Copyright � 2011 by the Genetics Society of AmericaDOI: 10.1534/genetics.110.125468

The Cik1/Kar3 Motor Complex Is Required for the ProperKinetochore–Microtubule Interaction After

Stressful DNA Replication

Hong Liu,*,1,2 Fengzhi Jin,†,1 Fengshan Liang,† Xuemei Tian† and Yanchang Wang*,†,3

*Department of Biological Sciences, Florida State University, Tallahassee, Florida 32306-4370 and †Department ofBiomedical Sciences, College of Medicine, Florida State University, Tallahassee, Florida 32306-4300

Manuscript received October 20, 2010Accepted for publication November 30, 2010

ABSTRACT

In budding yeast Saccharomyces cerevisiae, kinetochores are attached by microtubules during most of thecell cycle, but the duplication of centromeric DNA disassembles kinetochores, which results in a briefdissociation of chromosomes from microtubules. Kinetochore assembly is delayed in the presence ofhydroxyurea, a DNA synthesis inhibitor, presumably due to the longer time required for centromeric DNAduplication. Some kinetochore mutants are sensitive to stressful DNA replication as these kinetochoreproteins become essential for the establishment of the kinetochore–microtubule interaction aftertreatment with hydroxyurea. To identify more genes required for the efficient kinetochore–microtubuleinteraction under stressful DNA replication conditions, we carried out a genome-wide screen for yeastmutants sensitive to hydroxyurea. From this screen, cik1 and kar3 mutants were isolated. Kar3 is the minus-end-directed motor protein; Cik1 binds to Kar3 and is required for its motor function. After exposure tohydroxyurea, cik1 and kar3 mutant cells exhibit normal DNA synthesis kinetics, but they display asignificant anaphase entry delay. Our results indicate that cik1 cells exhibit a defect in the establishment ofchromosome bipolar attachment in the presence of hydroxyurea. Since Kar3 has been shown to drive thepoleward chromosome movement along microtubules, our data support the possibility that thischromosome movement promotes chromosome bipolar attachment after stressful DNA replication.

THE attachment of chromosomes by microtubulesis essential for chromosome segregation and the

kinetochore is a protein complex that associates withcentromeric DNA to mediate this attachment. In highereukaryotic cells, the establishment of kinetochore–microtubule (KT–MT) interaction occurs during meta-phase when DNA has been duplicated and compacted;however, budding yeast kinetochores are associated withmicrotubules during most of the cell cycle ( Janke et al.2002; Li et al. 2002). The KT–MT interaction in buddingyeast must be disrupted when centromeric DNA is beingduplicated. Indeed, previous data indicate that centro-meric DNA shows a transient detachment from micro-tubules during S-phase (Tanaka et al. 2007). Therefore,the reestablishment of the KT–MT interaction in bud-ding yeast takes place in S-phase just after centromereduplication and the subsequent kinetochore assembly.

The establishment of KT–MT interaction is a multi-step process. First, one of the sister kinetochores iscaptured by the side of a microtubule to establish side-on binding. In budding yeast, the captured kinetochoremoves toward the spindle poles with the assistance of theminus-end-directed motor protein Kar3 (Tanaka et al.2005). During this movement, the side-on bindingcould be switched to end-on binding, which dependson the DASH/Dam1, a kinetochore complex thatassociates with microtubules before the establishmentof KT–MT interaction ( Janke et al. 2002; Li et al. 2002;Tanaka et al. 2007). Recent data suggest that theaccumulated Stu1 protein on unattached kinetochoresmay facilitate chromosome capture, and Stu1 could bethe protein responsible for the initial KT–MT interac-tion on the basis of its microtubule-binding nature(Ortiz et al. 2009). After chromosomes are moved closeto the spindle pole, they become bipolar attached, but itis less clear whether this poleward movement facilitatesbipolar attachment. One possibility is that chromo-somes close to one spindle pole are easily captured bymicrotubules emanating from the other pole.

The duplication of centromeric DNA results in dis-sociation of kinetochore proteins from the centromere.Hydroxyurea (HU) slows down DNA synthesis by de-pleting the pool of dNTPs, the basic unit of DNA. The

Supporting information is available online at http://www.genetics.org/cgi/content/full/genetics.110.125468/DC1.

1These authors contributed equally to this work.2Present address: Department of Pharmacology, University of Texas

Southwestern Medical Center, Dallas, TX 75390.3Corresponding author: Department of Biomedical Sciences, College of

Medicine, Florida State University, Tallahassee, FL 32306-4300.E-mail: yanchang.wang@med.fsu.edu

Genetics 187: 397–407 (February 2011)

examination of the interaction of kinetochore proteinswith centromeric DNA in budding yeast revealed thatHU treatment delays kinetochore reassembly, presum-ably due to slower centromeric DNA replication (Liu

et al. 2008). Because of this delay, we expect more de-tached chromosomes in HU-treated yeast cells. Afterkinetochore assembly, one of the sister kinetochores iscaptured by a microtubule and moved close to the spin-dle pole (Tanaka et al. 2007). Therefore, this polewardchromosome transport might become critical for the re-establishment of KT–MT interaction after HU treatment.

We have found that mutation in a kinetochore pro-tein Ask1 leads to sensitivity to HU and ask1-3 mutantcells show difficulty in establishing correct KT–MTinteraction after HU treatment (Li et al. 2002; Liu

et al. 2008). To identify additional yeast genes requiredfor the efficient KT–MT interaction after HU treatment,we performed a genome-wide screen for HU-sensitivemutants. Interestingly, cik1D and kar3D mutants werefound to be sensitive to HU. Kar3 is one of the sixkinesin proteins in budding yeast and it was first foundto be essential for yeast nuclear fusion during mating(Meluh and Rose 1990; Molk et al. 2006). Unlike otherkinesins, Kar3 protein contains a motor domain at itscarboxy terminus that possesses minus-end-directedmotility (Endow et al. 1994). Two proteins, Cik1 andVik1, associate with Kar3. Cik1 is required for thelocalization of Kar3 to microtubule-associated struc-tures, whereas Vik1 aids in the localization of Kar3 atspindle poles (Page et al. 1994; Manning et al. 1999;Barrett et al. 2000). Kar3 protein forms a heterodimerwith either Vik1 or Cik1, which is required for associa-tion of Kar3 with microtubules and its motility (Chu

et al. 2005; Allingham et al. 2007). Recent evidenceindicates that Kar3 is also responsible for the polewardchromosome movement (Tanaka et al. 2005). In ad-dition to its motor function, Cik1/Kar3 complex local-izes at the spindle midzone as an interpolar microtubulecrosslinker to prevent spindle collapse (Gardner et al.2008b). In other eukaryotic cells, the homologs of Kar3promote the bundling of parallel microtubules as wellas the sliding of antiparallel microtubules (Braun et al.2009; Fink et al. 2009). This function may explain theabnormal dot-like spindle structure in cik1 and kar3 mu-tants (Meluh and Rose 1990; Page and Snyder 1992).Together, these observations indicate that Kar3 formsdistinct complexes with Cik1 and Vik1 to participate indifferent microtubule-mediated events, such as mating,spindle morphogenesis, and chromosome segregation.

