The F658G substitution in Saccharomyces cerevisiae cohesin ...

15
European Journal of Cell Biology 87 (2008) 831–844 The F658G substitution in Saccharomyces cerevisiae cohesin Irr1/Scc3 is semi-dominant in the diploid and disturbs mitosis, meiosis and the cell cycle Agata Cena a , Ewa Kozlowska b , Danuta Plochocka a , Marcin Grynberg a , Takao Ishikawa c , Jan Fronk c , Anna Kurlandzka a, a Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawinskiego 5A, 02-106 Warsaw, Poland b Department of Immunology, Institute of Zoology, Faculty of Biology, Warsaw University, Warsaw, Poland c Institute of Biochemistry, Faculty of Biology, Warsaw University, Warsaw, Poland Received 27 June 2007; received in revised form 29 April 2008; accepted 2 May 2008 Abstract The sister chromatid cohesion complex of Saccharomyces cerevisiae includes chromosomal ATPases Smc1p and Smc3p, the kleisin Mcd1p/Scc1p, and Irr1p/Scc3p, the least studied component. We have created an irr1-1 mutation (F658G substitution) which is lethal in the haploid and semi-dominant in the heterozygous diploid irr1-1/IRR1. The mutated Irr1-1 protein is present in the nucleus, its level is similar to that of wild-type Irr1p/Scc3p and it is able to interact with chromosomes. The irr1-1/IRR1 diploid exhibits mitotic and meiotic chromosome segregation defects, irregularities in mitotic divisions and is severely affected in meiosis. These defects are gene-dosage dependent, and experiments with synchronous cultures suggest that they may result from the malfunctioning of the spindle assembly checkpoint. The partial structure of Irr1p/Scc3p was predicted and the F658G substitution was found to induce marked changes in the general shape of the predicted protein. Nevertheless, the mutant protein retains its ability to interact with Scc1p, another component of the cohesin complex, as shown by coimmunoprecipitation. r 2008 Elsevier GmbH. All rights reserved. Keywords: Saccharomyces cerevisiae; Sister chromatid cohesion; Mitosis; Meiosis Introduction The maintenance of proper ploidy during cell division is crucial for the cell functioning. It requires both an accurate replication of chromosomes and their faithful segregation during mitosis and meiosis. In Saccharo- myces cerevisiae the majority of cell cycle events are similar to the processes in other eukaryotes. The major differences are that in yeast, unlike in higher eukaryotes, DNA replication and spindle assembly are initiated simultaneously and the nuclear envelope remains intact throughout mitosis. It is now generally accepted that the proper physical association of sister chromatids is maintained by the sister chromatid cohesion complex (SCC), and the unperturbed functioning of this complex is critical for genome stability (Hirano, 2000; Nasmyth, 2001, 2005; Uhlmann, 2004). The SCC, conserved in structure and function from yeast to mammals, is called cohesin in S. cerevisiae. It is proposed that cohesins form a ring around sister chromatids (for a review, see Nasmyth and ARTICLE IN PRESS www.elsevier.de/ejcb 0171-9335/$ - see front matter r 2008 Elsevier GmbH. All rights reserved. doi:10.1016/j.ejcb.2008.05.002 Corresponding author. Tel.: +48 2 2592 1318; fax: +48 2 2658 4636. E-mail address: [email protected] (A. Kurlandzka).

Transcript of The F658G substitution in Saccharomyces cerevisiae cohesin ...

Page 1: The F658G substitution in Saccharomyces cerevisiae cohesin ...

ARTICLE IN PRESS

European Journal of Cell Biology 87 (2008) 831–844

0171-9335/$ - se

doi:10.1016/j.ej

�Correspondfax: +482 2658

E-mail addr

www.elsevier.de/ejcb

The F658G substitution in Saccharomyces cerevisiae cohesin Irr1/Scc3 is

semi-dominant in the diploid and disturbs mitosis, meiosis and the cell cycle

Agata Cenaa, Ewa Kozłowskab, Danuta Płochockaa, Marcin Grynberga,Takao Ishikawac, Jan Fronkc, Anna Kurlandzkaa,�

aInstitute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawinskiego 5A, 02-106 Warsaw, PolandbDepartment of Immunology, Institute of Zoology, Faculty of Biology, Warsaw University, Warsaw, PolandcInstitute of Biochemistry, Faculty of Biology, Warsaw University, Warsaw, Poland

Received 27 June 2007; received in revised form 29 April 2008; accepted 2 May 2008

Abstract

The sister chromatid cohesion complex of Saccharomyces cerevisiae includes chromosomal ATPases Smc1p andSmc3p, the kleisin Mcd1p/Scc1p, and Irr1p/Scc3p, the least studied component. We have created an irr1-1 mutation(F658G substitution) which is lethal in the haploid and semi-dominant in the heterozygous diploid irr1-1/IRR1. Themutated Irr1-1 protein is present in the nucleus, its level is similar to that of wild-type Irr1p/Scc3p and it is able tointeract with chromosomes. The irr1-1/IRR1 diploid exhibits mitotic and meiotic chromosome segregation defects,irregularities in mitotic divisions and is severely affected in meiosis. These defects are gene-dosage dependent, andexperiments with synchronous cultures suggest that they may result from the malfunctioning of the spindle assemblycheckpoint. The partial structure of Irr1p/Scc3p was predicted and the F658G substitution was found to inducemarked changes in the general shape of the predicted protein. Nevertheless, the mutant protein retains its ability tointeract with Scc1p, another component of the cohesin complex, as shown by coimmunoprecipitation.r 2008 Elsevier GmbH. All rights reserved.

Keywords: Saccharomyces cerevisiae; Sister chromatid cohesion; Mitosis; Meiosis

Introduction

The maintenance of proper ploidy during cell divisionis crucial for the cell functioning. It requires both anaccurate replication of chromosomes and their faithfulsegregation during mitosis and meiosis. In Saccharo-

myces cerevisiae the majority of cell cycle events aresimilar to the processes in other eukaryotes. The major

e front matter r 2008 Elsevier GmbH. All rights reserved.

cb.2008.05.002

ing author. Tel.: +482 2592 1318;

4636.

ess: [email protected] (A. Kurlandzka).

differences are that in yeast, unlike in higher eukaryotes,DNA replication and spindle assembly are initiatedsimultaneously and the nuclear envelope remains intactthroughout mitosis.

It is now generally accepted that the proper physicalassociation of sister chromatids is maintained by thesister chromatid cohesion complex (SCC), and theunperturbed functioning of this complex is critical forgenome stability (Hirano, 2000; Nasmyth, 2001, 2005;Uhlmann, 2004). The SCC, conserved in structure andfunction from yeast to mammals, is called cohesin in S.

cerevisiae. It is proposed that cohesins form a ringaround sister chromatids (for a review, see Nasmyth and

Page 2: The F658G substitution in Saccharomyces cerevisiae cohesin ...

ARTICLE IN PRESSA. Cena et al. / European Journal of Cell Biology 87 (2008) 831–844832

Haering, 2005). The cohesin complex is composed of aheterodimer of SMC (structural maintenance of chro-mosomes) proteins and of two non-SMC subunits:Mcd1/Scc1 (YDL003W) and Irr1/Scc3 (YIL026C). Allgenes encoding cohesins are essential – their deletioncauses cell death. Mutations in genes encoding theproteins forming cohesin are either lethal or cause severedefects in chromosome segregation (Michaelis et al.,1997; Guacci et al., 1997; Klein et al., 1999; Kurlandzkaet al., 1995; Uhlmann, 2003). The dissolution of thecohesion complex is precisely regulated by mechanismsthat remain only partially known.

In mitosis, the cohesin complex holds sister chroma-tids together and, simultaneously, sister kinetochoresare pulled to opposite cell poles by microtubules of theelongating mitotic spindle. This is probably the basis ofthe tension which signals proper bipolar attachment ofkinetochores. This bipolar attachment is required for theproper assembly and functioning of the proteinsengaged in the spindle assembly checkpoint (SAC).The correct functioning of this checkpoint is indispen-sable for mitosis exit (for reviews, see Lew and Burke,2003; Pinsky and Biggins, 2005). Once all chromatidsare aligned, the inhibitory signal from SAC thatmonitors bi-orientation is relieved. The anaphase-promoting complex (APC) then targets Pds1/securinfor ubiquitin-mediated degradation. Pds1 proteolysisallows Esp1/separin to cleave the Mcd1/Scc1 cohesinsubunit, triggering chromatid separation (Cohen-Fix etal., 1996; Ciosk et al., 1998).

