INTRODUCTION

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The establishment of sister chromatid cohesion during S phase is a critical step in the series of events leading to high-fidelity cell division. By holding sisters together, cohesion proteins enable kinetochores to face opposite poles of the mitotic spindle, facilitating capture by microtubules from opposite poles (99). The sister chromatid association is sufficient to resist the separating force of the mitotic spindle until each kinetochore has been captured, at which time sister chromatid associations are released at the initiation of anaphase (reviewed in references 50, 72, 77, and 88). Because cohesion tightly binds sisters together from their synthesis to their separation, it must be properly established and maintained in a flexible environment supporting chromatin alterations that permit transcription, replication, repair, and condensation of the genome.

Cohesion between sister chromatids is carried out by at least four classes of proteins. The core particle, cohesin, is composed of at least four subunits encoded in budding yeast by the SMC1, SMC3, MCD1 (SCC1), and SCC3 (IRR1) genes (33, 68). Fully assembled cohesin binds chromatin in vitro and in vivo (9, 68, 97, 101). Orthologs of cohesins have been identified in Xenopus laevis, Drosophila melanogaster, Schizosaccharomyces pombe, Arabidopsis thaliana, Mus musculus, and Homo sapiens (6, 18, 58, 59, 82, 94, 100, 109, 110). Interestingly, although Mcd1p is required for both cohesion and chromosome condensation in budding yeast, these processes are carried out by distinct protein complexes in the Xenopus experimental system (33, 40, 41, 58). In addition, Pds5p, which is also required for the maintenance of sister chromatid cohesion, genetically and physically interacts with the cohesin complex (36, 78, 94). Thus, interactions between the cohesin complex and Pds5p are required to mediate sister chromatid cohesion. A highly conserved mechanism governs sister chromatid separation at anaphase initiation, mediated by the action of a CDC20-associated form of the anaphase-promoting complex (APC^CDC20), which provides a ubiquitin-conjugating activity directing the degradation of the anaphase inhibitor protein Pds1p (reviewed in reference 70). Upon release from a Pds1p-Esp1p complex, active Esp1p promotes the proteolysis of Mcd1p (94, 104, 107). This event is associated with loss of cohesion between sister chromatids and with poleward movement of the chromosomes (reviewed in references 73 and 116).

Scc2p and Scc4p, members of the second class of proteins, direct the binding of cohesin proteins to chromatin (16, 101). SCC2 and SCC4 associate with each other in coimmunoprecipitation experiments but are not core components of the cohesin particle (16). Scc2p may mediate cohesin complex interaction with chromatin via associations with Mcd1p and Scc3p. While the localizations of both Scc2 and Scc4 proteins on chromatin spreads are similar to one another, the two proteins seem to occupy different chromosomal loci from Mcd1p (16, 101). Furthermore, both Scc2p and Scc4p associate with chromatin in a nuclease- and salt-resistant manner, suggesting that they are tightly bound in a higher-order chromatin structure. Scc2p and Scc4p are required for establishment of cohesion early in the cell cycle but are not required for maintenance of cohesion in metaphase arrested cells (16). This function appears to be conserved, since a fission yeast homologue of Scc2p (Mis4p) is also required in S phase (26). SCC2 homologues have also been identified as the Coprinus RAD9 (83) and Drosophila Nipped-B (81) genes.

A third class of molecules, defined by CTF7 (ECO1) function, is required to render the cohesin complex competent to mediate siser chromatid cohesion during S phase. Budding yeast are inviable in the absence of CTF7, and conditional alleles lead to precocious sister separation (87, 101). Although Ctf7p associates with chromatin, it does not stably associate with the core cohesin particle, nor is it requred for cohesin association with chromatin (87, 101). Execution point studies indicate a requirement for CTF7 in S phase (87, 101). Interestingly, the chromosome instability and temperature-sensitive lethality of ctf7 alleles are suppressed by high-copy-number expression of POL30, encoding budding yeast proliferating-cell nuclear antigen (PCNA) (87). One hypothesis is that Ctf7p is required at the replication fork to activate interactions between cohesins on sister chromatids for a functional "glue" to be formed. Budding yeast Ctf7p is homologous to a C-terminal domain of the fission yeast Eso1 protein, whose N-terminal segment is homologous to the budding yeast DNA repair polymerase RAD30 (96). The Eso1⁺ gene functions similarly during S phase and is required for sister chromatid cohesion, pointing to conservation of this activity as well as its association with replication of DNA.

