in DNA replication.
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, so it was not surprising that a null allele of this gene was synthetically lethal with orc-ts mutants. Likewise, earlier work established that replication mutants become dependent on double-strand-break repair by homologous recombination (HARTWELL and SMITH 1985). Hence the synthetic lethality between orc-ts mutants and rad52, rad55, and hpr5 presumably reflected an inability to rescue stalled or collapsed replication forks by recombination mechanisms. MRC1 encodes a mediator of the DNA replication checkpoint and binds to Cdc45p as does the Tof1 protein (KATOU et al. 2003; OSBORN and ELLEDGE 2003); hence the synthetic lethal interactions between mutations in MRC1 and TOF1 and orc-ts mutants may reflect the contribution of the replication checkpoint to cell survival when origin firing becomes limiting. On the other hand, the genetic interaction of orc-ts with mrc1 could be caused by an active role of Mrc1p in the replication process (OSBORN and ELLEDGE 2003). Strong interactions of orc-ts mutations were also found with a deletion of CSM3 that encodes a newly identified component of the replication checkpoint (TONG et al. 2004). Compared with the replication checkpoint, DNA damage checkpoint mutants that exhibit reduced viability in combination with other replication mutants (see Figure 1C) were found to be only slightly sick in combination with orc2-1 (rad9 and rad24; Table 2) or were not picked up in the orc-ts screens (chk1, mec3, rad17, and ddc1).
Other double-mutant combinations with orc-ts mutants revealed connections that were not anticipated. For example, FUN30 is a member of the Swi/Snf2 family, whose other members play a role in chromatin remodeling. Another possible link between replication and chromatin remodeling was revealed by the interaction with ISW1, an Snf2-related chromatin-remodeling factor. Factors that have a role in the deposition of nucleosomes onto newly replicated DNA were also prominent in the screens with orc2-1, orc5-1, and other replication mutants. Furthermore, different genes that have a role in histone modification were also identified, thus strengthening a link between replication and chromatin assembly or function. Growth defects in orc-ts strains were observed in combination with a null allele of the NAD+-dependent histone deacetylase gene HST3 and its paralog HST1. Interestingly, a synthetic sick interaction was also found when orc5-1 and orc2-1 were combined with the sum1 mutation. SUM1 encodes a repressor of middle meiotic genes and requires the deacetylase activity encoded by HST1 for its function (XIE et al. 1999). Several genes that have no known role in the replication process but have established roles in other cellular processes [e.g., LSM1 in mRNA capping (THARUN et al. 2000) and CIK1 and BIM1 in microtubule function (PAGE and SNYDER 1992; SCHWARTZ et al. 1997)] also caused synthetic sickness or lethality when combined with orc2-1 and orc5-1. The synthetic lethal screens also revealed interactions of cdc45-1 and cdc7-1 with mad1 and bfa1, which are involved in the spindle checkpoint mechanism (see LEW and BURKE 2003). Activation of the spindle checkpoint at restrictive temperature by orc-ts and other replication mutations has been shown previously (GARBER and RINE 2002).
Links between ORC and sister chromatid cohesion:
Among the especially strong interactors with orc5-1 or orc2-1 alleles were null alleles of genes that are involved in the establishment of sister chromatid cohesion during S phase. These genetic interactions included CTF4 and three genes, CTF8, CTF18, and DCC1, that encode subunits of the alternate RFC complex that is important for sister chromatid cohesion (Figure 2A)
. The double-mutant segregants grew to microcolonies that consisted of large budded cells. Strong genetic interactions of CTF sister chromatid cohesion genes were also found with cdc6-1 and mcm2-1 (Figure 2B) and cdc45-1 and cdc7-1 (Figure 1C and data not shown). Given the strong genetic interactions of orc-ts alleles with components of one alternative RFC complex that replaces the canonical Rfc1 subunit (MAYER et al. 2001), we also analyzed the genetic interaction between ORC and the canonical Rfc1 subunit gene, RFC1. The combination of the cdc44-5 mutation in RFC1 with orc5-1 was lethal (Figure 2C).
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and ctf18 strains. Plasmid-loss rates for pDK243 and pDK368-7 were 9.4 and 13.1% for ctf4, 16.3 and 18.8% for ctf18, 16.5 and 1.8% for orc5-1, and 0.3 and 0.07% for wild-type control strain. Thus, by this assay, no link was found between CTF4 and CTF18 and replication initiation.