Although we found that cik1D and kar3D mutants arevery sensitive to HU, deletion of another Kar3 interact-ing protein Vik1 and other microtubule motor proteinsdid not result in any HU sensitivity. Our evidenceindicates that the HU sensitivity is not a consequenceof impaired DNA replication. Instead, the cik1D andkar3D mutant cells fail to establish proper KT–MT in-teraction after exposure to HU, as indicated by abnor-

mally distributed kinetochore proteins. Moreover, wegenerated cik1HU mutants that exhibit similar HU sensi-tivity to cik1D but show relatively normal spindle struc-ture, indicating the abnormal spindle structure may notbe the cause of HU sensitivity. Therefore, Cik1/Kar3-dependent chromosome transport might be required forthe efficient establishment of proper KT–MT interactionafter stressful DNA replication.

MATERIALS AND METHODS

Yeast strains, growth, and media: The relevant genotypesand sources of the yeast strains used in this study are listed inTable 1. All the strains listed are isogenic to Y300, a W303derivative. To arrest yeast cells in G1 phase, 5 mg/ml a-factorwas added into cell cultures in midlog phase in YPD (pH¼ 3.9)for 2.5 hr. The cells were centrifuged and washed once withwater to release from G1 arrest. Hydroxyurea was purchasedfrom ACROS Organics.

Protein techniques: The preparation of yeast protein sam-ples was described previously (Liu and Wang 2006). Proteinsamples were resolved by 10% SDS–PAGE. Primary antibodies(anti-myc) were purchased from Covance (Madison, WI), andanti-Pgk1 antibody was from Molecular Probes (Eugene, OR).The HRP-conjugated secondary antibody was purchased fromJackson ImmunoResearch (West Grove, PA).

Fluorescence microcopy: Collected cells were fixed with3.7% formaldehyde for 5 min at room temperature. The cellswere washed once with 13 PBS (pH 7.2) and then resus-pended in 13 PBS buffer to examine fluorescence signals witha microscope (Zeiss Axioplan 2) (Carl Zeiss, Thornwood, NY).

Screen of HU-sensitive mutants: Yeast cells of the �4700deletion strains from American Type Culture Collection(ATCC; Rockville, MD) were spotted onto YPD plates with andwithout 100 mm of HU. After a 3-day incubation at 25�, thegrowth of these yeast cells was examined and the strains thatshowed slow or no growth were selected. All the HU-sensitivemutants are listed in supporting information, Table S1. ‘‘1’’means that the mutant cells show slow growth on HU plates.‘‘11’’ means no growth.

The construction of pRS416-CIK1 plasmid: Yeast genomicDNA was used as template for the PCR reaction with a pair ofCIK1 gene-specific primers (forward primer, 59-GAT TCC CCGCGG GGA CTG TTA GTC CCG TAA CAT T-39; reverse primer,59-GCT ACC ATC GAT GGT GCG GTT GAT TCG TTT TAT A-39). The forward and reverse primers contain SacII and ClaIrestriction enzyme sites, respectively. The PCR products andpRS416 vector were digested with both SacII and ClaI(Sikorski and Hieter 1989). After recovery from the agarosegel, the fragments were ligated for the transformation toEscherichia coli.

Mutagenesis PCR of CIK1: The PCR reaction mixture withless dATP (�A) is 25 ml that contains 0.5 ml of 20 ng/mlpRS416-CIK1 plasmid template; 0.5 ml 20-mm primers (thesame primers used for the construction of pRS416-CIK1plasmid); 1 ml 12.5 mm dTTP, dGTP, and dCTP and 1 ml 2.5 mm

dATP; 2.5 ml 103 Mg21 free Taq DNA polymerase buffer; 2.5 ml25 mm MgCl2; 0.94 ml 20 mm MnCl2; 0.5 ml Taq DNApolymerase; and H2O. Similarly, the �T, �G, and �C PCRmixtures contain less concentrated dTTP, dGTP, and dCTP,respectively. After PCR reaction, the four PCR products weremixed together. The constructed pRS416-CIK1 plasmid wasdigested with PacI (one single cut within the CIK1 gene) andfollowed by the treatment with calf intestinal phosphatase(CIP) to prevent self ligation. The treated pRS416-CIK1

398 H. Liu et al.

plasmid was transformed into a cik1D strain along with the PCRmixture and the transformants were selected on URA dropoutplates. The mutated cik1 genes through the biased PCRreaction would insert into the linearized plasmid by recombi-nation. The growing colonies were replica copied onto HU(100 mm) and YPD plates and incubated at 25� and 37�,respectively. The HU- and temperature-sensitive mutants wereselected. The plasmids containing the mutated cik1 gene wererecovered and reintroduced into cik1D mutant to confirm thephenotype. Then the plasmids were sequenced to determinethe mutation sites.

To construct cik1HU mutant strains, the pRS416-cik1HU

plasmids were digested with ClaI and SacII and the cik1HU

genes were inserted into an integration vector pRS403(Sikorski and Hieter 1989). The resulting pRS403-cik1HU

plasmids were digested with NdeI and transformed into a cik1Dstrain. The transformants were selected on HIS dropout platesand the HU sensitivity was confirmed after transformation.