Recent studies have identified proteins more directly involved in DNA replication as members of the fourth class of cohesion proteins. This category includes PCNA by genetic interaction with CTF7 as described above (87) and a new DNA polymerase family, designated polymerase , exemplified by budding yeast gene TRF4 (108). PCNA forms a homotrimeric ring structure (clamp) which encircles DNA and supports processive DNA replication by associated DNA polymerases  and  (reviewed in references 39 and 47). A "clamp-loader," replication factor C (RF-C), is required to facilitate association of PCNA with DNA (reviewed in reference 71). RF-C is composed of five essential subunits that have a common core region of homology that may facilitate interactions with PCNA (1, 42, 65), as well as interact with DNA (103). RF-C may also mediate a switch from polymerase -directed replication initiation to processive replication by polymerases  and  through competitive interactions with PCNA and the budding yeast single-stranded binding protein replication protein A (RPA) (reviewed in reference 19).

TRF4 and its paralog TRF5 are both members of the -polymerase superfamily as defined by protein alignment (3). While neither the TRF4 nor TRF5 gene is essential, in combination they exhibit synthetic lethality. Recent work has provided direct evidence that Trf4p encodes a novel polymerase and that a trf4 trf5 double mutant exhibits highly inefficient S-phase DNA replication (108). Like other budding yeast genes that function in cohesion, TRF4 is required for both chromosome condensation (14) and sister chromatid cohesion (108). The chromosomal defects present in a trf4 mutant cell influence the maintenance of sister chromatid cohesion under mitotic arrest conditions, probably through an uncharacterized mechanism that operates during DNA replication (108).

In this work, we present an analysis of two genes, CTF4 and CTF18, that exhibit genetic and physical interactions with components of the replication fork and that are required for sister chromatid cohesion. Analysis of these genes provides independent evidence that cohesion-related functions are indeed carried out by proteins associated with the DNA synthesis machinery. Previous genetic analyses have indicated that neither CTF4 nor CTF18 is essential; however, absence of either gene increases chromosome instability and mitotic recombination rates and induces a strong preanaphase delay (51, 53, 69). CTF4 was first identified in concurrent studies as CTF4 (CHL15) (52) and POB1 (69) and encodes a 104-kDa polypeptide with motifs suggestive of three zinc finger structures, as well as a helix-loop-helix region (35, 51). Ctf4p exhibits high-affinity binding to DNA polymerase  in vitro (69), and ctf4 mutants show genetic interactions with conditional alleles of DNA polymerase  (encoded by POL1 [CDC17]) and other genes intimately associated with DNA synthesis (24, 112). A ctf4 mutation also causes synthetic lethality with a null allele of CTF18, the other gene investigated in this study (24). CTF18 (CHL12) was independently identified in two screens for chromosome loss mutants (52, 91). CTF18 encodes a predicted 84-kDa polypeptide with homology to all five subunits of the RF-C complex from yeast and other organisms, with the most significant similarity to the large subunit Rfc1p (17, 53).

Here we find that both CTF4 and CTF18 are required for sister chromatid cohesion. This, rather than the presence of damaged DNA, is likely to be the major mechanism underlying chromosome loss in both ctf4 and ctf18 null mutants. The preanaphase delay exhibited by each of these mutants is due to a spindle assembly checkpoint arrest. Because we and others have observed that CTF4 and CTF18 exhibit genetic interactions with genes that function in DNA replication, we suggest that CTF4 and CTF18 act in association with the replication fork complex(es) to facilitate the establishment of robust sister chromatid cohesion. We propose a model in which Ctf18p, an Rfc1p paralog, may act within a complex similar to the previously characterized RF-C, consistent with physical and genetic evidence presented in this report.

The replication fork plays numerous roles in the chromosome cycle, including duplication of genomic DNA, detection and repair of lesions (85, 93), and regulation of transcriptional states (20, 89). The properties of CTF4 and CTF18 highlight a new function of the replication fork machinery that is essential for high-fidelity segregation of the genome. The characterization of these proteins, which interact with residents of the replication fork and whose loss of function compromises cohesion, presents new opportunities for investigation of this novel role of the replication machinery.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Strains and media. Yeast transformation (29, 49) and genetic manipulations (34) were performed by published methods. rYPD is rich medium adjusted to pH 4 and supplemented with additional adenine hemisulfate (30 ng/ml), uracil (20 ng/ml), and L-tryptophan (30 ng/ml). Strain sets (Table 1) within each single experiment are composed of laboratory stocks related by transformation or genetic crosses maintaining background isogenicity. Complete oligonucleotide sequences will be provided on request.