When mitotically dividing yeast cells are induced toenter meiosis, cells exit the mitotic cell cycle at the G1phase (Kupiec et al., 1997; Neiman, 2005). The switchfrom mitosis to meiosis has been thoroughly investi-gated at the transcriptional level (Kassir et al., 2003;Pnueli et al., 2004), and the mechanisms of chromosomesegregation during meiosis are quite well known(Nasmyth, 2005). It has been established that accuratemeiotic segregation requires replacement of the mitoticcohesin subunit Scc1 by the meiosis-specific kleisinRec8. At the first meiotic division (meiosis I), Rec8 iscleaved along chromosome arms but is protected at thecentromeres, where it is only cleaved during the seconddivision (meiosis II) (Lee and Orr-Weaver, 2001;Watanabe, 2005).

The yeast Irr1/Scc3 protein is the least studiedelement of the cohesion complex, although homologuesof this protein have been identified in other eukaryotesfrom fungi to plants and mammals (Wang et al., 2003;Valdeolmillos et al., 2004; Chelysheva et al., 2005; Haufet al., 2005). In mammals, three homologues, calledSTAG1-3 or stromalins, were described, two of thembeing involved in mitotic cohesion and one being specificto meiosis (Carramolino et al., 1997; Prieto et al., 2001).The aminoterminal part of Irr1 (first 496 amino acids),conserved among eukaryotes, is 18–25% identical to

members of the stromal antigen protein family (Tothet al., 1999), whereas the C-terminal part is variable.

The proposed models of S. cerevisiae cohesin do notspecify the position of Irr1p in the complex, although itis assumed that the primary role of Irr1p consists inclosing the cohesin ring, and this protein is usuallydepicted as an element attached to the Mcd1/Scc1kleisin subunit (Haering and Nasmyth, 2003; Nasmyth,2005). Recent data obtained from proteome-widepurification of yeast protein complexes (Krogan et al.,2006) confirmed the previously identified interactionsamong Irr1p, Mcd1p and Smc3p, but also detected newIrr1p interactions with Hta2p (histone H2A subtype)and Yra1p (a nuclear protein required for export ofmRNA from the nucleus).

The role of Irr1p/Scc3p in meiosis is poorly known. Ameiosis-specific variant of Irr1p – STAG3 – has beendescribed in mammalian cells (Pezzi et al., 2000). InCaenorhabditis elegans, the localization of a Rec8homologue to chromosomes has been shown to dependon the presence of an Irr1p homologue – SCC-3 (Wanget al., 2003; Pasierbek et al., 2003). It has also beenshown that Arabidopsis cohesins, AtREC8 (homologueof Rec8p) and AtSCC3 (homologue of Irr1p), arenecessary for the monopolar orientation of kinetochoresat meiosis I and for the maintenance of centromericcohesion at anaphase I (Chelysheva et al., 2005).

Here we present data on an irr1-1 mutation (F658Gsubstitution in the Irr1p/Scc3p cohesin) of S. cerevisiae

which is lethal in the haploid and semi-dominant indiploid yeast cells. In our recent paper (Cena et al.,2007), we described a rather unexpected influence of thepresence of this mutated copy of IRR1 on the cell wallintegrity. Here we show that the heterozygous diploidirr1-1/IRR1 exhibits significant irregularities in mitoticdivisions: chromosome segregation errors, disturbancesin segregation of nuclei and in cytokinesis. Moreover,this diploid is severely affected in meiosis. Our datasuggest that these irregularities could result frommalfunctioning of the spindle assembly checkpoint.These defects exhibit incomplete penetrance, in whichthey resemble phenotypes observed in pre-cancerousmammalian cells. Thus, our yeast irr1-1/IRR1 diploidmay serve as a model to investigate the general aspectsof genome integrity maintenance.

Materials and methods

Strains and media

Yeast strains used in the present study, isogenic withthe strain W303, are listed in Table 1. Escherichia coli

XL1-Blue MRF0 (Stratagene, Saint Quentin en Yve-lines, France) was used for molecular manipulations.

Page 3: The F658G substitution in Saccharomyces cerevisiae cohesin ...

ARTICLE IN PRESS

Table 1. S. cerevisiae strains

Strain Genotype Source

W303 MAT a ade2-1 trp1-1 leu2-3,112 his3-11 ura3-1 can1-100 Rothstein

collection

IRR1/IRR1

(DW303)

MAT a/a ade2-1/ade2-1 trp1-1/trp1-1 leu2-3,112/leu2-3,112 his3-11/his3-11 ura3-1/

ura3-1 can1-100/can1-100

Rothstein

collection

irr1aD/IRR1

(AKD11)

MAT a/a ade2-1/ade2-1 trp1-1/trp1-1 leu2-3,112/leu2-3,112 his3-11/his3-11 ura3-1/

ura3-1 can1-100/can1-100 irr1DHkanMX4/IRR1

This study

irr1-1/IRR1

(AKD45)

MAT a/a ade2-1/ade2-1 trp1-1/trp1-1HTRP1-irr1-1 leu2-3,112/leu2-3,112 his3-11/his3-

11 ura3-1/ura3-1 can1-100/can1-100 irr1DHkanMX4/IRR1

This study

ACD2 MAT a/a ade2-1/ade2-1 trp1-1/trp1-1HTRP1-irr1-1-Myc13 leu2-3,112/leu2-3,112 his3-

11/his3-11 ura3-1/ura3-1 can1-100/can1-100 irr1DHkanMX4 /IRR1-HA3HHIS3

This study

458 MAT a ade2-1 trp1-1 leu2-3,112 his3-11,15 ura3-1 can1-100 SCC3-HA3HHIS3 F. Uhlmann

615 MAT a ade2-1 trp1-1 leu2-3,112 his3-11,15 ura3 can1-100 SCC3-Myc18HTRP1 SCC1-

HA6HHIS3

F. Uhlmann

K7518 MAT a scc3-1 ade2-101 his3D200 leu2D1lys2-801 trp1-1 CAN+ ura3H3�URA3 tet0112

leu2HLEU2tetR-GFP

F. Uhlmann

ACD5 MAT a/a ade2-1/ade2-1 trp1-1/trp1-1HTRP1-irr1-1 leu2-3,112/leu2-3,112 his3-11/his3-

11 ura3-1/ura3-1 can1-100/can1-100 irr1DHkanMX4/IRR1 ura3H3�URA3tet0112/

ura3H3�URA3tet0112 leu2HLEU2tetR-GFP/leu2HLEU2tetR-GFP

This study

ACD7 MAT a/a ade2-1/ade2-1 trp1-1/trp1-1HTRP1-irr1-1 leu2-3,112/leu2-3,112 his3-11/his3-

11 ura3-1/ura3-1 can1-100/can1-100 irr1DHkanMX4/IRR1 ura3H3�URA3tet0112/

ura3H3�URA3tet0112 leu2HLEU2tetR-GFP/leu2HLEU2tetR-GFP

This study

ACD8 MAT a/a ade2-1/ade2-1 trp1-1/trp1-1 leu2-3,112/leu2-3,112 leu2HLEU2tetR-GFP his3-

11/his3-11 ura3-1/ura3-1 can1-100/can1-100 IRR1/IRR1 ura3H3�URA3tet0112/ura3

This study

MW9C/9D MAT a/a ade2-1/ade2-1 his3-11/his3-11 trp1-1/trp1-1 IRR1/IRR1 ura3H3�URA3/

ura3H3�URA3 tet0112/ura3H3�URA3tet0112 leu2HLEU tetR-GFP/

leu2HLEU2tetR-GFP

Wysocka et al.

(2004)

A. Cena et al. / European Journal of Cell Biology 87 (2008) 831–844 833

Yeast culture media were prepared as described (Rose etal., 1990). YPD contained 1% Bacto-yeast extract, 2%Bacto-peptone and 2% (all w/v) glucose. SD contained0.67% yeast nitrogen base without amino acids (Difco,Detroit, MI, USA) and 2% glucose. For auxotrophicstrains, the media contained appropriate supplements.Standard methods were used to genetically manipulateyeast cells (Rose et al., 1990). Routinely, wheneverpossible strains were backcrossed at least twice. Fordrop tests, cells were grown overnight in YPD orminimal media and adjusted to a density OD600 ¼ 1.Growth was analyzed by plating 5-ml drops of 10-foldserial dilutions of cell suspensions onto solid media.Reagents were tested in concentrations recommended byRieger et al. (1999).