RESULTS

cik1D and kar3D mutants exhibit HU sensitivity: ask1-1was isolated as a HU-sensitive mutant and Ask1 turnedout to be a kinetochore protein (Alcasabas et al. 2001;Li et al. 2002). To identify more kinetochore mutantsthat exhibit HU sensitivity, we carried out a genome-wide screen for mutants that are sensitive to HU fromthe ATCC collection of yeast deletion mutants, and.100 HU-sensitive mutants were identified (Table S1).The cik1D mutant was found to exhibit dramatic HUsensitivity (Figure 1A). Kar3 is one of the kinesinproteins in budding yeast and both Cik1 and Vik1 forma complex with Kar3 (Manning et al. 1999), but ourATCC collection does not include the kar3D deletion

strain. Nevertheless, deletion of VIK1 did not show anyHU sensitivity. We also examined the HU sensitivity ofother kinesin and dynein mutants, cin8D, kip1D, kip2D,kip3D, and dyn1D (Saunders et al. 1995), and none ofthem showed HU sensitivity (Figure 1A). To determinethe HU sensitivity of kar3 mutants, we generated cik1D

and kar3D mutants with the Y300 strain, a W303 derivative,by using a one-step PCR approach (Longtine et al. 1998).Interestingly, both cik1D and kar3D mutants exhibited HUsensitivity. Moreover, a kar3-1 mutant was also sensitive toHU (Figure 1A). Therefore, we conclude that the loss offunction of the Cik1/Kar3 complex leads to sensitivity tostressful DNA synthesis.

One possibility for the HU sensitivity of cik1 and kar3mutants is that DNA replication is compromised and thetreatment with HU exacerbates the defect. Thus, we firstcompared the DNA replication kinetics in synchronouswild-type (WT) and cik1D mutant cells by fluorescence-activated cell sorting (FACS) analysis, but no differencewas observed (data not shown). Then we compared theDNA synthesis kinetics in WT and cik1D mutant cellsafter HU treatment. G1-arrested cells were released into200 mM HU medium for 100 min and DNA replicationwas monitored by using pulsed-field gel electrophoresis(PFGE) after HU was washed off (Liu and Wang 2006).The chromosomal DNA from both WT and cik1D cellswas able to run into the agarose gel after release from HUfor 80 min, indicating the completion of DNA synthesis(Figure 1B). Therefore, cik1D mutants showed no DNAsynthesis defects with or without HU treatment.

Another possibility for the HU sensitivity is that Cik1and Kar3 function in the S-phase checkpoint and the

TABLE 1

Strain list

Strain Relevant genotypes Source

Y300 MATa ura3-1 his3-11,15 leu2-3,112 trp1-1 ade2-1 can1-100 Lab stockYYW144 MATa cik1DTKanMX This studyYYW142 MATa kar3DTSphis51 This studyJBY649 MATa PDS1-18myc-LEU2 Lab stockYHL001 MATa cik1DTKanMX PDS1-18myc-LEU2 This study2346-2-3 MATa kar3DTSphis51 PDS1-18myc-LEU2 This study683-15-3 MATa MTW1-3GFP-HIS3 This study681-3-4 MATa cik1DTKanMX MTW1-3GFP-HIS3 This studyYYW115 MATa TUB1-GFP-LEU2 This study2184-3-2 MATa cik1DTKanMX TUB1-GFP-LEU2 This studyYYW277-1 MATa cik1DTKanMX cik1HU5-HIS3 PDS1-18myc-LEU2 This studyYYW141 MATa promURA3TtetRTGFP-LEU2 CENIVTtetOX448-URA3 TUB1-mCherry-URA3 This studyYYW276-1 MATa cik1DTKanMX cik1HU5-HIS3 promURA3TtetRTGFP-LEU2 CENIVTtetOX448-URA3

TUB1-mCherry-URA3This study

2348-7-1 MATa cik1DTKanMX cik1HU5-HIS3 MTW1-3GFP-HIS3 TUB1-mCherry-URA3 This study2348-20-4 MATa MTW1-3GFP-HIS3 TUB1-mCherry-URA3 This studyMY997 MATa kar3-1 Rose laboratoryAY202 MATa rad53-21 in Y300 Elledge laboratory2201-5-2 MATa scc1-73 CENV-tetO-HIS3 tetRTGFP-LEU2 TUB1-mCherry-URA3 This study2201-4-1 MATa scc1-73 cik1DTKanMX CENVTtetO-HIS3 tetRTGFP-LEU2 TUB1-mCherry-URA3 This study

Cik1/Kar3 Motor Complex 399

impaired checkpoint function contributes to the HUsensitivity. Thus, we determined the viability of cik1D

and kar3D mutant cells in the presence of HU. After cellswere incubated in 200 mm HU for 6 hr, we did notobserve significant viability loss; 60% of cik1D and 80%of kar3D cells were able to form colonies, suggesting thatthe S-phase checkpoint is proficient in cik1D and kar3D

mutant cells (Figure 1C). As a control, most of the rad53-21 mutant cells lost viability after incubation for 2 hr(Sanchez et al. 1996; Wang and Elledge 1999).Therefore, it is unlikely that the HU sensitivity of cik1D

and kar3D mutants is a consequence of impairedS-phase checkpoint.

Although we did not observe obvious DNA synthesisdefects with FACS and PFGE methods, we could notexclude the possibility that cik1 and kar3 mutant cellsshow specific defects in the duplication of centromericDNA, which could contribute to their HU sensitivity. Ifthat is the case, we expect delayed centromere separa-tion in cik1D mutants as the separation of centromericDNA depends on centromere duplication. A CEN5-GFPstrain was generated in the Nasmyth laboratory, whereinthe tetO array was inserted into the genome that is 1.4 kbaway from the centromere on chromosome V. Theexpressed tetR-GFP fusion protein binds to the tetOarray to mark CEN5 (Tanaka et al. 2000; Zhang et al.2006). Therefore, the separation of Cen5-GFP willindicate the completion of CEN5 duplication. cik1D

mutant cells exhibit mitotic defects, which delay theseparation of Cen5-GFP by activating of the spindleassembly checkpoint, but the absence of sister chroma-tid cohesion will allow the generation of two Cen5-GFPdots immediately after CEN5 duplication even when thecheckpoint is active (Michaelis et al. 1997). Thus, wecompared the separation of Cen5-GFP in scc1-73 andscc1-73 cik1D cells after G1 release into 37� medium,which inactivates cohesin Scc1 and prevents the gener-ation of sister chromatid cohesion. We found that thekinetics of the appearance of two GFP dots was identicalin WT and cik1D mutant cells (Figure 1D), a strongindication that there is no delay in centromere dupli-cation in cik1D cells. Therefore, we conclude that theHU sensitivity of cik1 and kar3 mutants is not due to thedefects in DNA replication.