Vector construction. A SpeI-NotI fragment from BFS66, containing the CTF18 ORF and flank, was cloned into pBluescript II to give pJH28. pJH28::TRP was created by removal of the internal two-thirds of CTF18 ORF by digestion with NsiI and MluI, and its replacement by ligation of a PCR product from primers OLFS264 plus OLFS265, which amplified the TRP1 gene from pRS404 (10). pJH72.1 was constructed by cloning the entire CTF18 ORF into pCR2.1 (Invitrogen TOPO-TA) using primers OLFS274 plus OLFS362 and LTI Pfx Taq; the CTF18 ORF was then released using NcoI-EcoRI digestion and cloned into the same sites in pAS2-1 (Clontech), which created an in-frame fusion with the GAL4 DNA binding domain (pJH74.4). pJH73.2 was constructed from a PCR product (using LTI Pfx Taq, primers OLFS274 plus OLFS345, and YJH40.4 genomic DNA) digested with PvuI to liberate CTF18-9myc from the coamplified TRP1 marker. Taq polymerase modified the ends, and the product was TA cloned into pCR2.1. pJH76.1 was constructed by cloning the CTF18-9myc allele on an XhoI-HindIII fragment from pJH73.2 into the same sites in pRS316GU (74).

Flow cytometry. A 1-ml volume of cells grown in rYPD was harvested and fixed in 500 µl of 0.2 M Tris (pH 7.5)-70% ethanol. After being washed in 1 ml of 0.2 M Tris (pH 7.5), samples were resuspended in 1 ml of 0.2 M Tris (pH 7.5) for >30 min, incubated in 100 µl of 0.2 M Tris (pH 7.5)-3 mg of RNase A per ml for 2.5 h at 37°C, washed with 1 ml of 0.2 M Tris (pH 7.5), and resuspended in 100 µl of 0.05% trypsin (Gibco 25300-054) at 37°C for 5 min. After a 1-ml wash in 0.2 M Tris (pH 7.5), samples were resuspended in 1 ml of 0.2 M Tris (pH 7.5)-9 µg of propidium iodide per ml.

Sister chromatid cohesion assay. Log-phase cultures were resuspended in SC-HIS medium-40 mM 3-aminotriazole for 40 min to induce green fluorescent protein (GFP)-LacI expression, incubated in YPD-15 µg of nocodazole per ml for 3 h, fixed in 4% paraformaldehyde for 30 min, washed in 1 ml of SK (1 M sorbitol, 50 mM KPO₄, pH 7.5), and resuspended in 50 µl of SK.

Chromosome spreads. Chromosome spreads were prepared on slides essentially as described in references 48 and 68. Samples on cured slides were washed in phosphate-buffered saline (PBS) for 20 min, preincubated in PBS-1% bovine serum albumin (BSA) for 1 h, and incubated in polyclonal rabbit anti-myc antibody (sc-789; Santa Cruz Biotechnology) in PBS-1% BSA (at a 1:250 dilution for CTF18-9myc and a 1:500 dilution for CTF4-9myc) for 2 h. Samples were then washed three times with PBS, incubated for 2 h with fluorescein isothiocyanate (FITC)-conjugated anti-rabbit antibody (Sigma) for 2 h (1:1,000 in PBS-1% BSA), washed three times with PBS, and mounted in Fluorsave (Calbiochem)-2 µg of 4',6-diamidino-2-phenylindole (DAPI) per ml.

Protein analysis. Samples prepared as described previously (49) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10% polyacrylamide) (SDS-PAGE) and transferred to a nitrocellulose membrane. Polyclonal rabbit anti-myc antibody (sc-789) and a polyclonal goat anti-rabbit antibody-horseradish peroxidase (111-035-144; Jackson ImmunoResearch) were used for detection. Protein loading was determined by standardization to -tubulin (polyclonal antibody R43; a gift of D. Koshland). Anti-Orc3p monoclonal antibody (SB3) was a gift of B. Stillman (57). Film scans were obtained with a Hewlett-Packard 4c/T scanner and analyzed using NIH Image 1.61.

Antisera against CTF18. A C-terminal 18-kDa peptide of CTF18 expressed from pRSETA (InVitrogen) in bacterial strain BL21(DE3)/pLysE was affinity purified on a Ni²⁺-containing column (Qiagen), solubilized in 8 M urea, and used to immunize two New Zealand rabbits (Hazelton, Inc.). Antibodies from one (rabbit 10C) were purified against ctf18 protein extract bound to cyanogen bromide-activated Sepharose beads. The Western blot signal from purified bacterial peptide in serial dilution indicated that the 10C antibody was capable of detecting at least 2 ng of Ctf18p in the experiment in Fig. 6.

Yeast two-hybrid. pJH74 (BD-CTF18) does not autoactivate reporters in strain AH109 (Clontech). AH109 containing pJH74 was transformed with a Saccharomyces cerevisiae cDNA library (gift of S. Elledge) cloned into pACT2-1. A total of 180,000 transformants yielded 162 His⁺-positive strains, of which 65 were also Ade⁺. PCR analysis and plasmid isolation indicated the presence of four distinct genes. Two of these, RFC3 and RFC4, also conferred -galactosidase activity by plate assay (Clontech).