Plasmid construction, gene tagging, site-directed

mutagenesis

Plasmids used in this study are listed in Table 2. Toconstruct the IRR1-Myc13 fusion (pAC1) the 30-terminal fragment of IRR1 was PCR-amplified usingprimers: F1: 50-GGAGAGTCTTTTCGCTAAGC-30

and R1: 50-ATCTTGTGTGATTTCGTCGC-30. TheMyc13-TADH1 fragment was amplified using pFA6a-

13Myc-kanMX6 as a template (Longtine et al., 1998)and primers F2: 50-GCGACGAAATCACACAA-GATTTAATTAACGGTGAACAAAAGC-30 (the se-quence encoding a fragment of IRR1 is underlined) andR2: 50-GTCCCGGGATATTACCCTGTTATCC-30

complementary to TADH1 and introducing an XmaIrestriction site (CCCGGG). Products of both PCRswere combined and served as a hybrid template fromwhich a third PCR product was obtained using primersF1 and R2. This product was introduced into pAK11/3.Site-directed mutagenesis was carried out using theAltered Sites in vitro mutagenesis system (Promega).Details of the F658G replacement have been describedbefore (Cena et al., 2007). The plasmid pAC1/2, bearingthe irr1-1 allele 30-fused with 13 repetitions of Myc, wasa derivative of pAC1.

Western blotting and co-immunoprecipitation

To visualize chimeric HA- or c-Myc-tagged proteinson Western blots, protein samples (100 mg/lane) weresubjected to 8% SDS–PAGE. Electrophoresis wasfollowed by blotting onto Hybond-C extra membraneand probing with an anti-HA monoclonal antibody(HA.11, clone 16B12) (BabCO) or anti-c-Myc 9E10

Page 4: The F658G substitution in Saccharomyces cerevisiae cohesin ...

ARTICLE IN PRESS

Table 2. Plasmids used in this study

Plasmid Relevant plasmid genotype Reference

pAK11/1 PIRR1-IRR1-TIRR1 URA3 CEN This study, based on pRS316

pAK11/3 PIRR1-IRR1-TIRR1 TRP1 CEN This study, based on pRS314

pAK11/3F-G PIRR1-irr1-1-TIRR1 TRP1 CEN This study, as above

pAK11/17 PIRR1-irr1-1-TIRR1 TRP1 INT This study, derivative of pRS305

pAC1 PIRR1-IRR1-Myc13-TADH1 TRP1 CEN This study

pAC1/2 PIRR1-irr1-1-Myc13-TADH1 TRP1 CEN This study

pAC1/3 PIRR1-irr1-1-Myc13-TADH1 TRP1 INT This study

A. Cena et al. / European Journal of Cell Biology 87 (2008) 831–844834

(BabCO). For co-immunoprecipitation, cells expressingchimeric Irr1p-Myc and Scc1p-HA or Irr1-1p-Myc andScc1p-HA proteins were harvested at an OD600 ¼ 1 andresuspended in 50mM sodium phosphate buffer (pH 7.5),50mMNaCl, 2mMMgCl2, 0.2% Triton X-100, contain-ing protease inhibitor cocktail Complete (Roche) and25U/ml Benzonase (Merck). Cells were homogenized withglass beads in a bead-beater, the homogenate was spundown in a microfuge. The whole procedure was carriedout on ice or at 4 1C. Soluble protein extracts wereincubated with Protein G-agarose (Sigma) covered withanti-HA (Covance, monoclonal antibody HA.11 clone16B12) or anti-Myc (Covance, monoclonal antibodyclone 9E10) overnight at 4 1C. The immunoprecipitatewas analyzed by SDS–PAGE, blotted and probed withanti-HA and anti-Myc antibodies, as above.

Microscopy and chromosome spreads

To localize nuclei, a Nikon Eclipse E800 fluorescencemicroscope with a 63� objective was used. DAPI (40,60-diamino-2-phenylindole dichloride, Sigma) was used tostain DNA. To visualize c-Myc-tagged Irr1-1p, an anti-Myc antibody from Invitrogen was used. Chromosomesegregation was monitored by following segregation ofchromosome V carrying tandem repeats of the Tetoperator integrated in the centromeric region whichcould be visualized by GFP because the cells expressedalso the tet-GFP fusion, as described by Michaelis et al.(1997). For quantitative assays, cells were grown inYPD medium to log phase, stained with DAPI, andexamined by fluorescence microscopy. Over 400 cellswere counted for each strain. Experiments were done induplicate, and the percentage of cells representingindicated morphology classes was determined. Chromo-some spreads were prepared as described (Klein et al.,1992; Michaelis et al., 1997), DNA was stained withDAPI.

Cell synchronization

For nocodazole (NZ, metaphase arrest) treatmentexperiments, diploid cells were grown in YPD complete

medium to OD600 ¼ 1 (2� 107 cells/ml), correspondingto the exponential part of the growth curve (data notshown). Subsequently, cells were incubated in YPDmedium with 15 mg/ml NZ at 30 1C for 3 or 5 h. Afterincubation, cells were washed with water twice andtransferred into fresh YPD to allow the cell cycle toresume. Aliquots were withdrawn at 30-min intervalsand transferred into meiosis-inducing medium (1%potassium acetate) or prepared for FACS analysis. Forthe 0-h time point the cells, after washing off thesynchronizing agent, were directly suspended in meiosis-inducing medium or prepared for FACS. Meiosis wasassayed after 3 days of incubation by observation in alight microscope.

FACS analysis

Flow cytometric DNA quantification was performedaccording to Mark Winey’s lab on-line protocols (http://mcdb.colorado.edu/labs/winey/protocols.html). Cellswere fixed in 70% ethanol and kept at �20 1C untiluse. For staining, the cells were incubated overnight in50 mg/ml propidium iodide in the dark at 4 1C andsonicated for 15 s before the measurement.

Results

The F658G substitution does not affect Irr1p level,

localization or ability to associate with chromosomes

Our main purpose was a systematic investigation ofthe role of various parts of the Irr1p/Scc3p cohesin,since functional domains of this protein (or its homo-logues) were not known. To achieve this we introducedamino acid substitutions in parts of the Irr1p moleculewhich were deemed, based on homology studies, to beimportant as potential functional or structural sites. Asit was mentioned in our previous paper (Cena et al.,2007), the introduction of an allele encoding the F658Gsubstitution, named irr1-1, into the irr1D/IRR1 diploidcaused incorrect morphology in mitosis and meiosis,indicating a role of the mutated residue in Irr1p

Page 5: The F658G substitution in Saccharomyces cerevisiae cohesin ...

ARTICLE IN PRESS

Fig. 1. Wild-type Irr1/Scc3 and mutated Irr1-1 protein are

synthesized and properly localized in the heterozygous irr1-1/

IRR1 diploid. Haploid strains expressing wild-type Scc3/Irr1-

HA3 or Scc3/Irr1-Myc18 protein were obtained from F.

Uhlmann. Myc13-tagged Irr1-1p was constructed as described

in Materials and methods. (A) Immunoblots of whole-cell

extracts were probed with anti-Myc and anti-HA antibodies.

Lane 1: Haploid strain expressing Scc3/Irr1-3HA, lane 2:

haploid strain expressing Scc3/Irr1-18Myc, lane 3: hetero-

zygous diploid expressing Irr1-1-Myc13 and Irr1-3HA. (B)

The mutated Irr1-1 protein is present in the nucleus. The

irr1D/IRR1 hemizygote was transformed with a plasmid

bearing the irr1-1-Myc13 gene, leading to its integration into

the TRP1 locus. Localization of the Myc13-tagged Irr1-1

protein was followed by indirect immunofluorescence; cells

were probed with anti-Myc primary antibodies and anti-mouse

Cy3-conjugated secondary antibodies. Nuclei were stained

with DAPI. (C) Irr1-1p localizes to chromosomes. Chromo-

some spreads from the heterozygous strain expressing Irr1-1-

Myc13p protein were analyzed by indirect immunofluores-

cence as detailed above.

A. Cena et al. / European Journal of Cell Biology 87 (2008) 831–844 835

functioning, and numerous phenotypes suggestingchanges in the cell wall integrity. The irr1-1 mutationwas lethal in the haploid state. To further assay the roleof the F658G substitution and to facilitate furtherexperiments, the irr1-1 gene was integrated into theTRP1 locus (chromosome IV, whereas IRR1 is onchromosome IX) of the irr1D/IRR1 hemizygote givingthe strain irr1-1/IRR1 (Cena et al., 2007).