cik1 and kar3 mutants exhibit anaphase entry delayafter HU treatment: To further address why cik1D andkar3D mutants are sensitive to HU treatment, weexamined the cell cycle progression in synchronousWT, cik1D, and kar3D cells by determining the buddingindex and Pds1 protein level, as the degradation of Pds1marks anaphase entry (Cohen-Fix et al. 1996). After G1

release for 140 min, the majority of WTcells divided andbecame unbudded, indicating the completion of thecell cycle. However, �40% of cik1D and kar3D cells werestill large budded after G1 release for 180 min. Pds1protein disappeared 80 min after G1 release in WT cells,

Figure 1.—cik1D and kar3D mutants ex-hibit HU sensitivity without notable DNAsynthesis defects. (A) cik1D and kar3D mu-tants are sensitive to HU. Stationary phasecell cultures with indicated genotypes were10-fold diluted and spotted onto YPDplates containing 0 or 100 mm HU. Theplates were incubated at 25� for 3–4 daysbefore being scanned. (B) cik1D cells shownormal DNA synthesis kinetics after treat-ment with HU. G1-arrested WT and cik1Dcells were released into YPD medium con-taining 200 mm HU for 100 min at 25�. Af-ter HU was washed off, the cells werereleased into YPD medium, collected atthe indicated time points, and subjectedto PFGE after being fixed with 70% etha-nol. (C) cik1D and kar3D mutants do notshow dramatic viability loss after treatmentwith HU. WT, cik1D, kar3D, and rad53-21cells in midlog phase were released intoYPD medium containing 200 mm HU.Cells were spread onto YPD plates at the in-dicated times and incubated at 25� over-night. The formation minicolony wasexamined under a microscope and thepercentage of viable cells is shown (n .300). (D) cik1D cells show normal centro-mere duplication. G1-arrested scc1-73 andscc1-73 cik1D cells with CEN5-GFP and

TUB1-mCherry were released into YPD medium at 37�. The cells were collected every 30 min to examine the separation ofCen5-GFP dots by fluorescence microscopy. The percentage of large-budded cells (solid markers) and cells with two GFP foci(open markers) is shown.

400 H. Liu et al.

but cik1D and kar3D mutant cells displayed high levels ofPds1 even after G1 release for 180 min (Figure 2A). Thefailure of Pds1 protein degradation in cik1D and kar3D

mutant cells indicates an anaphase entry defect in thesemutants.

Because cik1D and kar3D mutant cells are HU sensitive,we speculated that cik1D and kar3D mutants would showmore significant cell cycle delay after HU treatment.Therefore, we examined Pds1 protein levels in cik1D andkar3D mutants after HU treatment. G1-arrested cells werereleased into medium containing 200 mm HU and in-cubated for 2 hr. After HU was washed off, the cells werereleased into YPD medium to determine the buddingindex and Pds1 protein levels. WT cells exited mitosisafter release from HU for 140 min; however, almost 80%of cik1D and kar3D mutants were still large budded at180 min. Consistently, Pds1 protein disappeared after120 min release from HU in WT cells, but cik1D andkar3D mutants showed persistent Pds1 protein levels(Figure 2B), suggesting the failure of anaphase entryin cik1D and kar3D mutants after HU treatment.

To further confirm the anaphase entry delay in cik1D

and kar3D mutants after HU exposure, we examinedkinetochore distribution in WT and cik1D mutant cellsafter HU treatment by visualizing GFP-tagged Mtw1, akinetochore protein (Goshima and Yanagida 2000;Pinsky et al. 2003). WT and cik1D cells in log phase weretreated with 200 mm HU for 2 hr at 25�, and we thenwashed off HU. Both WT and cik1D cells exhibited anunseparated cluster of Mtw1-GFP in HU-arrested cells.After 3 hr release from HU, the majority of WT cellsalready finished chromosome segregation as judged bythe appearance of separated Mtw1-GFP clusters in twodaughter cells. However, most of the cik1D mutant cellsstill showed a single Mtw1-GFP cluster (Figure 3A).Because the establishment of chromosome bipolarattachment results in two separated kinetochore clus-

ters (Goshima and Yanagida 2000; He et al. 2000), thisobservation indicates the potential failure of chromo-some bipolar attachment. We also compared the spindlemorphology in WT and cik1D mutant cells after HUexposure. Most WT cells either were in G1 phase or hadan elongated spindle after 3 hr release from HU, while themajority of cik1D cells showed a dot-like spindle structure(Figure 3B), raising a possibility that the abnormalspindle morphology could contribute to the singlekinetochore cluster in cik1D mutants after exposure toHU. It is also possible that the defect in Cik1/Kar3-dependent chromosome movement contributes to thesingle kinetochore cluster phenotype after HU treatment.

Isolation of HU-sensitive and temperature-sensitivecik1 mutants: Previous data demonstrate the spindledefects in cik1D and kar3D cells during vegetativegrowth. Moreover, both cik1D and kar3D mutants aretemperature sensitive for growth and the mutant cellsarrest as large-budded cells with a dot-like spindlestructure (Meluh and Rose 1990; Page and Snyder

1992; Manning et al. 1999). We noted that cik1D mutantcells showed dot-like spindle morphology after HUtreatment, but it remains unclear whether this abnor-mal spindle structure leads to their HU sensitivity.Therefore, we performed a PCR-based mutagenesis inan attempt to isolate temperature-sensitive (TS) andHU-sensitive cik1 mutants. With these HU-sensitivemutants, we might be able to address whether the HUsensitivity of cik1 and kar3 mutants correlates with theabnormal spindle structure.

