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Molecular and Cellular Biology, May 2001, p. 3144-3158, Vol. 21, No. 9
McKusick-Nathans Institute of Genetic
Medicine, Johns Hopkins University School of Medicine, Baltimore,
Maryland1; Molecular Sciences Institute,
Berkeley, California2; and Department of
Molecular and Human Genetics, Baylor College of Medicine, Houston,
Texas3
Received 4 December 2000/Returned for modification 17 January
2001/Accepted 8 February 2001
CTF4 and CTF18 are required for
high-fidelity chromosome segregation. Both exhibit genetic and physical
ties to replication fork constituents. We find that absence of either
CTF4 or CTF18 causes sister chromatid cohesion
failure and leads to a preanaphase accumulation of cells that depends
on the spindle assembly checkpoint. The physical and genetic
interactions between CTF4, CTF18, and core components of
replication fork complexes observed in this study and others suggest
that both gene products act in association with the replication fork to
facilitate sister chromatid cohesion. We find that Ctf18p, an
RFC1-like protein, directly interacts with Rfc2p, Rfc3p,
Rfc4p, and Rfc5p. However, Ctf18p is not a component of biochemically
purified proliferating cell nuclear antigen loading RF-C, suggesting
the presence of a discrete complex containing Ctf18p, Rfc2p, Rfc3p,
Rfc4p, and Rfc5p. Recent identification and characterization of the
budding yeast polymerase 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
(APCCDC20), 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 TRF4 and its paralog TRF5 are both members of the
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 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.
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.
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.9.3144-3158.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Saccharomyces cerevisiae CTF18 and CTF4
Are Required for Sister Chromatid Cohesion
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
, encoded by TRF4, strongly
supports a hypothesis that the DNA replication machinery is required
for proper sister chromatid cohesion. Analogous to the polymerase
switching role of the bacterial and human RF-C complexes, we propose
that budding yeast RF-CCTF18 may be involved in a
polymerase switch event that facilities sister chromatid cohesion. The
requirement for CTF4 and CTF18 in robust
cohesion identifies novel roles for replication accessory proteins in
this process.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
, 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).
-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 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).
MATERIALS AND METHODS
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
Yeast strains used in this study
allele was transferred from YPH1238 using primers
OLFS365 plus OLFS366; detection was performed using PCR primers OLFS367 plus OLFS368. Epitope tagging of CTF18, CTF4, RFC1, RFC2, RFC3, RFC4, and RFC5 was performed as described previously
(49) using primer pairs OLFS359 plus OLFS360, OLFS373 plus
OLFS374, OLFS491 plus OLFS492, OLFS510 plus OLFS511, OLFS506 plus
OLFS507, OLFS508 plus OLFS509, and OLFS512 plus OLFS513, respectively;
detection of integration used primer pairs OLFS344 plus OLFS345,
OLFS371 plus OLFS372, OLFS493 plus OLFS494, OLFS514 plus OLFS515,
OLFS437 plus OLFS438, OLFS439 plus OLFS440, and OLFS516 plus OLFS517.
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).
pJH78 was constructed using primers OLFS437 plus OLFS438 and genomic DNA from YJH40.4; the product was cloned into pCR2.1. pJH79 was constructed in similar fashion using primers OLFS439 plus OLFS440. p414GEU1/12 was created by cloning a 2,236-bp SalI-EcoRI PCR fragment containing CTF18 into SalI- and EcoRI-cut p414GEU1 (54), placing the CTF18 ORF in frame with the vector ATG and double E1 epitope tag. The resulting plasmid conferred wild-type chromosome stability and growth at low temperature to a ctf18 strain in galactose-dependent fashion.
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.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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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).
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and wild-type controls revealed a dramatic increase
in the number of cells with an intermediate-length spindle (Fig. 1B,
class III), representing 20% of mutant cells versus 1% of wild-type
cells. The abundant class III morphology indicates the presence of many
cells with altered progression through metaphase or anaphase. We
conclude that an early mitotic delay underlies the G2
accumulation observed by flow cytometry.
Early mitotic delay is often due to activation of either the DNA damage
checkpoint or the spindle assembly checkpoint (reviewed in references
86 and 111). To investigate further, we constructed rad9 and mad2 mutants in a
ctf18 background to remove the DNA damage and spindle
assembly checkpoints, respectively. DNA content analysis of log-phase
cultures grown at 30°C was used to monitor the cell cycle
distributions in these double mutants (Fig. 1C). The results indicated
that the preanaphase delay in the ctf18 mutant was
dependent on the spindle assembly checkpoint and not on the DNA damage
checkpoint. Similar analysis of ctf18 mec1 double
mutants, in an sml1-1 mutant background which renders
mec1 mutants viable (119), also indicated
that the DNA damage checkpoint was not responsible for the
ctf18 G2 peak accumulation (data not shown).