Given the strong genetic interactions of orc5-1 and orc2-1 alleles with ctf4 and mutants in the CTF18-Rfc complex, the orc5-1 allele was also tested for genetic interactions with two genes, TRF4 and TRF5, that have overlapping roles in sister chromatid cohesion (WANG et al. 2000; EDWARDS et al. 2003). The trf4 orc5-1 segregants grew only slightly less well than the trf4 single mutants (Figure 2D). No genetic interaction was evident between orc5-1 and trf5 (Figure 2D). The weaker or nonexistent interactions between orc-ts mutations and trf4 or trf5 mutations may reflect a less direct role for Trf4p and Trf5p in the coupling of sister chromatid cohesion with DNA replication.
ORC function affects sister chromatid cohesion:
Given that the synthetic interaction between orc-ts mutations and mutations in sister chromatid cohesion genes did not seem to reflect roles for the cohesion proteins in replication initiation, we explored whether ORC might contribute in some way to efficient sister chromatid cohesion. The orc5-1 allele was combined with the scc1-73 mutation in the core cohesin complex. The scc1-73 mutation by itself leads to a precocious separation of sister chromatids before the onset of anaphase at its restrictive temperature (MICHAELIS et al. 1997). Both orc5-1 and scc1-73 have a restrictive temperature at 32°–33°. At 31°, both single mutants showed robust growth. However, growth of the orc5-1 scc1-73 double mutant was compromised at this temperature, whereas at 23° and 26°, the orc5-1 scc1-73 double mutant grows as well as the orc5-1 single mutant (Figure 3A)
. Thus, the combination of orc5-1 and scc1-73 revealed a synthetic growth defect at the maximum permissive temperature.
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8%. In an scc1-73 strain, an elevated loss of sister chromatid cohesion (16% cells with two spots) was evident at the semipermissive temperature of 31° (Figure 3C). The orc5-1 scc1-73 double mutant exhibited a cohesion defect substantially greater than that of the scc1-73 single mutant (29% of cells with two spots). Thus, reduced ORC function enhanced the cohesion defect of scc1-73 cells. A potential source of artifacts in this analysis would be an extra copy of the chromosome containing the tet operator repeats. To determine whether the elevated level of cells with two spots resulted from a cohesion defect or from the presence of an extra chromosome, chromosomes marked at the URA3 locus were scored in cells that were arrested in G1 phase by -factor. These cells are expected to contain only a single fluorescent dot, unless they have a second copy of the marked chromosome. The frequency of G1 cells containing two marked chromosomes was low in all cases. Some chromosome missegregation was observed in scc1-73 and scc1-73 orc5-1 strains (<5%; Figure 3D). However, the few G1 cells with two marked chromosomes could not account for the large number of chromosomes with two spots in G2/M phase that were therefore the consequence of a sister chromatid cohesion defect. A subtle cohesion defect was evident in the orc5-1 single mutant at semipermissive temperature (31°–32°) compared to wild type. This small difference was reproducible but not statistically significant. A similar result was obtained for orc2-1 cells arrested at semipermissive temperature (not shown). Furthermore, we observed no increase in the number of separated sister chromatids in orc5-1 single mutants at the restrictive temperature of 36° (not shown). Thus, defective sister chromatid cohesion is not a major phenotype of orc-ts mutations. However, the reduction in the permissive temperature and the enhanced cohesion defect in the orc5-1 scc1-73 double mutant revealed some role of ORC in sister chromatid cohesion.
Sister chromatid cohesion genes and transcriptional silencing:
Silencing functions have been established for different DNA replication genes, including POL30, CDC44, POL2, CDC45, DPB4, and DPB11 (EHRENHOFER-MURRAY et al. 1999; ZHANG et al. 2000). Therefore, genes with new roles in DNA replication that were identified in our screens could, in principle, also have potential functions in silencing. Ctf4p and the alternative RFC complex are thought to help establish sister chromatid cohesion during the replication process and could connect establishment of cohesion with silencing. To study transcriptional repression in ctf4 and ctf18 strains at the HMR locus, strains with ADE2 inserted into HMR were used (HMR::2EDA; Figure 4)
. Complete repression of the ADE2 gene results in red colonies, whereas derepression of ADE2 produces white colonies, and partial derepression produces pink colonies. The colony color in ctf4 colonies with the HMR::2EDA reporter was pink, whereas wild-type colonies were red (Figure 4A). Colonies were also predominantly pink in a ctf18 mutant strain (Figure 4A). Derepression of ADE2 was also observed when ctf4 and ctf18 strains contained the ADE2 gene in the opposite orientation with a weakened HMR E silencer (HMRB::ADE2; not shown). Thus, CTF4 and CTF18 contributed to silencing at the HMR locus.