In vitro immunoprecipitations. Portions (1 µg) of circular pJH73.2 (CTF18-9myc), pJH78 (RFC3), or pJH79 (RFC4) were used as templates in a 50-µl linked transcription-translation system (Promega). A 24-µl volume of each product was used per immunoprecipitation in a final volume adjusted to 150 µl with 1× PBS-1% Triton X-100, and the mixtures were incubated for 2 h at 4°C. A 1-µl volume of anti-myc (sc-789) was added for a further 2-h incubation, followed by immune complex precipitation for 1 h using 10 µl of protein A-agarose beads (sc-2001; Santa Cruz). Following three washes (each with 1 ml of PBS-1% Triton X-100), the beads were resuspended in 20 µl of HU buffer (49) and boiled for 5 min. The entire supernatant was analyzed by SDS-PAGE (10% polyacrylamide).

Whole-cell extract immunoprecipitations. A total of 5 × 10⁸ logarithmically growing cells were washed twice with water and resuspended in 1.5 ml of ice-chilled buffer B60 (50 mM HEPES-NaOH [pH 7.3], 0.1% Triton X-100, 20 mM -glycerophosphate, 10% glycerol, 0.5 mM phenylmethylsulfonyl fluoride, 2 µg of leupeptin per ml, and 2 µg of pepstatin per ml 2× Complete [Roche Biochemicals], 60 mM potassium acetate), and 1.5 g of ice-chilled glass beads (400 to 600 µm in diametet was added. The tubes were vortexed eight times for 30 s with 30-s intervals on ice. After 10 min on ice, the lysate was decanted into ice-chilled 15-ml Corex tubes and centrifuged for 20 min at 18,000 × g at 4°C.

Identification of candidate CTF18 orthologues. The protein sequence of budding yeast CTF18 was used as query for a TBLASTN search of the GenBank nonredundant protein database, yielding a significant match to human genomic clone HS321D2. GeneSCAN analysis of the genomic clone predicted three genes, including candidate HsCTF18, as depicted in Fig. 8. Verification between amino acids 356 and 1220 of the predicted protein is provided by sequence from the cDNA clone pJH3 (data not shown). The GenBank accession numbers for Rfc1p homologs and Ctf18p homologs in other species identified through PSI-BLAST are as follows: C. elegans Ctf18p, T23478; C. elegans Rfc1p, T20230; D. melanogaster Ctf18p, AAF51072.1; D. melanogaster Rfc1p, AAB58311.1; S. pombe Ctf18p, CAB62096.1; S. pombe Rfc1p, CAA18875; H. sapiens Rfc1p, NP_002904.1; S. cerevisiae Ctf18p, NP_013795.1; S. cerevisiae Rfc1p, NP_014860.1.

	RESULTS

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The G₂/M delay of ctf4 and ctf18 mutants is dependent on the spindle assembly checkpoint. Previous work has shown that cells lacking CTF18 accumulate a large-budded morphology (53). In agreement, we found that analysis of log-phase cultures of ctf18 cells by flow cytometry revealed a substantial accumulation of cells with G₂ DNA content. Time course analysis in synchronous cultures was performed to address the nature of the delay. After synchronization in G₁, cells were released into rich medium and samples were taken every 10 min for flow cytometry (Fig. 1A). In comparison with the wild-type control, the progression of ctf18 cells was not detectably different for the first 80 min. New G₁ cells appeared in wild-type and ctf18 strains at 90 and 100 min, respectively, indicating that a second cycle occurred with a ~10-min delay in the mutant. Moreover, a large proportion of the ctf18 population remained in the G₂ peak until the end of the experiment (150 min).

View larger version (41K): [in this window] [in a new window]   FIG. 1.   Absence of CTF4 or CTF18 leads to a MAD2-dependent preanaphase delay. (A) Log-phase cultures grown in rYPD were arrested in -factor, released into rYPD, and processed for flow cytometry. The strains were ctf18 (YE105) and CTF18 (YPH499). (B) Log-phase cultures of ctf18/ctf18 (YE77) or wild-type (YPH501) cells were fixed in formaldehyde and stained to visualize the DNA (DAPI) and microtubules (-tubulin indirect immunofluorescence). Class III was defined as cells with a single DNA mass that crossed the neck and a spindle that did not extend beyond the center of either mother or bud. (C) Early-log-phase cells were processed for flow cytometry. Strains were CTF18 (YPH499), mad2 (YPH1238), rad9 (YCB624), ctf18 (YE105), ctf18 mad2 (YJH18.3), ctf18 rad9 (YJH35.1), ctf4 (s65), and ctf4 mad2 (YJH41.1). Similar results were obtained in five independent experiments; representative results are shown. (D) Formaldehyde-fixed log phase cells were stained with DAPI. Large-budded cells with a single nucleus located within the bud neck were scored (6% in wild type, n = 100). Strains were CTF18 (YPH499), ctf18 (YE105), and ctf18 mad2 (YJH18.3).