Since the phenotype of irr1-1/IRR1 was different fromthat of the irr1DHkanMX4/IRR1 hemizygote, which infact did not differ from the wild type, except for sporegermination (Cena et al., 2007), we expected that themutated irr1-1 gene was expressed in the cell. To checkwhether Irr1-1p was indeed present, and also whether itaffected the level of the original Irr1 protein, weconstructed a diploid bearing the wild-type genomic copyof IRR1 30-terminally fused with a triple HA epitope-encoding sequence, and the irr1-1 allele 30-terminally fusedwith Myc13 (both under the native IRR1 promoter) andintegrated into the TRP1 locus. Due to the various lengthsof the epitopes, we could distinguish the wild-type and themutated protein and we found that both proteins wereindeed present in the irr1-1/IRR1 heterozygote and hadthe expected sizes (Fig. 1A). Importantly, the levels of bothproteins were similar to the level of Irr1p in wild-typehaploids (compare lane 3 with lanes 1 and 2). The Myc-tagged Irr1-1 mutant protein was also correctly localizedto the nucleus (Fig. 1B). To check whether Irr1-1p retainedits chromosome-binding properties, we examined chromo-some spreads of cells expressing the Irr1-1-Myc fusion. Asunsynchronized mid-log cultures were used for thespreads, they must have contained cells in all phases ofthe cell cycle. In almost all cases the fluorescence signalscoming from the Myc-fusion protein and from DAPI-stained DNA overlapped (Fig. 1C). This indicated that theIrr1-1 protein retained its ability to interact with chromo-somes. Further in this paper we show in more detail thatIrr1-1p also retained its ability to interact with a specificcomponent of the cohesin complex, Scc1p.

In conclusion, we found that the phenotypic effects ofthe irr1-1 allele in the heterozygous irr1-1/IRR1 diploidwere not due to problems with Irr1-1p synthesis,stability, subcellular distribution or general properties.

The strain with both the wild-type and mutated copiesof Irr1p tagged was constructed only to follow the fateof the proteins in the heterozygous diploid, but in allfurther studies the irr1-1/IRR1 strain with non-taggedversions of the genes was used to exclude possible side-effects of the tags.

The irr1-1 mutation causes irregularities in cell

divisions and affects meiosis

In the next step we characterized the defects caused bythe presence of Irr1-1p. In Fig. 2 we show examples of

irr1-1/IRR1 cells with nuclear segregation defects, inwhich we observed two nuclei in one cell or whichunderwent irregular divisions. Here, binucleate cellsconstituted ca. 15–20% of all cells, whereas in wild-typecontrol the percentage of aberrant cells did not exceed3%. Although the presence of the F658G substitution inIrr1-1p caused aberrations in mitotic divisions of irr1-1/IRR1, it did not affect the strain growth rate on solidmedia (Fig. 3).

Subsequently, we checked how the presence of themutated Irr1-1p cohesin influenced meiosis. We foundthat meiosis of an asynchronous culture of irr1-1/IRR1

was initiated ca. 24 h after transferring to 1% potassiumacetate (the same was observed in the wild-type diploidIRR1/IRR1), and after 48 h ca. 64% of cells sporulated(52% in the control). However, we observed that in irr1-

1/IRR1 asci were occasionally formed from cells thatstill had a bud attached, or asci containing more thanfour spores were present; the aberrant asci representedca. 11% of all. Fig. 4 shows examples of such cells (seealso Table 4). This aberration suggests that the irr1-1/IRR1 cells were entering meiosis before having com-pleted mitotic cell division.

Page 6: The F658G substitution in Saccharomyces cerevisiae cohesin ...

ARTICLE IN PRESS

Fig. 2. The presence of the irr1-1 mutation in the heterozygous

irr1-1/IRR1 diploid causes irregularities in mitotic divisions

and nucleus transmission to the progeny cell. Defects are

observed in ca. 15–20% of all cells. Cells were viewed under

fluorescence microscopy, nuclei were stained with DAPI.

Fig. 3. Strain irr1-1/IRR1 does not show growth defects on

YPD solid medium.

Fig. 4. Meiosis in irr1-1/IRR1 diploids is frequently observed

in cells which have not completed the mitotic cell cycle.

Examples of asci containing six or eight spores. DNA was

stained with DAPI.

A. Cena et al. / European Journal of Cell Biology 87 (2008) 831–844836

In the population of sporulating cells we noticed ascicontaining four, six or eight spores. Eighteen asci weredissected and the viability of spores was low (28germinated). None, one or two or three spores wereviable in asci containing six or eight spores and none orone spore in those containing four spores. The ploidy of

the obtained spore clones was estimated by FACSanalysis and, with one exception, the DNA content percell was equivalent to haploid (not shown). The sporeclones were able to mate exclusively with either a MATaor a MATa wild-type sex tester haploid strain; nonemated with both. DAPI staining of nuclei showed that,with the exception of one clone, the cells had only onenucleus. The spores did not contain the irr1-1 allele asneither of them was Trp+. One spore gave progeny ofcells with two nuclei which were Trp+ (but not G418R),thus they bore both irr1-1 (whose integration restoredwild-type TRP1) and the wild-type copy of IRR1 (as it isabsolutely required for cell viability).

To check whether the mutated Irr1-1p could becompeted out by an excess of wild-type Irr1p weintroduced a wild-type copy of IRR1 on the centromericplasmid pAK11/3 to the irr1-1/IRR1 heterozygote. Wefound that the presence of the extra copy of IRR1

decreased the number of bi-nucleate mitotically dividingcells and the frequency of irregular tetrads to ca. 2–3%,i.e. to the wild-type level. This indicates that theadditional wild-type copy of IRR1 phenotypically‘‘cured’’ the majority of cells, most likely by decreasingthe ratio of Irr1-1p to wild-type Irr1p.

Page 7: The F658G substitution in Saccharomyces cerevisiae cohesin ...

ARTICLE IN PRESS

Table 3. Mutation irr1-1 causes chromosome segregation

errors in mitotically dividing cells

Type of aberration Frequency (%)

IRR1/IRR1 irr1-1/IRR1

0.2 3.0

0 0.9

0 0.2

0 0.5

0 2.2

0 1.0

0 0.5

0 0.5

0 0.2

0 0.2

A. Cena et al. / European Journal of Cell Biology 87 (2008) 831–844 837

The F658G substitution in Irr1-1p causes a

chromosome segregation defect in mitosis and

meiosis

Since the main function ascribed to cohesins is sisterchromatid cohesion, we checked whether the irr1-1mutation caused defects in chromosome segregation.First we investigated mitotically dividing cells. To detectgross changes in cell ploidy we performed a FACSanalysis of the DNA content of irr1-1/IRR1 cells fromunsynchronized mid-log cultures. This analysis revealedmainly cells of 2C and 4C DNA content, similar tonormal IRR1/IRR1 (Fig. 5). To verify the range ofaneuploidies we introduced a GFP-labeled chromosomeV into irr1-1/IRR1. To analyze mitosis, we used strainACD5 with only one copy of chromosome V labeledand the other not, and to follow meiosis, strain ACD7was used, with both chromosome V copies labeled.Tables 3 and 4 summarize the results of microscopicobservations, and in Fig. 6 we show examples ofchromosome missegregation. We found that the pre-sence of Irr1-1p increased chromosome segregationerrors of mitotically dividing cells from 0.2% (controlIRR1/IRR1) to 9.4%. However, the chromosomalsegregation errors were not coupled to defects in nucleisegregation since they were frequently observed in cellsexhibiting regular nuclear divisions (Table 3). Thechromosome segregation errors were also elevated inmeiosis, both in asci of correct and incorrect morphol-ogy (Table 4). Basing on the above observations weconclude that the presence of Irr1-1p decreases chromo-some segregation fidelity in mitosis and meiosis.

Chromosome V segregation (black dots) was followed in IRR1/IRR1

and irr1-1/IRR1 ACD5 strains bearing one copy of chromosome V

labeled with tetR-GFP close to the centromere. Irregular chromosome

segregation was observed in 0.2% of the IRR1/IRR1 cells and in 9.4%

of the irr1-1/IRR1 cells (415 cells were counted for each strain).

The irr1-1 mutation influences the response to

nocodazole and the induction of meiosis

Since meiosis was frequently observed in cells whichhad not completed mitotic cell division, an influence ofthe irr1-1 mutation on the progression of the cell cyclewas implicated. To check whether this defect could becoupled to the malfunctioning of the spindle assembly

Fig. 5. The presence of the irr1-1 mutation in the heterozygous

irr1-1/IRR1 diploid does not cause significant changes in

ploidy. DNA content was estimated in non-synchronized

cultures by FACS.

checkpoint we subjected cells to nocodazole (NZ, amicrotubule-destabilizing agent causing metaphase ar-rest following activation of the spindle assemblycheckpoint). After 3 h of incubation of IRR1/IRR1

and irr1-1/IRR1 with NZ (see Materials and methodsfor details), the drug was washed off and the cultureswere allowed to resume the cell cycle. To follow itscourse, samples were removed every 30min for 2.5 h.Two samples were withdrawn at each time point: onewas subjected to FACS analysis and the other wastransferred into meiosis-inducing medium. Fig. 7Ashows the DNA content estimated by FACS, andmeiosis data are presented in Table 5.