Mutated cik1 genes in a centromere plasmid wereintroduced into cik1D mutant cells and the transform-ants were copied onto HU (100 mm) and YPD plates.The HU and YPD plates were incubated at 25� and 37�,respectively, to screen HU-sensitive and TS mutants.Some mutants (cik1HU) were sensitive to HU but grewwell at 37�, while others (cik1TS) grew well on HU plates

Figure 2.—cik1D andkar3D mutants exhibit de-layed anaphase entry afterHU treatment. (A) DelayedPds1 protein degradationin cik1D and kar3D mu-tants. G1-arrested PDS1-myc, cik1D PDS1-myc, andkar3D PDS1-myc cells werereleased into 25� YPD me-dium. a-factor was addedback after budding to blockthe second round of cell cy-cle. Cells were collected atthe indicated time pointsand protein samples wereprepared for the analysisof Pds1 protein levels by

Western blotting. Pgk1 protein levels are shown as a loading control. (B) cik1D and kar3D mutant cells fail to enter anaphaseafter exposure to HU. G1-arrested PDS1-myc, cik1D PDS1-myc, and kar3D PDS1-myc cells were released into YPD medium containing200 mm HU and incubated at 25� for 120 min. HU was then washed off and the cells were released into YPD medium containinga-factor. Cells were collected at the indicated time points for the preparation of protein samples. Pds1 protein levels are shown inthe bottom panel after Western blotting. The budding index is shown at the top.

Cik1/Kar3 Motor Complex 401

but failed to grow at 37� (Figure 4A). The plasmids withmutated cik1 genes were recovered from the trans-formants and sequenced, and the mutation sites areillustrated in Figure 4B. Interestingly, the two HU-sensitive mutants are frameshift mutations, which resultin truncation of part of the carboxyl-terminal end. In con-trast, the mutations in the two cik1TS mutants localizewithin the coiled-coil domain, which is required forCik1-Kar3 interaction (Barrett et al. 2000). As cik1HU5

exhibited similar HU sensitivity to cik1HU3, but with a lesstruncated C terminus, only this strain was used tocharacterize the mutant phenotype and it was namedcik1HU.

We first compared the cell cycle progression and thespindle morphology in WT, cik1HU, and cik1D cells.Compared to that in WT cells, we observed a slightlyincreased accumulation of large-budded cells in the

cik1HU mutant (from 34 to 40%), but cik1D deletionmutants showed many more large-budded cells (58%)(Figure 4C, left). Among the large-budded cells, 72% ofcik1D mutant cells showed a dot-like spindle structure,but this number reduced to 26% in cik1HU mutants andto 3% in WT cells (Figure 4C, right). Therefore, thecik1HU mutant exhibited much less spindle defect thanthe cik1D mutant. Although cik1HU mutants grew betteron YPD plates than cik1D mutants did, they exhibitedsimilar sensitivity to various concentrations of HU,ranging from 5 to 50 mm (Figure 4D). On the basis ofthese observations, we reason that defects other thanthe abnormal spindle structure contribute to the HUsensitivity in cik1HU mutants. The truncation in cik1HU

mutants may compromise the motor activity of theCik1/Kar3 complex, which is likely the cause of HUsensitivity.

cik1HU mutants exhibit a chromosome biorientationdefect in the presence of low concentrations of HU:Since WTand cik1HU mutant cells show distinct sensitivityto 20 mm HU (Figure 4D), we examined the chromo-some segregation in WT and cik1HU mutant cells withmCherry-tagged Tub1 and GFP-marked Mtw1 after treat-ment with 20 mm HU (Pinsky et al. 2003; Khmelinskii

et al. 2007). After incubation in 20 mm HU medium for6 hr at 25�, 63% cik1HU mutant cells arrested as large-budded cells, compared to 44% in WT cells. Among thelarge-budded cells, 45% of cik1HU mutant cells exhibiteda dot-like spindle structure. Therefore, we comparedthe distribution of Mtw1-GFP in WTand cik1HU cells witha normal short spindle structure. Interestingly, 78% ofWT cells showed two separated Mtw1-GFP clusters,indicating the establishment of chromosome bipolarattachment (Goshima and Yanagida 2000; He et al.2000). In contrast, only 39% cik1HU mutant cells with ashort spindle displayed two clearly separated Mtw1-GFPclusters (Figure 5A, middle panel). In some cik1HU

mutant cells, the Mtw1-GFP signals distributed alongthe entire spindle (Figure 5A, bottom panel, arrow),while some cells showed multiple Mtw1 clusters.

We have shown that the treatment of yeast cells withHU slows down kinetochore reassembly (Liu et al.2008), which may result in more detached chromo-somes. We reason that the Kar3-mediated chromosometransport becomes more critical for the reestablishmentof KT–MT interaction after HU treatment. Therefore,we examined the centromere localization relative to thespindle by using strains with mCherry-tagged TUB1 andGFP-marked centromere of chromosome IV (Cen4-GFP) (D’Amours et al. 2004; Tang and Wang 2006).The relative localization of Cen4-GFP to the spindle canbe grouped into three categories (Liu et al. 2008): cellswith one or two close GFP dots colocalized with themiddle part of the spindle (middle), a single GFP dot atone end of the spindle (end), and a single GFP dot thatis away from the spindle (out). After 6 hr incubation in20 mm HU, the accumulation of large-budded cells in

Figure 3.—cik1D mutants fail to establish bipolar attach-ment after HU treatment. (A) HU treatment results in dra-matically delayed chromosome segregation in cik1D mutants.G1-arrested MTW1-GFP and cik1D MTW1-GFP cells were re-leased into YPD medium containing 200 mm HU for 2 hrat 25�. HU was then washed off and the cells were releasedinto YPD medium. a-Factor was added to block the secondround of the cell cycle. Cells were collected after releasefor 3 hr for fluorescence microscopy. The Mtw1-GFP signalin some representative cells is shown in the left panel. Theright panel presents the percentage of large-budded cells withone Mtw1-GFP cluster before and after 3 hr release from HUarrest (n . 300). Black bars (WT); grey bars (cik1D).(B) cik1Dcells exhibit abnormal spindle morphology. TUB1-GFP andcik1D TUB1-GFP cells in midlog phase were released intoYPD medium containing 200 mm HU for 2 hr at 25�. HUwas then washed off and the cells were released into YPD me-dium with a-factor for 3 hr. The spindle morphology beforeand after HU treatment is shown.