ctf18 mec1

ctf18 mad2

Sister chromatid cohesion failure in ctf18 and ctf4 mutants. Most inducers of the spindle assembly checkpoint cause preanaphase delay or arrest associated with a short spindle morphology (reviewed in reference 86). However, an appreciable proportion of ctf18 cells contain partially elongated spindles and stretched DNA masses. Interestingly, a MAD2-dependent G₂/M delay with an intermediate spindle morphology had recently been described for ctf7 mutants, which also exhibit a defect in sister chromatid cohesion. One hypothesis (87) is that precocious sister separation results in a loss of tension at the kinetochore-microtubule interface (triggering the spindle assembly checkpoint), allowing an increase in pole separation at metaphase arrest due to altered balance between opposing forces within the spindle. Based on the phenotypic similarity to ctf7, we were encouraged to assay ctf18 and ctf4 mutants for a sister chromatid cohesion defect.

View larger version (22K): [in this window] [in a new window]   FIG. 2.   CTF4 and CTF18 are required for sister chromatid cohesion. (A) Early-log-phase cultures were treated to induce GFP-LacI expression, incubated for 3 h in rYPD containing nocodazole, and fixed in paraformaldehyde. For each strain, 100 cells exhibiting GFP fluorescence were scored within each experiment. The histogram shows the mean and standard deviation for a minimum of three trials. Strains were CTF18 (AFS173), ctf18 (YJH17.2), ctf4 (YJH37), and mad2-1 (AFS387). Note that approximately 5% of ctf4 or ctf18 cells have >1 chromosome III as determined during G1 arrest. (B) Kinetics of sister chromatid separation in ctf18 mutants. Log-phase cultures were arrested in -factor and released into rYPD-nocodazole. After -factor release, both strains sychronously completed S phase at 60 min postrelease as determined by flow cytometry (data not shown). YJH17.2 data were normalized using the -factor arrest time point, to remove the contribution of cells containing >1 copy of chromosome III. A total of 100 informative cells were counted at each time point. (C) A ctf18 strain containing GAL-CTF18 on a plasmid (YJH48) was grown in selective medium containing either galactose or glucose. Log-phase cells were then shifted to rYPD-nocadozole for 4 h and then plated onto SC-URA glucose medium. CFU were counted after 24 h. The experiment was performed twice with similar results (mean values are shown).

CTF18 is required for mitotic condensation of the rDNA array. In budding yeast, the cohesion defect observed in mcd1, pds5, and trf4 mutants is accompanied by defective chromosome condensation (33, 36, 68, 108). To test whether CTF18 protein is required for chromosome condensation, wild-type and ctf18 cells were arrested in nocodazole-containing medium to obtain a uniformly staged culture at the point when condensation is complete. Formaldehyde-fixed cells were subjected to fluorescence in situ hybridization (32) with a digoxigenin-labeled DNA probe against the repetitive rDNA region. Bulk DNA was visualized with propidium iodide, and the rDNA probe was detected with antidigoxigenin and fluorescein isothiocyanate-conjugated secondary antibodies. Wild-type condensation of the rDNA region resulted in a distinct loop structure, as illustrated in the wild-type examples (Fig. 3A). However, 32% of ctf18 nuclei exhibited a decondensed rDNA staining pattern, indicating a condensation defect (Fig. 3). Thus, like the three other cohesion proteins for which mitotic chromosome condensation has been tested, Ctf18p is required for wild-type condensation of the rDNA as well as for sister chromatid cohesion.

View larger version (46K): [in this window] [in a new window]   FIG. 3.   Absence of CTF18 leads to a condensation defect. (A) Nocodazole-arrested CTF18 (YPH499) and ctf18 (YE105) cells were assayed by fluorescence in situ hybridization using an rDNA probe. rDNA is detected by immunofluorescence (green); chromosomal DNA is stained with propidium iodide (red). A nucleus with decondensed rDNA is indicated by the arrow. (B) A total of 100 nuclei were scored for rDNA condensation status in four categories. Data were collected twice (averages are shown).

Ctf4p and Ctf18p are stable proteins that associate with chromatin throughout the cell cycle. To determine whether the steady-state levels or chromatin associations of Ctf4p or Ctf18p were regulated, epitope-tagged alleles of both genes were generated by integration of a 9myc epitope in frame after the last codon (Fig. 4A). Both epitope-tagged alleles conferred wild-type stability to a test chromosome, a sensitive indicator of protein function (data not shown).