Morphological analysis also indicated a MAD2-dependent
mitotic delay. A nuclear morphology in which the DAPI-stained
chromosomal mass crossed the neck between mother and bud was present in
higher levels in ctf18 cells than in wild-type cells
(Fig. 1D). The frequency of this class of cells was reduced to
wild-type levels in the ctf18 mad2 strain. Thus, in
cultures grown at 30°C, the absence of ctf18 protein leads
to activation of the spindle assembly checkpoint.
This conclusion suggested that a similar phenomenon could explain a
non-RAD9-dependent delay observed in ctf4 mutant
cells (69). Cells lacking CTF18 cannot survive
in the absence of CTF4, consistent with the idea that these
gene products separately contribute to the same essential function. To
determine whether ctf4 mutant delay was dependent on the
spindle assembly checkpoint, ctf4 mad2 double mutants
were created and analyzed by flow cytometry. Again, the accumulation of
ctf4 cells of G2 DNA content was dependent on
MAD2 (Fig. 1C).
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.
and ctf18 cultures were
arrested in nocodazole for 3 h, fixed, and scored for the number
of GFP spots per cell. Parallel analysis of wild-type and
mad2-1 strains served as positive and negative controls for
cohesion. Compared to wild-type cells, ctf4 and
ctf18 mutants exhibited a high level of precociously separated sister chromatids (Fig. 2A).
The number of cells with two GFP spots was comparable to that seen in
mad2-1 cells, which cannot respond to the loss of spindle
integrity and inappropriately proceed to anaphase (92). A
time course experiment in which the number of GFP spots was observed
through a synchronous cycle indicated that the number of cells
containing separated sisters exhibited a steady accumulation over time
(Fig. 2B). Wild-type and ctf18 strains were arrested with
-factor for 2.5 h, released into nocodazole-containing medium,
and sampled every 10 min. The number of cell bodies containing two GFP
signals was small for both wild-type and mutant strains early in the
cycle, and increased as the cells traversed S phase and entered
nocodazole arrest. The gradual, and early, appearance of cohesion
failure suggests that mutant cells perform faulty cohesion
establishment that results in slow decay of sister association.
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cells bearing a galactose-inducible copy of
CTF18 were grown in repressing (glucose) or inducing
(galactose) medium to early log phase and subjected to a 3-h nocodazole
arrest. The cells were then spotted onto solid medium without
nocodazole, and the ability to form microcolonies was scored the
following day (Fig. 2C). Approximately 90% of ctf18
cells not expressing CTF18 failed to recover. This result is
predicted by the data in Fig. 2A. If the GFP-marked chromosome
separates with kinetics representative of other chromosomes, the
appearance of ~30% of cells with two GFP spots predicts that ~5 of
the 16 sister chromatid pairs in an average cell will have
disassociated. Because dissociated sister chromatids are expected to
segregate randomly, the mitotic division following drug removal is
expected to result in very high inviability.
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.
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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).
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-factor, released into pheromone-free medium, and sampled every 10 min for 1.5 cell cycles. In
a comparison with a -tubulin standard, Ctf18p abundance varied over
a three- to fivefold range, reaching a maximum during S phase and
minimum during G2/M (Fig. 4B). These results are consistent with published mRNA levels (90), which show a shallow peak
accumulation of CTF18 transcript near the G1/S
boundary, and with the presence of two degenerate MluI cell
cycle (MCB) boxes in the promoter region of CTF18
(53). Arrest release experiments did not reveal significant fluctuations in CTF4 protein levels in the cell
cycle (data not shown).
Previous studies have shown that association between cohesion proteins
and chromatin in budding yeast can be visualized by indirect
immunofluorescence on spread chromosomes (48, 68). We
detected both Ctf4p and Ctf18p association with chromatin in cells
arrested in G1, S, or M phase (Fig. 5A and
B) in this assay. Cells expressing
epitope-tagged alleles were spheroplasted, lysed on glass slides in the
presence of detergent, and washed to remove loosely adherent cellular
contents. The presence and location of Ctf18p-9myc or Ctf4p9-myc
proteins was compared with the location of DAPI-stained chromatin.
Visible signals were observed in all samples of similar exposure,
suggesting that some Ctf4p and Ctf18p is chromosome associated at each
of these arrest points.
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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.
