identification and characterization of new elements involved in

Molecular Biology of the CellVol. 5, 147-160, February 1994

Identification and Characterization of New ElementsInvolved in Checkpoint and Feedback Controlsin Fission YeastF. Al-Khodairy, E. Fotou, K.S. Sheldrick, D.J.F. Griffiths,A.R. Lehmann, and A.M. Carr

MRC Cell Mutation Unit, Sussex University, Falmer, Sussex BN1 9RR, United Kingdom

Submitted October 13, 1993; Accepted December 16, 1993Monitoring Editor: Mitsuhiro Yanagida

To investigate the mechanisms that ensure the dependency relationships between cell cycleevents and to investigate the checkpoints that prevent progression through the cell cycleafter DNA damage, we have isolated mutants defective in the checkpoint and feedbackcontrol pathways. We report the isolation and characterization of 11 new loci that definedistinct classes of mutants defective in one or more of the checkpoint and feedback controlpathways. Two mutants, rad26.T12 and rad27.T15, were selected for molecular analysis.The null allele of the rad26 gene (rad26.d) shares the phenotype reported for the "checkpointrad" mutants radl, rad3, rad9, radi7, and husl, which are defective in the radiation check-point and in the feedback controls that ensure the order of cell cycle events. The null alleleof the rad27 gene (rad27.d) defines a new class of Schizosaccharomyces pombe mutant. Therad27 complementing gene codes for a putative protein kinase that is required for cell cyclearrest after DNA damage but not for the feedback control that links mitosis to the completionof prior DNA synthesis (the same gene has recently been described by Walworth et al.(1993) as chkl). These properties are similar to those of the rad9 gene of Saccharomycescerevisiae. A comparative analysis of the radiation responses in rad26.d, rad26.T12, andrad27.d cells has revealed the existence of two separable responses to DNA damage con-trolled by the "checkpoint rad" genes. The first, G2 arrest, is defective in rad27.d andrad26.d but is unaffected in rad26.T12 cells. The second response is not associated with G2arrest after DNA damage and is defective in rad26.d and rad26.T12 but not rad27.d cells.A study of the radiation sensitivity of these mutants through the cell cycle suggests thatthis second response is associated with S phase and that the checkpoint rad mutants, inaddition to an inability to arrest mitosis after radiation, are defective in an S phase radiationcheckpoint.

INTRODUCTION

The arrest of cell cycle progression in response to in-complete DNA synthesis is a general phenomenon thatis under genetic control in both Schizosaccharomycespombe and Saccharomyces cerevisiae (reviewed in Enochand Nurse, 1991; Murray, 1992; Sheldrick and Carr,1993). The pathway that mediates this response, the Sphase feedback control, helps maintain ordered pro-gression through the cell cycle by making mitosis de-pendent on the prior completion of DNA synthesis.Feedback control pathways can detect a wide variety

of cell cycle events. For example, in both budding andfission yeast the majority of cdc mutants block the cellcycle at a specific point. Such cells continue to accu-mulate mass but do not attempt to continue progressionthrough the cell cycle and therefore do not enter mitosisor attempt cell division.

Exposure to DNA-damaging agents (such as ionizingradiation) also prevents progression through the cellcycle (reviewed in Hartwell and Weinert, 1989). Thefirst genetic link between the response to DNA-dam-aging agents and the cell cycle was established by anal-ysis of the rad9 mutant of S. cerevisiae (Weinert and

© 1994 by The American Society for Cell Biology 147

F. Al-Khodairy et al.

Hartwell, 1988). Cells harboring this mutation failed toprevent mitosis after irradiation and lost viability as aresult of segregating damaged chromosomes. Analysisof cell cycle arrest caused by a variety of cdc mutantsat the restrictive temperature in a rad9 genetic back-ground indicated that RAD9 function was required toprevent cell cycle progression when a block was im-posed in late S and G2 phases of the cycle, but notwhen the block was imposed during early S phase orin G1 phase (Weinert and Hartwell, 1993). rad9 mutantsin S. cerevisiae do not attempt mitosis when DNA syn-thesis is prevented by the addition of hydroxyurea tothe growth medium.