View larger version (35K): [in this window] [in a new window]   FIG. 4.   Ctf18p throughout the cell cycle. (A) -factor arrest-release time course for CTF18-9myc cells (YJH40.4). CTF18-9myc was detected using anti-myc antibody and compared with -tubulin (Tub 2p) on the same blot. (B) (Left) Fluctuation in Ctf18p-9myc accumulation was determined in normalized units by comparison of band intensity relative to -tubulin. (Right) Separate aliquots from the same time course were prepared for flow cytometry and analyzed for DNA content (data not shown) as well as being scored for nuclear morphologies. These analyses indicated the execution of S phase by the majority of cells between 30 and 60 min and the initiation of M phase between 70 and 80 min.

View larger version (31K): [in this window] [in a new window]   FIG. 5.   Ctf4p and Ctf18p are chromatin associated. Cells grown to early log phase in rich medium were arrested in -factor (G1), HU (S), or nocadozole (M) for 2.5 h. (A) Ctf18p-9myc (in YJH40.4) was detected using indirect immunofluorescence on chromosome spreads. For comparison, an untagged control strain (YPH277) in log phase is shown in the top row. Drug-induced arrests were verified by flow cytometry as shown. (B) Ctf4p-9myc (YJH38) cells were similarly analyzed. (C) Chromatin pellet fractions from cells containing Ctf18p-9myc (YJH40.4), Ctf4p-9myc (YJH38), and Orc3p (YJH62.6) were generated from arrested cell populations as above and analyzed by Western blotting. Similar results were obtained for Ctf18p-9myc and Ctf4p-9myc in four independent experiments. Results for Orc3p are consistent with previous data from Liang and Stillman (57). In these experiments, a pellet-to-supernatant cell equivalent loading ratio of 4:1 was used.

CTF18 exhibits genetic interaction with replication mutants. Genetic interaction between CTF18 and a subset of cell cycle genes involved in DNA metabolism as well as other aspects of the chromosome cycle was tested by using a synthetic dosage lethality phenotype. In synthetic dosage lethality, a process may be efficiently disrupted if one interacting factor is present at levels that interfere with complex formation in vivo and the function of another factor is limited by a hypomorphic mutation (25, 54, 55, 115). Since overexpression of CTF18 from a galactose-inducible promotor (in p414GEU1/12) did not cause a growth defect, it could be used in a synthetic dosage lethality screen.

Ctf18p can interact with components of the RF-C complex. To search for proteins with which Ctf18p interacts, we screened a yeast cDNA library using the two-hybrid system. The coding region of CTF18 was cloned in frame downstream of the GAL4 DNA binding domain (GAL4BD-CTF18). This fusion protein complemented the chromosome loss defect of a ctf18 mutant (data not shown). The screen identified two interacting genes whose fusion proteins were capable of conferring expression to three different GAL4-driven reporters: HIS3, ADE2, and the -galactosidase gene (data not shown). Sequence analysis of the library plasmids indicated that they contained either RFC3 or RFC4, each present as full-length fusions.

View larger version (33K): [in this window] [in a new window]   FIG. 6.   Ctf18p interacts with a subset of RF-C components but is not a component of purified RF-C. (a) Unlabeled Ctf18p-9myc was used to pull down [35S]methionine-labeled Rfc3p or Rfc4p. The slowest-migrating bands are approximately the size expected for full-length products; the faster-migrating bands are consistent with the positions of in-frame translational start sites. Similar results were obtained from four independent trials. (B) Immunoprecipitation experiments were performed in yeast whole-cell extracts from strains containing 6HA epitope-tagged alleles of Rfc2p, Rfc3p, Rfc4p, or Rfc5p in the presence and absence of a CTF18-9myc allele. Duplicate SDS-PAGE gels loaded as shown were transferred and probed with anti-myc and anti-HA antibodies. The slowest-migrating species detected by the anti-myc antibody corresponds to Ctf18p-9myc. Species detected by the -HA antibody are consistent with the expected migrations for RF-C proteins as indicated. Note that excess immunoprecipitate was loaded in the rightmost lane, accounting for the increased intensity of the background cross-reactive band. (C) A 100-ng portion of purified RF-C complex was probed for the presence of Ctf18p using affinity-purified antibody 10C. The antibody detected a strong band at 85 kDa in protein from wild-type cells (WT extract, from YPH499) that was absent from ctf18 cells (ctf18 extract, from YE105). Lanes 1 to 3 show the Western blot, and lane 4 is silver stained SDS-PAGE. Starred species are those identified by Fien and Stillman (23) and Cullmann et al. (17).

ctf18 mutant cells contain short telomeres. A screen for telomere length alteration in a collection of mutants exhibiting increased chromosome loss (91) identified a telomere length defect in all three alleles of ctf18 (V. Lundblad and F. Spencer, unpublished data). The screen assayed telomere length following cleavage of genomic DNA with XhoI, which cuts in both subtelomeric Y' elements, to generate a broad 1.2-to 1.5-kb band, as well as in non-Y'-containing termini, to generate bands ranging in size from 2 to >6 kb. Both types of terminal restriction fragments exhibited a moderate reduction in size in the ctf18 null strain, indicating that a loss of CTF18 function influenced telomere length (Fig. 7).