The treatment with NZ stopped IRR1/IRR1 at the 4Cstage, as expected, whereas irr1-1/IRR1 was not arrested

Page 8: The F658G substitution in Saccharomyces cerevisiae cohesin ...

ARTICLE IN PRESS

Table 4. Mutation irr1-1 causes irregularities in meiosis and

chaotic chromosome segregation

Type of asci Frequency (%)

IRR1/IRR1 irr1-1/IRR1

Tetrade (all) 95.5 93.7

0 4.0

0 0.4

Dyade 4.5 3.3

Others 0 3.0

0 0.4

0 0.4

0 0.8

0 0.4

0 0.4

0 1.0

Chromosome V segregation (black dots) was followed in IRR1/IRR1

and irr1-1/IRR1 ACD7 strains bearing two copies of chromosome V

labeled with tetR-GFP close to the centromere. The efficiency of

sporulation was 52% for IRR1/IRR1 cells and 63.7% for irr1-1/IRR1

cells (424 cells were counted for each strain). For asci type analysis, the

number of all sporulating cells was set as 100%. Small ovals in column

1 represent spores, nuclei are not marked.

Fig. 6. The presence of the irr1-1 mutation in the heterozygous

irr1-1/IRR1 diploid causes chromosome missegregation. Ex-

amples of (A) mitotically dividing cells, one chromosome

labeled and (B) irregular asci, two chromosomes labeled. In the

control and mutant strains, the centromeric region of

chromosome V was visualized by GFP (tetR-GFP), DNA

was stained with DAPI.

A. Cena et al. / European Journal of Cell Biology 87 (2008) 831–844838

by this agent since at time 0 cells of various DNAcontents were present. After being released from the NZtreatment, a small fraction of IRR1/IRR1 cells were ableto enter meiosis at 30min, and a significant number ofcells after 60 and 90min. The irr1-1/IRR1 strain treatedwith NZ entered meiosis throughout the 150min courseof the post-synchrony recovery, with a maximumefficiency after 30–90min. These results, summarizedin Table 5, supported the observation that in the irr1-1/IRR1 mutant the SAC could be compromised.

To exclude the possibility that the lack of the arrest ofirr1-1/IRR1 was caused by the experimental conditionsused, we prolonged the NZ treatment up to 5 h to ensurethat all cells had the chance to reach the point in their

cycle where the NZ action would stop its progress. Inthe above conditions, the majority of the irr1-1/IRR1

cells subjected to FACS analysis contained more than4C DNA (Fig. 7B). Cells were alive and under themicroscope we did not observe a significant increase ofthe number of binucleate cells. Thus, we assume thatduring prolonged NZ treatment, the majority of theirr1-1/IRR cells did not arrest at 4C but resumed DNAreplication thus producing polyploid cells. This observa-tion supported the claim of SAC malfunctioning.

To check whether the presence of Irr1-1p causes otherdefects associated with DNA metabolism we performeddrop tests on solid medium checking the sensitivity toHU (hydroxyurea), MMS (methyl methane sulfonate)and UV light, since they are well-known agentsinfluencing cells bearing mutations involved in DNAmetabolism. As expected, the irr1D/IRR1 strain did notexhibit an altered sensitivity to these agents compared toIRR1/IRR1. However, although we did not find analtered sensitivity of irr1-1/IRR1 to UV, this hetero-zygote exhibited a moderately increased sensitivity to

Page 9: The F658G substitution in Saccharomyces cerevisiae cohesin ...

ARTICLE IN PRESS

Fig. 7. The presence of the irr1-1 mutation in the heterozygous irr1-1/IRR1 diploid causes alterations of the cell cycle course. Cells

were treated with 1 mg/ml nocodazole (NZ) for 3 h (A) or 5 h (B) and released into YPD medium. Samples were assessed for DNA

content by FACS at the indicated time points.

A. Cena et al. / European Journal of Cell Biology 87 (2008) 831–844 839

HU and to MMS (Fig. 8). These phenotypes suggestthat irr1-1 may also affect DNA replication/repairsystems.

The F658G substitution is likely to change the

structure of Irr1p/Scc3p, but it does not abrogate its

ability to interact with Scc1p

Since the F658G substitution causes strong pheno-typic effects, we attempted to predict whether it might

introduce a significant change in the Irr1p/Scc3pstructure. The structure of this protein is not known,although the presence of HEAT repeats has beenclaimed (Neuwald and Hirano, 2000). We analyzed thesequence of the Irr1/Scc3 protein for possible structuralhomologs using three servers: MetaServer (Ginalski et al.,2003), PHYRE (http://www.sbg.bio.ic.ac.uk/phyre)and HHpred (Jones, 1999; Soding, 2005). Each of theapplied tools indicated the following proteins as Irr1p/Scc3p structural ‘‘neighbors’’: clathrin adapters AP2(1GW5:A, 1GW5:B) and AP1 (1W63), karyopherin

Page 10: The F658G substitution in Saccharomyces cerevisiae cohesin ...

ARTICLE IN PRESS

Table 5. Sporulation efficiency of diploids subjected to

nocodazole treatment

Time (min) Formation of asci (%)

IRR1/IRR1 irr1-1/IRR1

0 None 8–10

30 3–5 52

60 43 56

90 20–30 30–50

120 12 34

150 3–5 20–30

Yeast culture was treated with 15 mg/ml NZ for 3 h. At indicated

intervals after release from the synchronizing agent cells were

transferred into liquid meiosis-inducing medium, and sporulation

was assayed after 36 h. Numbers represent the estimated percentage of

cells forming asci in meiosis-inducing medium. The experiment was

performed three times, for each sample ca. 200 cells were scored.

Fig. 8. The heterozygous diploid irr1-1/IRR1 exhibits in-

creased sensitivity to HU and MMS. Cells were grown in YPD

medium and diluted to identical concentrations. Serial 10-fold

dilutions were spotted in 5-ml portions onto YPD plates

supplemented with HU (6mg/ml) or MMS (0.01%).

Fig. 9. Ribbon diagram of structural models of a fragment of

Irr1p/Scc3p (light blue) and Irr1-1p (salmon). To show

changes in the general shape of the protein bearing the

F658G substitution, the N-parts of the protein structures were

superimposed. F658 is shown as sticks (green). Hinge

fragments are highlighted in dark blue and red for the wild-

type and mutant proteins, respectively.

A. Cena et al. / European Journal of Cell Biology 87 (2008) 831–844840

beta-2 (1QBK:B), and importin beta (1QGR:A) (ProteinData Bank (Berman et al., 2000) entries are shown inbrackets). All three structure prediction algorithms usedconcurred that Irr1p/Scc3p belongs to the Armadillo(ARM) repeat superfamily in the all-alpha class ofproteins.

The ARM repeat is implicated in mediating protein–protein interactions and has a common origin with theHEAT repeat (Riggleman et al., 1989; Coates, 2003;Andrade et al., 2001), the presence of which in Irr1p/Scc3p was postulated earlier (Neuwald and Hirano,2000). The approximately 40-amino-acid ARM repeat,first identified in the Drosophila segment polarity geneproduct Armadillo, has since been identified in over 240different proteins of diverse cellular functions, fromprotists to mammals. ARM repeat proteins containseveral helices which are connected by variable-length

loops. The mutual orientation of the helices changes, sothe proteins assume a curved structure.

The whole amino acid sequence of Irr1p/Scc3p wassubmitted to the PHYRE server which generated aputative model only for a fragment of Irr1p comprisingamino acids 514–860, which, fortunately, included F658.To check the effect of the F658G substitution, the modelstructures of the wild-type and mutant protein weresubjected to energy minimization in vacuo usingconsistent valence force field as implemented in DIS-COVER ver.98 (Accelrys, San Diego, CA).

F658 is located in the central part of the Irr1/Scc3protein and the 514–860 fragment of Irr1p/Scc3p whichcould be modeled comprises several amino acid repeatssurrounding F658. These repeats form two armsconnected by a loop in which F658 is located. Themodel shows that the side chain of F658 is directed intothe cavity formed by P642, L653, S675, I679 and thehydrophobic part of the side chain of K673. Inconsequence, F658 is inaccessible from the outside. Acomparison of the wild-type and F658G mutantstructures (Fig. 9) shows a substantial change of therelative orientation of the N- and C-parts of the mutantprotein compared to that in the wild type. The shape ofthe individual repeats seems not to change, except forthe fragment 650-665 containing F658.

An overall root-mean-square (RMS) difference be-tween the Irr1-1p and Irr1p structures is about 7 (A,while when measured individually for the N- and C-parts of the two proteins included in the model thesedifferences are only about 2 (A. This suggests that the

Page 11: The F658G substitution in Saccharomyces cerevisiae cohesin ...