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mutation was combined with orc5-1R, the colonies were white, indicating complete derepression of ADE2 (Figure 4A). Therefore, the effect of the ctf4 null mutation on transcriptional silencing was independent of ORC. Similarly, ctf4 led to an enhanced derepression of HMR::2EDA in combination with the rap1-12 allele (Figure 4B). The pink and white colony color was caused by the growth defect in the mutants since single- and double-mutant colonies are red in strains without ADE2. Furthermore, silencing in an orc5-1R ctf4 double mutant at the HML locus was also partially defective as judged by -factor confrontation assay. About 10% of the double-mutant cells grew into small colonies after 14 hr at 23° in the presence of -factor, whereas all wild-type or single-mutant cells remained arrested by -factor (not shown).
To assay silencing at telomeres in cohesion mutants, strains were used that contained either the URA3::TRP1 reporter at telomere VII-L (Figure 5A)
or the ADE2 gene at telomere V-L (Figure 5B). When strains containing TELVII-L::URA3::TRP1 were assayed on 0.1% 5-FOA medium, selecting against URA3 function, ctf4 strains did not grow, whereas ctf18 and dcc1 strains formed small colonies. The wild-type control strain grew well, indicating that URA3 was silenced at this location, whereas the silencing defective sir2 strain did not. The TRP1 gene at telomere VII-L was also derepressed in ctf4, ctf18, and dcc1 mutants, although to a lesser extent than in the sir2 control strain. In wild-type cells, partial repression of the ADE2 gene at telomere V-L leads to a phenotype of red and white sectored colonies (Figure 5B). In contrast, ctf4 and ctf18 with this reporter were white. Thus, CTF4 and CTF18/DCC1 contributed to transcriptional silencing at two different telomeres.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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Temperature-sensitive orc-ts mutations in combination with mutations in sister chromatid cohesion genes resulted in reduced viability at temperatures permissive for the individual mutations. These interactions did not appear to reflect a defect of cohesion mutants in replication initiation. Rather the reduced viability in an orc5-1 scc1-73 double mutant was likely caused by an enhanced cohesion defect. Synthetic lethal interactions are also observed for cohesion mutants in combination with mcm2-1, cdc6-1, cdc7-1, and cdc45-1 mutations, and hence we favor the notion that the interaction reflects a dependence of robust sister chromatid cohesion on initiation of DNA replication. A link between initiation of DNA replication and sister chromatid cohesion was found in S. pombe. Here, a mutation in hsk1+, which is the homolog of the S. cerevisiae CDC7 encoded serine threonine kinase, leads to a cohesion defect (TAKEDA et al. 2001; BAILIS et al. 2003).
However, orc5-1 alone showed no obvious cohesion defect compared to wild-type strain. Furthermore, the majority of chromosomal cohesin binding sites do not co-localize with origins (BLAT and KLECKNER 1999; LALORAYA et al. 2000). Thus, ORC's involvement in cohesion is presumably not completed at replication origins. Cohesion between sister chromatids is established at the time of replication, presumably by processes that occur at the replication fork. At permissive temperature, the orc2-1 mutation leads to a 30% reduction in the number of replication forks (SHIMADA et al. 2002), and presumably this loss is greater the closer to the restrictive temperature the mutant is grown. We speculate that there may be a limit to the amount of cohesion that can be established at any given fork. In this model, the reduction in the number of replication forks in the mutants leads to less cohesion, which becomes growth limiting in cells with temperature-sensitive Scc1p.