View larger version (57K): [in this window] [in a new window]   FIG. 7.   CTF18 participates in telomere length control. Meiotic segregants from a single tetrad derived from a ctf18/CTF18 heterozygote are shown. The broad band at 1.2 kb [detected by a radiolabeled poly(CA)n/(GT)n probe as in reference 84] contains termini from chromosomes with Y' elements.

CTF18p homologues are detected in fission yeast and higher eukaryotes. Although Ctf18p has homology to RF-C subunits, the similarity is concentrated within an approximately 250-amino-acid region containing RF-C homology boxes II through VIII and falls off sharply outside of this region. This is clearly seen in a pairwise-alignment diagram with Rfc1p from budding yeast, the most homologous RF-C subunit (Fig. 8A). Interestingly, Ctf18p is also homologous to predicted C. elegans, D. melanogaster, S. pombe, and H. sapiens proteins. While these predicted proteins have significant homology over six of the eight RF-C boxes, the similarity to Ctf18p extends outward from the central region, indicating that the predicted proteins are more closely related to Ctf18p than to Rfc1p. This is clearly seen in an alignment between S. cerevisiae Ctf18p and a predicted H. sapiens Ctf18p (Fig. 8B). This observation is supported by cluster analysis (Fig. 8C) of a multiple alignment containing the Ctf18p homologs and Rfc1 proteins from S. cerevisiae, C. elegans, D. melanogaster, S. pombe, and H. sapiens. The Rfc1 proteins have been experimentally defined (71, 98) with the exception of the C. elegans predicted Rfc1p. In cluster analysis, the Ctf18p homologs form a group apart from the Rfc1 proteins. Additional work is required to determine if the candidate Ctf18p homologs are involved in sister chromatid segregation during mitosis.

View larger version (14K): [in this window] [in a new window]   FIG. 8.   Candidate CTF18 orthologues and conserved RF-C boxes. (A and B) Modified output of National Center for Biotechnology Information pairwise BLAST (BLOSUM62) of representative sequences to illustrate the distribution of similarity. Roman numerals indicate RF-C homology boxes, the hatched box represents the ligase homology domain, and black bars mark the relative positions of RF-C boxes. (A) ScCtf18p versus ScRfc1p (e-value = 1e15). (B) ScCtf18p versus HsCtf18p (e-value = 3e35). (C) Proteins from C. elegans, D. melanogaster, S. pombe, and H. sapiens were identified among the top hits from a PSI-BLAST search using S. cerevisiae Ctf18p as the query. Clustal X v1.8 (44) was used to make a multiple alignment of proteins from C. elegans, D. melanogaster, S. pombe, and H. sapiens. Using the multiple alignment, a bootstrapped neighbor-joining tree was produced. The analysis shows that Ctf18p homologs cluster away from Rfc1p homologs.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

In this study, we have identified a role for CTF4 and CTF18 in sister chromatid cohesion. Consistent with a cohesion defect, ctf4 and ctf18 mutants induce the spindle assembly checkpoint. An increased fraction of cellular Ctf4p and Ctf18p appears to associate with chromatin during early S- and early M-phase arrests. ctf18 mutants exhibit a chromosome condensation defect, similar to other cohesion mutants tested to date (33, 36, 108). CTF18 is putative paralog of RFC1, and its encoded protein physically interacts with Rfc2p, Rfc3p, Rfc4p, and Rfc5p. However, it is not a component of the canonical PCNA clamp-loading RF-C complex. We and others have observed that CTF4 and CTF18 interact physically and genetically with many genes that function in DNA replication. These results directly address the hypothesis suggested by recent identification of a requirement for budding yeast DNA polymerase  for the proper establishment of cohesion (108). The requirement for DNA polymerases and accessory factors in cohesion strongly supports the hypothesis that cohesion establishment is a job performed by proteins associated with replication fork complexes.

Checkpoints and cohesion. The MAD2-dependent preanaphase delay observed in both ctf4 and ctf18 mutants is predicted after cohesion failure, because dissociation of a sister chromatid pair should relieve tension on the kinetochore-microtubule junction, thereby activating the spindle assembly checkpoint. As noted previously (87), cohesion failure is not repaired following activation of the spindle assembly checkpoint but, rather, appears to be augmented during mitotic arrest. In agreement, we have observed a marked reduction in the viability of ctf18 cells after a 3-h checkpoint-induced arrest. While the classic model of checkpoint function emphasizes the opportunity allowed for repair of an inducing lesion, in this case a checkpoint-induced delay favors removal of damaged cells from the dividing population.