402 H. Liu et al.

the cik1HU mutant was more dramatic compared to thatof WT cells. In 76% of WT cells with a short spindle, theCen4-GFP was localized at the middle of the spindle, butthe number was reduced to 39% in cik1HU cells. MoreCen4-GFP dots localized at the end of the spindle incik1HU mutant cells (53%) compared to WT cells (23%),and 8% of mutant cells showed a Cen4-GFP dot that wasaway from the spindle (Figure 5B). One possibility isthat some cik1HU mutant cells fail to move the capturedchromosomes to the spindle. Moreover, even after achromosome has been moved close to the spindle polethrough one of the attached sister kinetochores, cik1and kar3 mutant cells may have difficulty in moving thesister kinetochore toward the other pole. Therefore, weobserved more cik1HU mutant cells showing a Cen4-GFPdot close to one end of the spindle.

cik1HU mutants exhibit delayed anaphase entry afterexposure to HU: In addition to the analysis of cell cycledefects in cik1HU mutant cells in a low concentration ofHU, we also examined cell cycle progression in cik1HU

mutant cells after a short exposure to a high concen-tration of HU. First, we compared undisturbed cell cycle

progression in WT, cik1D, and cik1HU mutant cells.Without HU treatment, the cell cycle progression ofthe cik1HU mutant was relatively normal as indicated bythe budding index and Pds1 protein level, which was inclear contrast to that of the cik1D mutant (Figure 6A).We next analyzed the cell cycle progression in these cellsafter exposure to a high concentration of HU. G1-synchronized cells with Pds1-myc were grown in 200 mm

HU medium for 2 hr at 25�. After we washed off HU, thecells were released into YPD medium and collected toanalyze cell cycle progression. After HU exposure, cik1HU

mutant cells showed an obvious cell cycle delay becausemore mutant cells remained large budded, althoughthe delay was not as dramatic as that of the cik1D mutant.Consistently, the Pds1 protein level was stabilized incik1HU mutant cells after release from HU arrest (Figure6B), suggesting that some cik1HU cells exhibit delay inanaphase onset after HU treatment.

We further assayed the relative localization of Cen4-GFP to the spindle in WT and cik1HU mutants after HUexposure. Similarly, G1-arrested WTand cik1HU cells withCen4-GFP and Tub1-mCherry were first grown in 200 mm

Figure 4.—Isolation of cik1HU and cik1TS mutants. (A) The growth of cik1HU and cik1TS mutants. cik1D cells harboring a vector andCIK1, cik1TS, and cik1HU plasmids were grown to saturation and then serial 10-fold diluted and spotted onto URA dropout plateswith or without 100 mm HU. The plates were incubated at 25� or 37� as indicated for 3 days before they were scanned. (B) Thediagram of mutation sites in the cik1TS and cik1HU mutants. (C) The cell cycle distribution and the spindle morphology in cik1mutant cells. WT, cik1D, and cik1HU mutant cells with TUB1-mCherry were grown to midlog phase. Cells were fixed to count the bud-ding index and to examine the spindle morphology. The left panel shows the percentage of unbudded, small, and large-buddedcells. The percentage of different spindle morphologies in large-budded cells is shown in the right panel (n . 300). (D) Thegrowth of WT, cik1HU, and cik1D mutants on YPD plates containing various concentrations of HU. Saturated cultures were 10-folddiluted and spotted onto YPD and HU plates. The plates were scanned after a 3-day incubation at 25�.

Cik1/Kar3 Motor Complex 403

HU for 2 hr and then released into YPD medium.After HU release, cik1HU mutant cells showed an obvi-ous anaphase entry delay as indicated by the budding in-dex and the appearance of elongated spindles (Figure6, C and D). After release from HU for 80 min, 59%of WT cells showed one or two Cen4-GFP dots thatlocalized in the middle part of the spindle, but thisnumber was only 28% in cik1HU mutant cells (Figure 6E).There were more cik1HU mutant cells with a Cen4-GFPdot that was either close to one spindle end or away fromthe spindle (Figure 6E, arrow), indicating a possiblefailure of the poleward chromosome transport. Theseresults support the notion that cik1HU mutants havedifficulty in establishing chromosome bipolar attach-ment after HU treatment. The impaired chromosometransport in cik1HU mutants may contribute to thisdefect.

DISCUSSION

In budding yeast, kinetochores are attached to microtu-bules throughout the cell cycle except when the DNAreplication machinery encounters the kinetochore thatassembles on centromeric DNA. The replication-inducedkinetochore disassembly allows the migration of the de-tached centromeres away from the spindle pole. Morechromosomes will be detached during stressful DNAreplication because of the longer time required to finish

the duplication of centromeric DNA. After the kineto-chore reassembles, a poleward transport mechanismmoves the captured chromosome close to the spindle.Thus, chromosome transport may become more criticalwhen yeast cells are treated with HU that slows downDNA synthesis. Kar3, a minus-end-directed motor pro-tein, has been found to be required for polewardchromosome transport (Tanaka et al. 2005). Here, weshow that yeast cells deficient in the Cik1/Kar3 motorcomplex are very sensitive to HU. On the basis of thesimilarity of the mitotic defects in cik1 and kar3 mutants(Page and Snyder 1992; Manning et al. 1999), wereason that the Cik1/Kar3 complex is responsible forchromosome transport. Our data suggest that thechromosome transport defect in cik1 and kar3 mutantscontributes to the HU sensitivity.

Because cik1D and kar3D mutants exhibit a dot-likespindle structure, especially when incubated at highertemperatures (Meluh and Rose 1990; Page andSnyder 1992), either the abnormal spindle structureor the chromosome transport defect could contributeto the HU sensitivity. To distinguish these possibilities,we isolated HU-sensitive cik1HU mutants that were nottemperature sensitive and showed relatively normalspindle structure. Interestingly, these mutants exhibitedsimilar HU sensitivity to cik1D. Although some cik1HU

mutant cells had abnormal spindles in the presence ofHU, we found that many cik1HU cells with a normal short

Figure 5.—cik1HU mutants exhibit abnormalkinetochore distribution in the presence of20 mm HU. (A) cik1HU mutant cells show abnor-mal kinetochore distribution. Asynchronous WTand cik1HU cells with Tub1-mCherry and Mtw1-GFP were released into YPD medium containing20 mm HU for 6 hr at 25�. Cells before and afterHU treatment were fixed to examine the bud-ding index and the distribution of Mtw1-GFP sig-nals. The percentage of large-budded cells isshown in the top panel. The percentage of cellswith two kinetochore clusters and a short spindleis shown in the middle. The experiment was re-peated three times. The arrow (bottom panel) in-dicates a cik1HU mutant cell with abnormallydistributed Mtw1-GFP signal. (B) cik1HU mutantcells show defects in chromosome bipolar attach-ment. WT and cik1HU cells with Tub1-mCherryand Cen4-GFP were treated as described in A.The percentage of large-budded cells is shownin the top panel. The percentage of cells with dif-ferent Cen4-GFP localization relative to the spin-dle is shown in the middle (n . 300). Therelative localization of Cen4-GFP to the spindlein some representative cells is shown in the bot-tom panel.