Although an enhanced cohesion defect provides an explanation for the reduced viability in the orc5-1 scc1-73 double mutant, other mechanisms might also contribute to the strong genetic interactions of ORC with cohesion genes. CTF4 and genes encoding components of the Ctf18-RFC are sensitive to different DNA-damaging agents (CHANG et al. 2002). Furthermore, cohesion is important for recombinational repair (SJOGREN and NASMYTH 2001). It is possible that the repair of broken replication forks could lead to a dependence of orc-ts mutants on cohesion genes. CTF18 also has an overlapping role with RAD24 in the replication checkpoint (NAIKI et al. 2001). Moreover, replication mutants strongly interact with the replication checkpoint genes MRC1, TOF1, and CSM3 (Figure 1C). Thus, proper replication checkpoint function could be important for viability of orc-ts and other replication mutants, and the cohesion phenotypes in the ctf mutants may be an indirect consequence of improper DNA replication or failure of the replication checkpoint. During the final stage of preparing this manuscript, work was in press that also demonstrated a cohesion function for other genes that were identified in our screens (MAYER et al. 2004; WARREN et al. 2004). Importantly, synthetic lethal screens for genes interacting with ctf4 and ctf8 also established a role in sister chromatid cohesion for the replication checkpoint genes MRC1, TOF1, and CSM3.
Only a slight interaction was observed between orc5-1 and trf4 and essentially none between orc5-1 and trf5, suggesting that the roles of Trf4p and Trf5p are mechanistically distinct from those of Ctf4p, Ctf18-RFC, and other cohesion factors. Thus, it seems likely that some cohesion factors are more intimately connected to the replication process than others. Plasmid-loss experiments suggested that Ctf4p and Ctf18p did not directly affect initiation of DNA replication. However, this does not exclude the possibility that these proteins somehow couple the initiation of DNA replication with the establishment of sister chromatid cohesion.
We demonstrated that the genes important for the establishment of sister chromatid cohesion, CTF4, CTF18, and DCC1, also contributed to silencing of HMR, HML, and telomeres. A partial defect in telomeric silencing has also been observed in an scc1-73 strain at semipermissive temperature (P. KAUFMAN, personal communication). In principle, indirect effects on chromosome organization that occur in these mutants could explain such links. A mutation in a cohesin subunit removed a boundary for the spread of heterochromatin at the HMR locus (DONZE et al. 1999). Spreading of heterochromatic factors into the adjacent euchromatin might dilute heterochromatin components and decreases transcriptional silencing (MENEGHINI et al. 2003). Thus, sister chromatid cohesion could be important for the proper separation and distribution of euchromatin and heterochromatin.
More direct models are supported by the temporal coincidence of the establishment of sister chromatid cohesion in S phase with chromatin assembly and the establishment of silencing (MILLER and NASMYTH 1984; SHIBAHARA and STILLMAN 1999; ZHANG et al. 2000; KIRCHMAIER and RINE 2001; LI et al. 2001). Recent work suggests that cohesins have to be removed before silencing is complete (LAU et al. 2002), which implies an inhibitory role for cohesins in silencing. Our data present the reciprocal view that cohesion establishment contributes positively to silencing, suggesting a more complex interplay between proteins involved in the two processes. Defects in transcriptional silencing have been shown for chromatin assembly factors that are associated with the replication fork (KAUFMAN et al. 1998; TYLER et al. 1999; SHARP et al. 2001). Similarly, Ctf4p and the Ctf18-RFC complex could also affect silencing by their possible function at the replication fork. This also raises the question of whether the cohesion phenotypes in ctf4 and cf18 are a consequence of a defective organization of newly replicated chromatin. The involvement of cohesion factors in transcriptional silencing demonstrates therefore an interdependence of DNA replication, chromosome structure, and proper chromosome segregation.
DNA replication, like other DNA-dependent processes, must contend with the organization of DNA into nucleosomes and higher-order chromatin structures. Previous work established the contribution of positioned nucleosomes to replication initiation at ARS1 (SIMPSON 1990; LIPFORD and BELL 2001). The identification of multiple genes with roles in nucleosome remodeling and histone modification in our screens suggests that specific aspects of chromatin structure influence replication. Mobilization of nucleosomes by chromatin-remodeling factors may promote origin firing or fork progression. The recovery of histone acetyltransferases, NAD+-dependent protein deacetylase paralogs, and chromatin-remodeling factors in the screen implies a deep and potentially complex relationship between chromatin structure and DNA replication.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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