ctf18 rad9

CTF18 as an RFC1 paralog. Similarity between Ctf18p and Rfc1p has been noted previously (Cullmann et al. 1995), and BLASTP alignment analyses using the entire predicted yeast proteome indicate that these two proteins exhibit the highest degree of homology to one another (e-value = 3e¹⁵ [Fig. 8]). In a two-hybrid screen, we identified strong interactions of Ctf18p with both Rfc3p and Rfc4p. In vitro immunoprecipitation experiments confirmed the ability of Ctf18p to directly interact with in vitro-generated Rfc3p and Rfc4p. Furthermore, in vivo coimmunoprecipitation studies indicate that Ctf18p interacts with Rfc2p, Rfc3p, Rfc4, and Rfc5p. Independent studies have also identified a complex which contains Ctf18p, Rfc2p, Rfc3, Rfc4p, and Rfc5p protein (M. Mayer and P. Hieter, personal communication). Recent studies indicate that RF-C homology box IV, a highly conserved -sheet found in both Rfc1p and Ctf18p, may be required to mediate interactions between Rfc1p and PCNA (1). In addition, the region between RF-C homology boxes IV and VIII has been implicated in RF-C^RFC1 subunit interactions (5). However, Ctf18p is not detected in a purified, biochemically active RF-C fraction. Together, these observations support the concept of a novel CTF18-containing RF-C like complex (RF-C^CTF18) distinct from PCNA-loading RF-C^RFC1.

Roles of CTF4 and CTF18 in association with DNA replication proteins. The synthetic dosage lethality results we report here, as well as the physical association of CTF18p with RFC3p and RFC4p, add to the growing list of genetic and physical interactions involving CTF4, CTF18, and components of the replication complex (Fig. 9). These interactions strongly suggest that the molecular functions of CTF4 and CTF18 are closely related to DNA synthesis. They also support a model in which sister chromatid cohesion defects may be among the consequences of mutations in other proteins that function at DNA replication forks. Although previous studies have suggested that Ctf4p and Ctf18p each play roles associated with DNA metabolism, these roles have not been defined. Intriguingly, CTF4 was isolated as a DNA polymerase  (Cdc17p) binding protein, and genetically interacts with with CDC17, RAD27, DNA2, and RFC1 (24). ctf4 mutants exhibited general properties consistent with DNA synthesis defects, including an elevated rate of mitotic recombination and an accumulation of preanaphase cells in log phase (51, 69). However, previous work indicated that the ctf4-induced preanaphase delay was not RAD9 or MEC1 dependent (69), and therefore it was unlikely that compromised DNA replication caused the observed preanaphase delay. The presence of a cohesion defect offers an explanation for the preanaphase delay and suggests a new direction for elucidation of the molecular role of Ctf4p.

View larger version (49K): [in this window] [in a new window]   FIG. 9.   Physical and genetic interactions among CTF4, CTF18, CTF7, and DNA synthesis proteins. The bold circles denote replication-associated proteins affecting sister chromatid cohesion. Physical associations are indicated by symbol overlap, synthetic lethality is indicated by solid lines with double arrows, and synthetic dosage lethality is indicated by dashed lines with single arrows. The interactions are described in this work [1] and references 87 [2], 112 [3], 24 [4], 69 [5], 11 [6], 56 [7], 4 [8], 28 [9], 67 [10], 114 [11], and 27 [12]. The physical interactions among RF-C subunits are described in references 17, 23, 75, and 104.

CTF18 and telomere structure. The short telomere length displayed by ctf18 null mutants, as well as the previously isolated ctf18 alleles, demonstrates that CTF18 is required for normal telomere structure. Since a ctf18 null mutant does not exhibit a telomere replication defect comparable to that of a telomerase null mutant, it is unlikely that this is due to an absence of telomerase. However, proteins important for full telomere replication may play structural rather than enzymatic roles. For example, the Mre11-Xrs2-Rad50 complex, which has been proposed to play a structural role in DNA repair of sister chromatids, may facilitate telomere replication by presenting chromosome ends to telomerase for replication (76). Ctf18p may also make a structural contribution to telomere length maintenance. Specifically, we suggest that loss of cohesion at chromosome ends in a ctf18 mutant decreases the efficiency of complete telomere replication. This hypothesis is attractive in light of the proposal that telomerase functions as a dimer at chromosome termini (79).

Cohesion and replication. A temporal linkage between DNA synthesis and sister chromatid cohesion is suggested by the tight association between sister chromatids observed from the time of their generation until anaphase (32). Furthermore, the specificity of sister association must be derived from something other than sequence similarity because the efficiency of pairing between homologous chromosomes in a diploid does not indicate a comparable intimate physical relationship (12, 13). The establishment of sister chromatid cohesion as a part of the chromosomal duplication process provides an attractive model that satisfies the specificity requirement, as well as the time of appearance, of the association between sisters.