A subset of cell cycle mutants were chosen that included genes involved in DNA metabolism as well as other aspects of the chromosome cycle. Each mutant was transformed under noninducing conditions with either a GAL-CTF18-expressing minichromosome (p414GEU1/12) or with empty vector (p414GEU1). After induction, growth of the transformants with or without overexpression of CTF18 was compared (Table 2). A temperature series ranging from 25 to 37°C was used to detect changes in maximum permissive growth temperature. A synthetic dosage lethal interaction was found with cdc2-1, cdc7-4, cdc17-1, and cdc46-1, each of which functions in an aspect of DNA synthesis. CDC2 and CDC17 encode catalytic subunits of polymerase and
polymerase , respectively (43). CDC7 encodes
a protein-kinase whose activity controls replication initiation,
probably through modification of targets in the prereplication complex
(64). CDC46 encodes an essential protein of the
prereplication complex, components of which are proposed to remain
associated with the replicative fork (2). These genetic
interactions extend the list of interactions already identified through
traditional synthetic lethality (24, 87) and support the
idea that Ctf18p interacts with replication fork proteins in vivo.
|
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.
|
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).
|
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.
|
| |
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.
Interestingly, Kouprina et al. (53) noted a RAD9-dependent arrest in a ctf18 null mutant at 11°C. In contrast, at the normal growth temperature (30°C) we detected no appreciable RAD9 or MEC1 dependence of the preanaphase delay in ctf18 cells; therefore we
propose that the 11°C observation reveals an additional physiological consequence. Perhaps at low temperature ctf18 cells
accumulate defects that are detected by the DNA damage checkpoint.
Paradoxically, Kouprina et al. (53) found that
ctf18 rad9 cells exhibit increased (rather than
decreased) viability at the nonpermissive temperature. We propose an
explanation based on the observed sister chromatid cohesion defect. A
delay induced by the RAD9-dependent checkpoint will probably
augment the frequency of cohesion failure in ctf18 cells,
similar to a nocadozole-induced preanaphase delay (Fig. 2B). Thus,
removal of the checkpoint may explain the enhanced cell survival.
If checkpoint arrest promotes cell death in cohesion-defective cells,
why do viable cohesion mutants exhibit aneuploidy? One potential
explanation is that adaptation of the spindle assembly checkpoint
allows at least some cells with separated chromosomes to progress
through mitosis. Cohesion-defective mutants are frequently isolated in
chromosome loss screens. Within a collection of viable chromosome loss
mutants encompassing approximately 60 genes (91), alleles
of six genes required for cohesion (CTF4, CTF7, CTF18, SMC1,
MCD1, and SCC2) have been identified to date. This
observation suggests that cohesion defects may underlie a significant
proportion of naturally occurring chromosome instability phenotypes.
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 = 3e
15 [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-CRFC1 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-CCTF18) distinct
from PCNA-loading RF-CRFC1.
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.
|
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.
Indirect evidence for a relationship between DNA synthesis and sister chromatid cohesion is found in several recent studies, indicating that known cohesion proteins possess S-phase roles. S. cerevisiae Mcd1p, Ctf7p, Scc2p, and Scc4p must all be functional during S phase (16, 87, 101, 102), as must fission yeast Mis4p, an Scc2p homologue (26). In addition, S. pombe Eso1p includes extensive homology to both Ctf7p and polymerase
(96) in distinct domains. It has been recently proposed
that this type of homology configuration, suggestive of an evolutionary
gene fusion or splitting event, may indicate that where the two
polypeptides are encoded separately, they nonetheless function together
in the cell (21, 63). This view suggests that Ctf7p in
S. cerevisiae acts together with a DNA polymerase activity.
Finally, the novel polymerase protein family in budding yeast not
only has a demonstrated requirement in replication of the genome but
also plays a poorly understood role in establishing and/or maintaining
robust cohesion between sisters (108). The independent
observation that both CTF4 and CTF18 possess
functions important for sister chromatid cohesion strongly supports the
concept that replication forks directly mediate molecular events
important for sister chromatid cohesion.
The proposed roles for CTF4 and CTF18 proteins in
coupling S-phase DNA replication and cohesion do not preclude potential activities that are also important for cohesion maintenance. Several observations are consistent with roles for each of these proteins in
cohesion establishment, such as genetic and physical interaction with
replication proteins, the early defect in sister association observed
for ctf18 cells, and the augmented chromatin association of both Ctf4p and Ctf18p during early S phase suggested by cell fractionation. However, a detailed analysis of rapid-response conditional alleles is required to evaluate functional execution points
for these genes. Whether replication-associated proteins will
contribute solely to the establishment of robust cohesion or will be
important for later steps in the chromosome cycle that support the
maintenance of cohesion until anaphase remains to be seen. We note that
neither CTF4 nor CTF18 is an essential gene, although cohesion and replication are essential functions. The synthetic lethality observed for double null cells is consistent with
each making a complementary but nonoverlapping molecular contribution
to the essential function they share. Whether this represents spatial,
temporal, or biochemical differentiation awaits further study.