ARTICLE IN PRESS

Fig. 10. The mutated Irr1-1 protein co-immunoprecipitates

with Scc1p. Proteins extracted from diploid yeast strains

expressing Scc1-HA6 fusion protein and Scc3/Irr1-Myc18 or

Irr1-1-Myc13 fusion proteins were precipitated with anti-HA

or anti-Myc antibodies. (A) Soluble protein extracts (I) and

anti-Myc (M) or anti-HA (H) precipitates were analyzed for

the presence of Irr1-Myc or Irr1-1-Myc and Scc1-HA fusion

proteins. (B) Control experiment showing the specificity of

binding of the fusion proteins to anti-Myc- or anti-HA-

covered beads.

A. Cena et al. / European Journal of Cell Biology 87 (2008) 831–844 841

655–675 fragment plays a role of a ‘‘hinge’’. Particularly,the arms of the F658G mutant protein fragment arecloser to each other than those of the wild-type Irr1p/Scc3p. Thus, replacing F658 by G changes the interac-tions with surrounding amino acids, which in turnresults in changes of the overall protein geometry.

Since it had been suggested that Scc3p (Irr1p)physically interacts with Scc1p and associates with thecohesin complex in an Scc1-dependent manner (Haeringet al., 2002; Ho et al., 2002; Krogan et al., 2006; McIntyre et al., 2007), we assumed that the predictedchange in Irr1p/Scc3p geometry might hamper itsinteraction with Scc1p. To check this, we performedco-immunoprecipitation of Scc1p and Irr1-1p. As shownin Fig. 10, despite the predicted structure changes, Irr1-1p retains its ability to interact with Scc1p. Thus, theintroduced substitution, although lethal in the haploidand causing strong effects in the heterozygote, does notexert its effects through abolishing the Scc1p-Irr1p/Scc3p interaction.

Discussion

The participation of the Irr1p/Scc3p cohesin inmitotic chromosome segregation has been documentedsince 1999 (Toth et al., 1999) but details of its role in thisand other processes are not available yet. Here weobserved that a single substitution in Irr1p not onlydisturbed chromosome segregation in mitosis and

meiosis, but also caused errors in segregation of nuclei,the course of meiosis and the cell cycle.

Since the hemizygote irr1D/IRR1 does not showsignificant mitotic aberrations, but the irr1-1/IRR1

heterozygote does, one is inclined to think that indeedthe phenotypes of irr1-1/IRR1 result from the presenceof the mutated Irr1-1p and not from an insufficientamount of wild-type Irr1p. Since the irr1-1/IRR1 strainsynthesizes two forms of Irr1p, the wild-type and themutated one, three types of cohesin complexes maypotentially be formed: those containing the wild-typeprotein only, those containing the mutated protein only,and mixed ones (assuming that two or more Irr1pmolecules are present per one cohesin complex). It is notknown whether both proteins have the same affinity forother elements of the complex, but since the mutantexhibits sporadic chromosome segregation errors itseems likely that cohesion complexes containing bothversions of Irr1p/Scc3 (wild-type and Irr1-1) are formedin a stochastic manner. It is highly unlikely that Irr1-1pdisplaces all wild-type Irr1p/Scc3 molecules from thecomplexes, as without the wild-type Irr1p/Scc3 the cellcannot function (the irr1-1 haploid is lethal). Besides,since introduction of an additional copy of wild-typeIRR1 to irr1-1/IRR1 almost fully reversed the pheno-types of the mutant strain, it probably did so byincreasing the ratio of Irr1p to Irr1-1p. This alsosuggests that the mutated Irr1-1 protein accesses thecohesin complex by competing with non-mutated Irr1p/Scc3.

Although the heterozygous irr1-1/IRR1 diploid dis-played numerous defects associated with mitotic andmeiotic divisions, the errors in chromosome segregationin mitosis seemed not to be coupled with defects insegregation of nuclei. Although there are data showingthat interfering with proper cohesion of chromatids maysomehow influence nuclear migration, mostly in meiosis(Pasierbek et al., 2003; Salah and Nasmyth, 2000), inyeast the mechanisms linking errors of chromosomesegregation with the aberrant transmission of thenucleus to the bud are far from being understood.

The observed irregularities in meiosis could be due toSAC malfunctioning. One should bear in mind thatinduction of meiosis in mitotically dividing cells requiresexit from the mitotic cell cycle at the G1 phase, whenchromosome segregation is completed (Kupiec et al.,1997). The irr1-1/IRR1 strain enters meiosis withoutcompleting cell division, which is manifested by progenycells forming asci while still attached to the mother.Such a phenotype could result from the mutant failingto recognize an un-completed mitotic division beforeentering meiosis. The experiment, in which we inducedmeiosis in cells treated with NZ in attempt tosynchronize them at metaphase, indicated that in factNZ failed to cause the G2/M arrest of the mutant. Alsothe FACS analysis after 5 h of NZ treatment showing

Page 12: The F658G substitution in Saccharomyces cerevisiae cohesin ...

ARTICLE IN PRESSA. Cena et al. / European Journal of Cell Biology 87 (2008) 831–844842

substantial population of cells with more than 4C DNA,indicated partial failure of the synchronization attempt.These data suggest malfunctioning of the mitotic spindlecontrol. It is well established that the cohesin complexesconcentrate in the pericentromeric domain and resist thetendency of the microtubules to pull chromatids apartduring their bi-orientation (Glynn et al., 2004). It isbelieved that in the absence of proper bipolar attach-ment or sufficient tension, the SAC is triggered to inhibitthe onset of anaphase. In case of spindle malfunction-ing, the SAC inhibits subsequent mitotic events, thushelping to maintain co-ordination of the cell cycle(reviewed in Lew and Burke, 2003; Gillett et al., 2004).Since properly assembled cohesin complexes are aprerequisite for subsequent correct assembling ofkinetochores, we assume that the presence of Irr1-1pin a fraction of cohesin complexes could perturb thesestructures. As a result, the malformed kinetochorescould not sufficiently support the correct assembling andfunctioning of the SAC. In consequence, it is likely thatirr1-1/IRR1 cannot be arrested in mitotic divisions byNZ treatment because of SAC malfunctioning. Unableto halt the progress of the cell cycle until a properlyorganized mitotic spindle is in place, the cells chaoticallyenter meiosis in response to a meiosis-inducing signal oraberrantly continue the mitotic cell cycle by synthesizingDNA without having completed the chromosomesegregation phase.

The role of cohesin in kinetochore assembly andfunctioning is poorly known. One report indicates anenhancement of cohesin association in pericentromericregions by kinetochores (Weber et al., 2004). Anotherstudy points to a role of cohesin in the kinetochore-microtubule interactions exemplified by prolongedBub1p binding to centromeres that could result fromerrors in these interactions (Toyoda et al., 2002). Severalobservations concern the role of Scc1 and its orthologsin centromere and kinetochore organization and func-tioning (Sonoda et al., 2001; Hoque and Ishikawa, 2002;Vass et al., 2003; Vagnarelli et al., 2004). Thus, sincecohesin constitutes the structural basis of the kineto-chore, on which elements of the SAC are assembled, weassume that the phenotypes of irr1-1/IRR1 discussedabove could likely be caused by SAC malfunctioning.

The increased sensitivity of the irr1-1/IRR1 mutant toHU and MMS implicates a defect in DNA replication/repair. It is known that DNA replication and chromatidcohesion are coupled, although the mechanisms whichlink them are far from being understood (Petronczkiet al., 2004; Wysocka et al., 2004; Skibbens, 2005; Lahaet al., 2006). Our observations suggest a possible role ofIrr1p/Scc3p in these processes.

To sum up, our results show that the presence of themutated Irr1-1 protein in the heterozygous diploid irr1-

1/IRR1 triggers numerous effects. Besides errors inchromosome segregation we observed aberrations of the

cell cycle which could cause meiotic defects. Thedescribed diploid yeast cell mimics to some extent amammalian cell bearing a semi-dominant mutation inthe genome-segregation machinery. Cells bearing such adefect are not eliminated and may lead to the develop-ment of tumors. The incomplete penetrance of thedefects could be due to the stochastic nature of Irr1-1pincorporation into the cohesion complexes, or toa significant proportion of errors caused by the pre-sence of Irr1-1p being compensated by an unknownmechanism.

Acknowledgments

This work was supported by the Ministry of Scienceand Higher Education, Grant 2 P04C01130. We thankF. Uhlmann (London Research Institute, London, UK)for yeast strains, and Teresa Zoladek for criticalcomments.

References

Andrade, M.A., Petosa, C., O’Donoghue, S.I., Muller, C.W.,

Bork, P., 2001. Comparison of ARM and HEAT protein

repeats. J. Mol. Biol. 309, 1–18.