In fission yeast several mutations have been identifiedthat result in an inability to prevent progression throughthe cell cycle when DNA synthesis is inhibited by hy-droxyurea. The cdc2.3w mutation, which was initiallyidentified as a mutation in the cdc2 gene that affectedthe timing of entry into mitosis, is an example of sucha mutation (Enoch and Nurse, 1990). The properties ofthe cdc2.3w mutant established that the feedback controlof mitosis is maintained by a genetic pathway and in-dicated that the signals generated by the cell in responseto incomplete DNA synthesis ultimately impinged onthe activity of the p34cdc2 mitotic kinase. It has recentlybeen established that cdc2.3w mutant cells show a nor-mal cell cycle arrest after treatment with DNA-damagingagents (Sheldrick and Carr, 1993). This separates thepathway responding to a block in S phase of the cyclefrom the pathway responding to DNA damage.A second class of mutants that abolish the replication

feedback controls (checkpoint rad mutants) has beenreported in S. pombe. The radl, rad3, rad9, radl7, andhusl mutants are unable to arrest the cell cycle whenDNA synthesis is inhibited by hydroxyurea (Al-Kho-dairy and Carr, 1992; Enoch et al., 1992; Rowley et al.,1992b). The checkpoint rad mutants are also unable toarrest mitosis after treatment with DNA-damagingagents (Al-Khodairy and Carr, 1992; Rowley et al.,1992b). The genetic overlap between the feedback con-trols that ensure the dependency of mitosis on the com-pletion of DNA synthesis and the radiation checkpointpathway suggests that several elements of these oth-erwise distinct mechanisms are shared.The feedback control pathways and the radiation

checkpoint pathway can each be considered to consistof three distinct parts: 1) a detection system, 2) a signaltransduction mechanism, and 3) a specific interactionwith the cell cycle machinery. To clarify the relationshipsbetween the various pathways, we have used a com-bination of genetic screens to identify new mutants de-ficient in the feedback control pathways and the radia-tion checkpoint. We have cloned and sequenced thegenes corresponding to two of these mutants and cre-ated null alleles. Analysis of the null mutants has clar-ified some of the complex interrelationships betweenthe feedback controls and the radiation checkpoint. Our

collection of mutants provides new tools for dissectingthe mode of action of these pathways.

MATERIALS AND METHODS

Molecular and Genetic TechniquesStandard genetic procedures were used as described in Gutz et al.(1974). Transformation of S. pombe was performed using the proto-plasting technique described in Beach and Nurse (1981). Routine mo-lecular biology techniques were performed as described in Sambrooket al. (1989). Bacterial strains DH5a and DH5aF' were used for cloningand generating single stranded DNA. Mutagenesis was performedusing 2.5% ethyl methanesulfonate (EMS) on either S. pombe strainspOll Ura4.D18 ade6.704 leul.32 h- (Murray et al., 1991) or sp867cdcl7.K42 Ura4.D18 leul.32 h- in YE media (Gutz et al., 1974) shakingat room temperature for 150 min.

Survival AnalysisUV survival was performed in one of two different ways. 1) For generalsurvival analysis, a known density of cells were plated onto appropriateagar plates and exposed to a dose of UV light determined by thesetting on a Stratagene Stratalinker (La Jolla, CA). 2) For survival ofrad-rad double mutants at low doses, cells were treated as above butirradiated under a UVC lamp at a dose rate of 6 J/m2. T-irradiationwas performed on growing cells in liquid culture in YE media at adensity of 1 X 104 cells ml-' using a Gammacell 1000 (Nordion, On-tario, Canada) '37Cs source (12 Gy min-'). Survival in hydroxyureawas performed on growing asynchronous cultures in supplementedminimal media in the presence of 10 mM hydroxyurea. Aliquots wereremoved, diluted, and plated at regular intervals, and after 2-3 dincubation at 27°C, colonies were scored and survival expressed as apercentage of colonies formed by samples plated immediately beforeaddition of hydroxyurea. Survival of cdc rad/hus double mutants aftera temperature shift from 27 to 35.5°C was measured by plating knowndensities of cells and incubating plates at the restrictive temperaturefor various periods of time followed by incubation at the permissivetemperature for 2-3 d. Colonies were counted, and survival was ex-pressed as a percentage of colonies formed on equivalent plates in-cubated directly at the permissive temperature.

Preparation of Synchronous CulturesCultures of synchronous cells were prepared on a 7-30% lactose gra-dient essentially as described in Barbet and Carr (1993) (modifiedfrom Mitchison and Carter, 1975). Cells in G2 were recovered fromthe top of the gradient and inoculated into fresh media. Samples werethen divided and subjected to appropriate treatment. Aliquots wereremoved at 15- or 30-min intervals and fixed in methanol for esti-mation of cell cycle position by 4,6-diamidino-2-phenylindole (DAPI)and calcofluor staining.