The identification of polymerase , a new class of polymerase with a
distinct function, is consistent with the previously observed division
of labor among other characterized polymerases in budding yeast
(reviewed in references 19 and 43). It has been proposed
that cohesion may be established by polymerase through a mechanism
whereby this specialized replication fork variant is required for
replication through particular chromosomal regions (108;
reviewed in reference 95). In support of this idea, the
distribution of cohesion on chromatin is observed to be nonrandom
(9, 30, 37, 66, 97), with evident areas of enrichment.
Interestingly, recent studies have led to an attractive model for
polymerase switching necessary for the transition from the primase
function of polymerase to the highly processive extension functions
of polymerases and (106, 117, 118). Under this
model, sequential competitions for common binding sites on
single-stranded binding protein and the PCNA sliding clamp support the
notion that the polymerase switch occurs in two steps: displacement of
polymerase by RF-CRFC1 followed by displacement of
RF-CRFC1 by polymerase . It is tempting to speculate
that RF-CCTF18 may promote a switch from the polymerase or complex to polymerase at sites of cohesion, in a role
analogous to that of RF-CRFC1. This switch might
incorporate chromatin association or activation of cohesion accessory
factors such as CTF7 (87, 101) or the SCC2-SCC4 complex
(16). We note that CTF18 is not an essential gene, although cohesion and replication are essential functions. Perhaps in a ctf18 null mutant, Rfc1p partially substitutes
for Ctf18p, similar to the proposed functional substitutions between Trf4p and Trf5p (15, 108).
Taking a broad view, it is not yet clear whether a cohesion role is
specific to polymerase or whether the regulation of sister
chromatid association is a general job of replication forks. In favor
of the general scenario, the tight association between Ctf4p and
polymerase large subunit (69) is consistent with a
cohesion role for polymerase -primase-mediated replication. Interestingly, bovine SMC1 and SMC3 candidate
orthologues encode proteins characterized within the recombination
protein complex RC-1, which also contains DNA polymerase (45,
46). Moreover, S. cerevisiae Mcd1p is required for
wild-type radiation resistance (38), as is its candidate
orthologue Rad21 in S. pombe (7, 8).
The involvement of cohesion subunit proteins in DNA recombination and
in repair suggests that these processes may incorporate cohesion remodeling activities in vivo. Perhaps the absence of a juxtaposed sister chromatid caused by compromised cohesion may lead to increased utilization of a homolog in recombinational DNA damage repair. This
hypothesis may explain increased rates of mitotic recombination in
cohesion-deficient mutants. Although not well understood, the association between cohesion and repair-recombination pathway activities strongly suggests a role for sister chromatid cohesion regulation in many aspects of DNA metabolism.
The requirement for CTF4 and CTF18 in cohesion
identifies a role for these replication accessory proteins in
high-fidelity chromosome segregation. The proposed intimate coupling
between DNA synthesis and the establishment of sister chromatid
cohesion by one or more DNA polymerase complexes provides a mechanism
for the specificity of this tight and essential chromatin association. However, it is not yet clear how many distinct steps or molecular complexes operate in cohesion, which must incorporate sufficient flexibility to support both local and global dynamic chromatin restructuring events, including transcriptional responses, DNA repair,
DNA replication, and mitotic chromosome condensation. It is to be
anticipated that the protein complexes operating within mechanisms
governing the establishment, maintenance, and release of sister
chromatid cohesion are just coming to light.
| |
ACKNOWLEDGMENTS |
|---|
J.H. and E.K. contributed equally to this work.
We thank P. Hieter and M. Mayer for valuable discussion and for sharing unpublished results. We also thank V. Larionov, J. Li, L. Hartwell, A. Straight, A. Murray, S. Gasser, D. Koshland, B. Lavoie, S. Gould, M. Basrai, S. Devine, R. Krishnan, and B. Stillman for sharing reagents and thoughts. We are additionally grateful to R. Skibbens and C. Grieder for comments on the manuscript.
This work was supported by grants from the American Cancer Society and the NIH to F.S.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Johns Hopkins University School of Medicine, 720 Rutland Ave./Ross 850, Baltimore, MD 21205. Phone: (410) 614-2536. Fax: (410) 955-0484. E-mail: fspencer{at}jhmi.edu.
| |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Molecular and Cellular Biology, May 2001, p. 3144-3158, Vol. 21, No. 9
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.9.3144-3158.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Lavoie, B. D., Hogan, E., Koshland, D.
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