404 H. Liu et al.

spindle failed to form two Mtw1-GFP foci after incuba-tion in 20 mm HU, an indication of the failure ofchromosome bipolar attachment. We also noted thatthe relative localization of the GFP-marked centromereto the spindle in cik1HU cells was different from that inWT cells. There were increased numbers of cells with aCen4-GFP dot either at one end of the spindle or awayfrom the spindle in cik1HU mutants after HU treatment.Therefore, we reason that a spindle-independent de-fect, likely the failure of chromosome transport, con-tributes to the HU sensitivity in cik1 and kar3 mutants,although we cannot exclude the possibility that theabnormal spindle structure contributes to their HUsensitivity as well.

The failure to transport captured chromosomes to thespindle pole can explain the increased population ofcik1HU cells with a Cen4-GFP dot away from the spindle.Nevertheless, we also observed that many cik1HU cells

showed a Cen4-GFP dot at one end of the spindle. Onepossibility is that, after one of the sister kinetochores iscaptured and transported toward the spindle, the othersister kinetochore will be captured by a microtubule fromthe other spindle pole and the Cik1/Kar3 motor complexmoves it to the spindle midzone. Therefore, defects inthis process may lead to the accumulation of cells with aCen4-GFP dot at one end of the spindle. If Cik1/Kar3is required to move sister kinetochores toward oppositedirections, this movement may generate tension acrosssister kinetochores, but further research is needed to testthis possibility.

We noted the abnormal distribution of kinetochoreproteins in cik1HU mutant cells incubated in mediumcontaining HU and we speculate that the chromosometransport defect leads to this phenotype. A plus-end-directed motor protein, Cin8, is required for chromo-some congression by regulating the spindle microtubule

Figure 6.—cik1HU mutants ex-hibit delayed establishment ofbipolar attachment after HUtreatment. (A) Comparison ofcell cycle progression in synchro-nized WT, cik1D, and cik1HU mu-tants. G1-arrested PDS1-myc,cik1D PDS1-myc, and cik1HU PDS1-myc cells were released into 25�YPD medium. Cells were col-lected at the indicated timepoints and protein samples wereprepared for the analysis ofPds1 protein levels by Westernblotting. Pgk1 protein levels areshown as a loading control. Thebudding index is shown in thetop panel. (B) cik1HU mutantsshow delayed anaphase entry af-ter exposure to HU. G1-arrestedPDS1-myc, cik1D PDS1-myc, andcik1HU PDS1-myc cells were re-leased into YPD medium contain-ing 200 mm HU and incubated at25� for 120 min. HU was thenwashed off and the cells were re-leased into YPD medium contain-ing a-factor. The cells weretreated as described in A. Thebudding index and the Pds1 pro-tein levels are shown. (C and D)The cell cycle progression ofWT and cik1HU mutant cells afterHU treatment. G1-arrested WTand cik1HU cells with Cen4-GFPand Tub1-mCherry were releasedinto 200 mm HU YPD and incu-bated at 25� for 120 min. HUwas then washed off and the cells

were released into YPD medium containing a-factor. Cells were collected at the indicated time points for budding index andfluorescence microscopy. The percentage of large-budded cells is shown in C, and the percentage of cells with elongated spindlesis shown in D. (E) cik1HU mutant cells exhibit defects in establishing chromosome bipolar attachment. The relative localization ofCen4-GFP to the spindle in the cells collected above was analyzed by fluorescence microscopy. The percentage of cells withindifferent categories is shown in the top panel (n . 300), and the representative cells are shown in the bottom panel. The arrowindicates a cell with Cen4-GFP away from the spindle.

Cik1/Kar3 Motor Complex 405

dynamics, and the defect in this regulation leads to thefailure of formation of two kinetochore clusters beforeanaphase entry (Gardner et al. 2008a). The similarkinetochore distribution pattern in cik1HU and cin8mutants raises the possibility that the Cik1/Kar3 complexalso plays a role in chromosome congression. Indeed, theCik1/Kar3 complex binds to the end of the microtubuleand promotes its depolymerization (Maddox et al. 2003;Sproul et al. 2005). However, we found that cin8 mu-tants are not sensitive to HU. Although we cannotexclude the role of Cik1/Kar3 in chromosome con-gression, the HU sensitivity of cik1 and kar3 mutants isunlikely due to the defect in chromosome congression.

Recent data from the Tanaka laboratory suggest thatkinetochores either can slide along the microtubulelateral surface, which is driven by Kar3, or are tetheredat the microtubule ends through the DASH/Dam1kinetochore complex and pulled poleward as micro-tubules shrink (end-on pulling) (Tanaka et al. 2007).Ask1, a component of the DASH/Dam1 complex, wasoriginally identified as a protein required for growth inthe presence of HU (Alcasabas et al. 2001; Li et al.2002). ask1-3 mutants also exhibit HU sensitivity and wespeculated that the partially elongated spindle contrib-utes to the HU sensitivity, because deletion of CIN8suppressed the spindle elongation and the HU sensitivityof the ask1-3 mutant (Liu et al. 2008). As both Kar3 andthe DASH/Dam1 complex are required for the polewardchromosome transport, it is possible that the defectivechromosome transport in ask1-3 mutants contributes tothe HU sensitivity as well. Our observation that ask1-3 andcik1D mutants are synthetically lethal supports thispossibility (data not shown). It will be interesting todetermine the chromosome transport defect in ask1-3and another HU-sensitive mutant, ask1-1.