Radiation Checkpoint MeasurementsThe extent of the radiation checkpoint defect after T-irradiation wasmeasured by exposing samples of a synchronous G2 culture to ionizingradiation of the appropriate dose. At 15-min intervals aliquots werefixed in methanol, and the percentage of cells that had passed throughmitosis was estimated by fluorescence microscopy after DAPI andCalcofluor staining. The extent of the radiation checkpoint defect fol-lowing UV irradiation was measured by first synchronizing cells usinga cdc25 genetic background (Al-Khodairy and Carr, 1992). Cells werearrested for 150 min at the restrictive temperature, and samples wereexposed to appropriate doses of UV light. After subsequent releasefrom the restrictive temperature (cells are in G2 immediately precedingmitosis), aliquots were fixed in methanol at 15-min intervals, and thepercentage of cells that had passed mitosis was estimated as above.

Molecular Biology of the Cell148

Checkpoint Mutants in S. pombe

Replication Feedback Control MeasurementsThe extent of the replication feedback control defect was measuredby incubating one-half of a synchronous culture in supplementedminimal media containing 10 mM hydroxyurea and the other half inmedia only. At 30-min intervals aliquots from both samples werefixed in methanol and the percentage of septated cells was scored byfluorescence microscopy after DAPI and Calcofluor staining.

Cloning of Genes Complementing rad26.T12 andrad27.T15Strains rad26.T12 ura4.D18 leul.32 ade6.704 h- and rad27.T15 ura4.D18leul.32 ade6.704 h- were transformed with pURSP1 and pURSP2 ge-nomic libraries described in Barbet et al. (1992). For each mutant,- 20 000 independent colonies were divided into four separate poolsand subjected to selection by irradiation as described previously(Murray et al., 1992). After three rounds of selection, individual col-onies were analyzed for coinstability of the radiation resistant andura+ phenotypes. For rad26.T12 two independent clones derived frompURSP2 were obtained and complementing plasmid was recovered.Restriction mapping showed these to be identical 3.8-kilobase (kb)fragments. For rad27.T15 two independent clones derived from eachlibrary were obtained and complementing plasmid recovered. Re-striction mapping showed these to be identical 4.4-kb fragments. Foreach mutant the complementing activity was localized, and the min-imum complementing fragment was subjected to sequence analysisby generating nested deletions in pGEM3 vectors.

cDNA Analysis of Complementing GenesSearching of the sequenced regions for open reading frames (ORFs)and intron specific sequences (Prabhala et al., 1992) indicated bothgenes contained multiple introns. An S. pombe pREP1 cDNA library(a gift from P. Nurse, ICRF, London, UK) was screened by hybrid-ization with fragments of both complementing genes. A cDNA wasobtained for the rad26-, but not for rad27-complementing gene. Therad26 cDNA indicated that three exons were spliced together to for atotal ORF of 615 aas. The entire coding region from the first methionineto the stop codon was amplified using polymerase chain reaction (PCR)with the primers 26-Nde (AACCATATGATGGCTGATGAAAGT)and 26-Bam (TTGGATCCTCTAAAAATTAGTGTACAA) doned intoa T vector, and the Nde I/BamHI insert subcloned into pREP1. Func-tional analysis indicated that this construct could complement theradiation sensitivity of a rad26.d (null) allele to approaching wild-typelevels.The original cDNA isolated contained a considerable upstream re-

gion that showed no evidence of splicing. To investigate whether thisregion was spliced in mRNA (several poor acceptor/donors could bepostulated), we used primer 26-up (GTGCGCATTTTGGAAAA-GACT) with both primer 26-Ilspan (TGCTAAAGTTCTAGAA-GAAAAATAC) or 26-I2span (AAGCGTTATCCATTCATTCAATT-CAG) to PCR amplify from two independent cDNA libraries. In allcases bands were obtained that indicated a splice event. The PCRproducts from 26-up/26-Ilspan reactions were cloned into a T vector.Six isolates from the pREP1 cDNA library and six from the cDNAlibrary described in Fikes et al. (1990) were sequenced. Eleven out of12 cloned PCR products contained one splice event (11/12, Figure 4)and one out of the 12 contained a second splice event, which utilizeda different donor sequence. In neither case did the splice event generatean additional ORF for the rad26 gene. Recent evidence (Murray et al.,1991; Walworth et al., 1993; Carr et al., 1994; unpublished data) sug-gests that S. pombe rad genes are expressed at a low level, often containnovel splice donor sequences, and can be incorrectly spliced in somecDNA products. In addition, several very small amino terminal exonsare seen in some DNA repair genes, and introns in excess of 700 basepairs have been detected (unpublished data). Introns have also beendetected in noncoding regions. Although we are unable to eliminate

the possibility that the rad26 gene contains additional upstream exons,the evidence presented here suggests that this is not the case.The rad27 cDNA was generated by PCR from the cDNA library

described in Fikes et al. (1990) using primers 27-Nde (CAACA-TATGGCTCAAAAATTAGATAAC) and 27-Sal (GATGTCGACT-TAATTTTGTGAAACATC). The PCR product was sequenced andcloned in pREP41. Functional analysis showed that this constructcould complement the radiation sensitivity of the rad27.d (null) alleleto wild-type levels. Sequence analysis of the resulting product dem-onstrated a single ORF of 496 aas generated by splicing together sevenexons.