Berman, H.M., Westbrook, J., Feng, Z., Gilliland, G., Bhat,

T.N., Weissig, H., Shindyalov, I.N., Bourne, P.E., 2000.

The protein data bank. Nucleic Acids Res. 28, 235–242.

Carramolino, L., Lee, B.C., Zaballos, A., Peled, A., Barthel-

emy, I., Shav-Tal, Y., Prieto, I., Carmi, P., Gothelf, Y.,

Gonzalez de Buitrago, G., Aracil, M., Marquez, G.,

Barbero, J.L., Zipori, D., 1997. SA-1, a nuclear protein

encoded by one member of a novel gene family: molecular

cloning and detection in hemopoietic organs. Gene 195,

151–159.

Cena, A., Orlowski, J., Machula, K., Fronk, J., Kurlandzka,

A., 2007. Substitution F659G in the Irr1p/Scc3p cohesin

influences the cell wall of Saccharomyces cerevisiae. Cell

Struct. Funct. 32, 1–7.

Chelysheva, L., Diallo, S., Vezon, D., Gendrot, G., Vrielynck,

N., Belcram, K., Rocques, N., Marquez-Lema, A., Bhatt,

A.M., Horlow, C., Mercier, R., Mezard, C., Grelon, M.,

2005. AtREC8 and AtSCC3 are essential to the monopolar

orientation of the kinetochores during meiosis. J. Cell Sci.

118, 4621–4632.

Ciosk, R., Zachariae, W., Michaelis, C., Shevchenko, A.,

Mann, M., Nasmyth, K., 1998. An ESP1/PDS1 complex

regulates loss of sister chromatid cohesion at the metaphase

to anaphase transition in yeast. Cell 93, 1067–1076.

Coates, J., 2003. Armadillo repeat proteins: beyond the animal

kingdom. Trends Cell Biol. 13, 463–471.

Cohen-Fix, O., Peters, J.M., Kirschner, M.W., Koshland, D.,

1996. Anaphase initiation in Saccharomyces cerevisiae is

controlled by the APC-dependent degradation of the

anaphase inhibitor Pds1p. Genes Dev. 10, 3081–3093.

Page 13: The F658G substitution in Saccharomyces cerevisiae cohesin ...

ARTICLE IN PRESSA. Cena et al. / European Journal of Cell Biology 87 (2008) 831–844 843

Gillett, E.S., Espelin, C.W., Sorger, P.K., 2004. Spindle

checkpoint proteins and chromosome–microtubule attach-

ment in budding yeast. J. Cell Biol. 164, 535–546.

Ginalski, K., Elofsson, A., Fischer, D., Rychlewski, L., 2003.

3D-Jury: a simple approach to improve protein structure

predictions. Bioinformatics 19, 1015–1018.

Glynn, E.F., Megee, P.C., Yu, H.G., Mistrot, C., Unal, E.,

Koshland, D.E., DeRisi, J.L., Gerton, J.L., 2004. Genome-

wide mapping of the cohesin complex in the yeast

Saccharomyces cerevisiae. PLoS Biol. 9, E259.

Guacci, V., Koshland, D., Strunnikov, A., 1997. A direct link

between sister chromatid cohesion and chromosome con-

densation revealed through the analysis of MCD1 in S.

cerevisiae. Cell 91, 47–58.

Haering, C.H., Nasmyth, K., 2003. Building and breaking

bridges between sister chromatids. Bioessays 25, 1178–1191.

Haering, C.H., Lowe, J., Hochwagen, A., Nasmyth, K., 2002.

Molecular architecture of SMC proteins and the yeast

cohesin complex. Mol. Cell 9, 773–788.

Hauf, S., Roitinger, E., Koch, B., Dittrich, C.M., Mechtler,

K., Peters, J.M., 2005. Dissociation of cohesin from

chromosome arms and loss of arm cohesion during early

mitosis depends on phosphorylation of SA2. PLoS Biol. 3,

e69.

Hirano, T., 2000. Chromosome cohesion, condensation, and

separation. Annu. Rev. Biochem. 69, 115–144.

Ho, Y., Gruhler, A., Heilbut, A., Bader, G.D., Moore, L.,

Adams, S.L., Millar, A., Taylor, P., Bennett, K., Boutilier,

K., Yang, L., Wolting, C., Donaldson, I., Schandorff, S.,

Shewnarane, J., Vo, M., Taggart, J., Goudreault, M.,

Muskat, B., Alfarano, C., Dewar, D., Lin, Z., Michalick-

ova, K., Willems, A.R., Sassi, H., Nielsen, P.A., Rasmus-

sen, K.J., Andersen, J.R., Johansen, L.E., Hansen, L.H.,

Jespersen, H., Podtelejnikov, A., Nielsen, E., Crawford, J.,

Poulsen, V., Sørensen, B.D., Matthiesen, J., Hendrickson,

R.C., Gleeson, F., Pawson, T., Moran, M.F., Durocher,

D., Mann, M., Hogue, C.W., Figeys, D., Tyers, M., 2002.

Systematic identification of protein complexes in Saccha-

romyces cerevisiae by mass spectrometry. Nature 415,

180–183.

Hoque, M.T., Ishikawa, F., 2002. Cohesin defects lead to

premature sister chromatid separation, kinetochore dys-

function, and spindle-assembly checkpoint activation. J.

Biol. Chem. 277, 42306–42314.

Jones, D.T., 1999. Protein secondary structure prediction

based on position-specific scoring matrices. J. Mol. Biol.

292, 195–202.

Kassir, Y., Adir, N., Boger-Nadja, E., Guttmann-Raviv, N.,

Rubin-Bejerano, I., Sagee, S., Shenhar, G., 2003. Tran-

scriptional regulation of meiosis in budding yeast. Int. Rev.

Cytol. 224, 111–171.

Klein, F., Laroche, T., Cardenas, M.E., Hofmann, J.F.,

Schweizer, D., Gasser, S.M., 1992. Localization of RAP1

and topoisomerase II in nuclei and meiotic chromosomes of

yeast. J. Cell Biol. 117, 935–948.

Klein, F., Mahr, P., Galova, M., Buonomo, S.B., Michaelis,

C., Nairz, K., Nasmyth, K., 1999. A central role for

cohesins in sister chromatid cohesion, formation of axial

elements, and recombination during yeast meiosis. Cell 98,

91–103.

Krogan, N.J., Cagney, G., Yu, H., Zhong, G., Guo, X.,

Ignatchenko, A., Li, J., Pu, S., Datta, N., Tikuisis, A.P.,

Punna, T., Peregrin-Alvarez, J.M., Shales, M., Zhang, X.,

Davey, M., Robinson, M.D., Paccanaro, A., Bray, J.E.,

Sheung, A., Beattie, B., Richards, D.P., Canadien, V.,

Lalev, A., Mena, F., Wong, P., Starostine, A., Canete,

M.M., Vlasblom, J., Wu, S., Orsi, C., Collins, S.R.,

Chandran, S., Haw, R., Rilstone, J.J., Gandi, K., Thomp-

son, N.J., Musso, G., St Onge, P., Ghanny, S., Lam, M.H.,

Butland, G., Altaf-Ul, A.M., Kanaya, S., Shilatifard, A.,

O’Shea, E., Weissman, J.S., Ingles, C.J., Hughes, T.R.,

Parkinson, J., Gerstein, M., Wodak, S.J., Emili, A.,

Greenblatt, J.F., 2006. Global landscape of protein

complexes in the yeast Saccharomyces cerevisiae. Nature

440, 637–643.

Kupiec, M., Byers, B., Esposito, R.E., Mitchell, A.P., 1997.

Meiosis and sporulation in Saccharomyces cerevisiae. In:

Pringle, J.R., Broach, J.R., Jones, E.W. (Eds.), The

Molecular and Cellular Biology of the Yeast Saccharo-

myces. Cell Cycle and Cell Biology. Cold Spring Harbor

Laboratory Press, Cold Spring Harbor, New York,

pp. 889–1036.

Kurlandzka, A., Rytka, J., Gromadka, R., Murawski, M.,

1995. A new essential gene located on Saccharomyces

cerevisiae chromosome IX. Yeast 11, 885–890.

Laha, S., Das, S.P., Hajra, S., Sau, S., Sinha, P., 2006. The

budding yeast protein Chl1p is required to preserve genome

integrity upon DNA damage in S-phase. Nucleic Acids Res.

34, 5880–5891.

Lee, J.Y., Orr-Weaver, T.L., 2001. The molecular basis of

sister-chromatid cohesion. Annu. Rev. Cell Dev. Biol. 17,

753–777.