We isolated two cik1HU mutants and both mutantgenes are truncated at the C termini. It remains unclearhow the truncation of the C terminus affects the func-tion of the Cik1/Kar3 motor complex. One possibilityis that the truncated fraction of the Cik1 protein me-diates the interaction of the Cik1/Kar3 complex withthe kinetochore. We can examine the association ofKar3 with centromeric DNA in the cik1HU mutants totest this possibility. Alternately, the C terminus of theCik1 protein binds to microtubules and allows themotility of the Kar3 motor, like Vik1, the other partnerof Kar3 (Chu et al. 2005; Allingham et al. 2007).Therefore, the truncation of the C terminus of Cik1may abolish the motility of the Cik1/Kar3 complex, butmore experiments are needed to clarify this issue.

In mammals, dynein and dynactin are responsible forthe minus-end-directed chromosome movement (Rieder

and Alexander 1990; King et al. 2000). In fission yeast,the homologs of Kar3 and Dam1 are required forchromosome transport through lateral sliding or theend-on pulling mechanism (Gachet et al. 2008), suggest-ing that the mechanism is conserved. Here we character-

ized the role of the Cik1/Kar3 motor complex inresponse to stressful DNA replication, but this functionis likely specific to budding yeast because only buddingyeast cells establish KT–MT interaction during S-phase.Nevertheless, this poleward chromosome movement mayfacilitate chromosome bipolar attachment in all eukary-otic cells. First, the poleward movement might beimportant to orient sister kinetochores to favor theestablishment of chromosome bipolar attachment. Ithas been shown that the abrogation of the polewardchromosome transport in higher eukaryotic cells leads tomisoriented kinetochores (Varma et al. 2008). Moreover,after a chromosome is attached by microtubules fromopposite spindle poles, poleward movement of sisterkinetochores could generate tension on this chromo-some, which further regulates KT–MT interaction andcell cycle progression. Further, we are interested incharacterizing the role of Cik1/Kar3 in the establishmentof chromosome bipolar attachment.

We thank Steve Elledge, Mark Rose, Sue Biggins, Uttam Surana, andElmar Schiebel for yeast strains. We also thank Akash Gunjan foradvice on PCR mutagenesis and Ruth Didier for the FACS analysis. Weare grateful to Daniel Richmond and Kelly McKnight who read thismanuscript. This work was supported by a Research Scholar Grant(RSG-08-104-010CCG) from the American Cancer Society, a Multi-Disciplinary Grant from the Florida State University (FSU) Council onResearch and Creativity, and a Research Enhancement Grant from theFSU College of Medicine (to Y.W.).

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Communicating editor: N. M. Hollingsworth

Cik1/Kar3 Motor Complex 407

GENETICSSupporting Information

http://www.genetics.org/cgi/content/full/genetics.110.125468/DC1

The Cik1/Kar3 Motor Complex Is Required for the ProperKinetochore–Microtubule Interaction After

Stressful DNA Replication

Hong Liu, Fengzhi Jin, Fengshan Liang, Xuemei Tian and Yanchang Wang

Copyright � 2011 by the Genetics Society of AmericaDOI: 10.1534/genetics.110.125468

H. Liu et al. 2 SI

TABLE S1

HU sensitive mutants from the ATCC yeast deletion strains

ORF Gene HU Sensitivity

YCR063W BUD31 +a

YCR066W RAD18 ++b

YCR077C PAT1 +

YFR041C ERJ5 +

YHR039C-B VMA10 +

YLR447C VMA6 +

YER111C SWI4 +

YER116C SLX8 +

YER090W TRP2 +

YHL025W SNF6 +

YHR168W MTG2 +

YNL250W RAD50 +

YML112W CTK3 +

YMR116C ASC1 +

YMR125W GCR3 +

YJR090C GRR1 ++

YJR104C SOD1 ++

YJR139C HOM6 +

YJL027C Putative protein ++

YJL140W RPB4 ++

YJL175W Dubious ++

YKL139W CTK1 +

YNR052C POP2 +

YPL268W PLC1 +

YRP024W YME1 +

YBR112C CYC8 +

YBR289W SNF5 ++

YCR047C BUD23 +

YCR053W THR4 ++

YPL078C ATP4 +

YNL059C ARP5 +

YOL148C SPT20 ++

YAL035W FUN12 +

YAL021C CCR4 +

YBL025W RRN10 ++

YBL058W SHP1 +

YBR035C PDX3 +

YBR036C CSG2 +

H. Liu et al. 3 SI

YBR069C VAP1 +

YBR097W VPS15 +

YBR127C VMA2 ++

YBR171W SEC66 +

YBR203W COS11 ++

YDL185W TFP1 ++

YDR004W RAD57 +

YDR028C REG1 ++

YDR076W RAD55 +

YDR126W SWF1 +

YDR158W HOM2 +

YDR159W SAC3 +

YDR245W MNN10 +

YDR264C AKR1 ++

YDR359C VID21 +

YDR364C CDC40 ++

YDR369C XRS2 +

YDR378C LSM6 ++

YDR386W MUS81 +

YDR432W NPL3 +

YDR433W Dubious +

YDR463W SPT1 ++

YDR470C UGO1 +

YDR532C KRE28 +

YEL044W IES6 +

YEL051W VMA8 ++

YER014W HEM14 +

YER068W MOT2 ++

YER070W RNR1 +

YER083C RMD7 +

YER086W ILV1 +

YGL012W ERG4 +

YGL024W SST3 +

YGL025C PGD1 +

YGL058W RAD6 ++

YGL070C RPB9 +

YGL148W ARO2 +

YGR104C SRB5 +

YGR105W VMA21 +

YHR060W VMA22 ++

YHR081W LRP1 +

H. Liu et al. 4 SI

YIL128W MET18 +

YJL124C LSM1 +

YJL115W ASF1 +

YKL054C DEF1 +

YKL120W OAC1 ++

YKL119C VPH2 ++

YKL080W VMA5 ++

YLR148W PEP3 +

YLR182W SWI6 +

YLR240W VPS34 ++

YLR268W SEC22 +

YLR369W SSQ1 +

YML032C RAD52 +

YML021C MAC1 +

YMR198W CIK1 +

YMR202W ERG2 +

YNL139C RLR1 +

YOR141C ARP8 +

YOL012C HTZ1 ++

YPL254W HFI1 ++

YPL234C TFP3 ++

YPL178W CDC2 +

YPL129W ANC1 +

YPL045W VPS16 +

YPR036W VMA13 +

YPR072W NOT5 ++

YPR099C Dubious ++

YPR135W POB1 +

YPR163C TIF3 +

a. Slow growth. b. No growth.