Lew, D.J., Burke, D.J., 2003. The spindle assembly and spindle

position checkpoints. Annu. Rev. Genet. 37, 251–282.

Longtine, M.S., McKenzie 3rd, A., Demarini, D.J., Shah,

N.G., Wach, A., Brachat, A., Philippsen, P., Pringle, J.R.,

1998. Additional modules for versatile and economical

PCR-based gene deletion and modification in Saccharo-

myces cerevisiae. Yeast 14, 953–961.

Mc Intyre, J., Muller, E.G., Weitzer, S., Snydsman, B.E.,

Davis, T.N., Uhlmann, F., 2007. In vivo analysis of cohesin

architecture using FRET in the budding yeast Saccha-

romyces cerevisiae. EMBO J. 26, 3783–3793.

Michaelis, C., Ciosk, R., Nasmyth, K., 1997. Cohesins:

chromosomal proteins that prevent premature separation

of sister chromatids. Cell 91, 35–45.

Nasmyth, K., 2001. Disseminating the genome: joining,

resolving, and separating sister chromatids during mitosis

and meiosis. Annu. Rev. Genet. 35, 673–745.

Nasmyth, K., 2005. How might cohesin hold sister chromatids

together? Philos. Trans. R. Soc. Lond. B Biol. Sci. 360,

483–496.

Nasmyth, K., Haering, C.H., 2005. The structure and function

of SMC and kleisin complexes. Annu. Rev. Biochem. 74,

595–648.

Neiman, A.M., 2005. Ascospore formation in the yeast

Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev. 69,

565–584.

Neuwald, A.F., Hirano, T., 2000. HEAT repeats associated

with condensins, cohesins, and other complexes involved in

Page 14: The F658G substitution in Saccharomyces cerevisiae cohesin ...

ARTICLE IN PRESSA. Cena et al. / European Journal of Cell Biology 87 (2008) 831–844844

chromosome-related functions. Genome Res. 10,

1445–1452.

Pasierbek, P., Fodermayr, M., Jantsch, V., Jantsch, M.,

Schweizer, D., Loidl, J., 2003. The Caenorhabditis elegans

SCC-3 homologue is required for meiotic synapsis and for

proper chromosome disjunction in mitosis and meiosis.

Exp. Cell Res. 289, 245–255.

Petronczki, M., Chwalla, B., Siomos, M.F., Yokobayashi, S.,

Helmhart, W., Deutschbauer, A.M., Davis, R.W., Wata-

nabe, Y., Nasmyth, K., 2004. Sister-chromatid cohesion

mediated by the alternative RF-CCtf18/Dcc1/Ctf8, the

helicase Chl1 and the polymerase-alpha-associated protein

Ctf4 is essential for chromatid disjunction during meiosis

II. J. Cell Sci. 117, 3547–3559.

Pezzi, N., Prieto, I., Kremer, L., Perez Jurado, L.A., Valero,

C., Del Mazo, J., Martinez, A.C., Barbero, J.L., 2000.

STAG3, a novel gene encoding a protein involved in

meiotic chromosome pairing and location of STAG3-

related genes flanking the Williams-Beuren syndrome

deletion. FASEB J. 14, 581–592.

Pinsky, B.A., Biggins, S., 2005. The spindle checkpoint:

tension versus attachment. Trends Cell Biol. 15, 486–493.

Pnueli, L., Edry, I., Cohen, M., Kassir, Y., 2004. Glucose and

nitrogen regulate the switch from histone deacetylation to

acetylation for expression of early meiosis-specific genes in

budding yeast. Mol. Cell. Biol. 24, 5197–5208.

Prieto, I., Suja, J.A., Pezzi, N., Kremer, L., Martinez, A.C.,

Rufas, J.S., Barbero, J.L., 2001. Mammalian STAG3 is a

cohesin specific to sister chromatid arms in meiosis I. Nat.

Cell Biol. 3, 761–766.

Rieger, K.J., El-Alama, M., Stein, G., Bradshaw, C.,

Slonimski, P.P., Maundrell, K., 1999. Chemotyping of

yeast mutants using robotics. Yeast 15, 973–986.

Riggleman, B., Wieschaus, E., Schedl, P., 1989. Molecular

analysis of the armadillo locus: uniformly distributed

transcripts and a protein with novel internal repeats are

associated with a Drosophila segment polarity gene. Genes

Dev. 3, 96–113.

Rose, M., Winston, F., Hieter, P., 1990. Methods in Yeast

Genetics. A Cold Spring Harbor Laboratory Course. Cold

Spring Harbor Laboratory Press, New York.

Salah, S.M., Nasmyth, K., 2000. Destruction of the securin

Pds1p occurs at the onset of anaphase during both meiotic

divisions in yeast. Chromosoma 109, 27–34.

Skibbens, R.V., 2005. Unzipped and loaded: the role of DNA

helicases and RFC clamp-loading complexes in sister

chromatid cohesion. J. Cell Biol. 169, 841–846.

Soding, J., 2005. Protein homology detection by HMM–HMM

comparison. Bioinformatics 21, 951–960.

Sonoda, E., Matsusaka, T., Morrison, C., Vagnarelli, P.,

Hoshi, O., Ushiki, T., Nojima, K., Fukagawa, T.,

Waizenegger, I.C., Peters, J.M., Earnshaw, W.C., Takeda,

S., 2001. Scc1/Rad21/Mcd1 is required for sister chromatid

cohesion and kinetochore function in vertebrate cells. Dev.

Cell 1, 759–770.

Toth, A., Ciosk, R., Uhlmann, F., Galova, M., Schleiffer, A.,

Nasmyth, K., 1999. Yeast cohesin complex requires a

conserved protein, Eco1p(Ctf7), to establish cohesion

between sister chromatids during DNA replication. Genes

Dev. 13, 320–333.

Toyoda, Y., Furuya, K., Goshima, G., Nagao, K., Takahashi,

K., Yanagida, M., 2002. Requirement of chromatid

cohesion proteins rad21/scc1 and mis4/scc2 for normal

spindle–kinetochore interaction in fission yeast. Curr. Biol.

12, 347–358.

Uhlmann, F., 2003. Chromosome cohesion and separation:

from men and molecules. Curr. Biol. 13, R104–R114.

Uhlmann, F., 2004. The mechanism of sister chromatid

cohesion. Exp. Cell Res. 296, 80–85.

Vagnarelli, P., Morrison, C., Dodson, H., Sonoda, E., Takeda,

S., Earnshaw, W.C., 2004. Analysis of Scc1-deficient cells

defines a key metaphase role of vertebrate cohesin in

linking sister kinetochores. EMBO Rep. 5, 167–171.

Valdeolmillos, A., Rufas, J.S., Suja, J.A., Vass, S., Heck,

M.M., Martinez, A.C., Barbero, J.L., 2004. Drosophila

cohesins DSA1 and Drad21 persist and colocalize along the

centromeric heterochromatin during mitosis. Biol. Cell 96,

457–462.

Vass, S., Cotterill, S., Valdeolmillos, A.M., Barbero, J.L., Lin,

E., Warren, W.D., Heck, M.M., 2003. Depletion of drad21/

scc1 in Drosophila cells leads to instability of the cohesin

complex and disruption of mitotic progression. Curr. Biol.

13, 208–218.

Wang, F., Yoder, J., Antoshechkin, I., Han, M., 2003.

Caenorhabditis elegans EVL-14/PDS-5 and SCC-3 are

essential for sister chromatid cohesion in meiosis and

mitosis. Mol. Cell. Biol. 23, 7698–7707.

Watanabe, Y., 2005. Shugoshin: guardian spirit at the

centromere. Curr. Opin. Cell Biol. 17, 590–595.

Weber, S.A., Gerton, J.L., Polancic, J.E., DeRisi, J.L.,

Koshland, D., Megee, P.C., 2004. The kinetochore is an

enhancer of pericentric cohesin binding. PLoS Biol. 9,

E260.

Wysocka, M., Rytka, J., Kurlandzka, A., 2004. Saccharomyces

cerevisiae CSM1 gene encoding a protein influencing

chromosome segregation in meiosis I interacts with

elements of the DNA replication complex. Exp. Cell Res.

294, 592–602.

Page 15: The F658G substitution in Saccharomyces cerevisiae cohesin ...

本文献由“学霸图书馆-文献云下载”收集自网络,仅供学习交流使用。

学霸图书馆(www.xuebalib.com)是一个“整合众多图书馆数据库资源,

提供一站式文献检索和下载服务”的24 小时在线不限IP

图书馆。

图书馆致力于便利、促进学习与科研,提供最强文献下载服务。

图书馆导航:

图书馆首页 文献云下载 图书馆入口 外文数据库大全 疑难文献辅助工具