Gene Deletion of rad26 and rad27The disruption construct for rad26 was created by replacing a 1.4-kbApaI to HpaI fragment with the 1.7-kb ura4+ gene using the methoddescribed in Barbet et al. (1992). Similar methods were used to generatethe rad27 disruption construct by replacing the 0.65-kb SpeI to NcoIIfragment with the ura4+ gene. Both disruption constructs were isolatedas linear fragments consisting of the ura4 gene flanked by rad-specificsequences. The linear DNAs were used to transform an S. pombe diploidura4.D18 strain (splOl) (Grimm et al., 1988) to uracil prototropy.Twelve colonies for each fragment were tested for integration. Forrad26 11 stable diploids were obtained. For rad27 9 stable diploidswere obtained. Four representative diploids for rad26 and four forrad27 were used to isolate h+/h' derivatives. These were sporulatedand subjected to random spore analysis. In all cases a rad phenotypewas always associated with a ura4+ marker in haploids. Southem blotanalysis using probes specific to the rad sequences and to the ura4gene showed that in three out of four cases for rad26 and four out offour cases for rad27 a single ura4 gene had replaced the appropriaterad specific fragment. In the one remaining case, multiple ura4-specficsequences appeared to have integrated at the rad26 locus.

For both rad26 and rad27 a representative single integrant diploidwas chosen and subjected to tetrad analysis. Six individual tetradsfor each strain were dissected, and the spores were allowed to ger-minate. In all cases the ura4+ phenotype always cosegragated withan obvious radiation-sensitive phenotype in the expected ratio. Therad26.d (null) allele was crossed to the rad26.T12 mutant, and therad27.d (null) allele was crossed to the rad27.T15 mutant. In both casesno radiation-resistant progeny were detected from >1000 spores. Thisindicated that the cloned and deleted genes were equivalent to therespective mutant loci originally used in the cloning events.

Radiation Sensitivity Through the CycleGrowing cells (YE) were synchronized and the resulting culture was in-cubated at 27°C. At 15-min intervals for 7 h a sample was taken anddiluted to 1 X 104 cells mr1. One aliquot was plated and irradiated withthe appropriate dose of UV. A second aliquot was T-irradiated and plated.A third aliquot was plated without treatment (a control for each timepoint). Two plates were prepared for each treatment. After incubation at27°C for 3 d, the numbers of colonies were counted, and the percentagesurvival at each time for both treatments was calculated. To follow thecourse of the cell cyde, every 30 min for the duration of the experimenta sample was fixed in methanol and subjected to DAPI and Calcofluorstaining. Graphs of the percentage septated cells against time and ofmitotic index against time give a reference for position in the cell cydeat any given point. In fission yeast, S phase almost immediately followsmitosis (Gl is very short), and a septum is present during this period.Thus, the percentage of septated cells is a reasonable estimate of thenumber of cells in S phase.

RESULTS

Genetic Screens for New MutantsTo screen for new mutants we utilized our knowledgeof the phenotype of known checkpoint and feedback

Vol. 5, February 1994 149


Table 1. Complementation/linkage analysis of new mutants

CIs T3 T9 T12 T15 T21 T39 T45 164 173 TiN T2N T3N T4N R/H

A Ti Y Y Y Y Y Y Y Y Y Y - - r24B T3 Y Y Y Y Y Y Y N Y - r34B T9 Y Y Y Y Y Y Y Y r33A T12 Y Y Y Y Y Y Y r26B T15 Y Y Y Y Y Y r27C T21 Y Y Y Y Y h6C T39 Y Y Y Y h7B T45 Y Y Y - r29C 164 Y Y h8B 173 Y r34A TiN N N N r30

Cls, class to which mutant is assigned. R/H, gene name assigned to locus r for rad and h for hus. Class A, radiation and hydroxyurea sensitive;class B, radiation sensitive; class C, hydroxyurea sensitive. Y, .10% wild-type recombinants upon free spore analysis; N, <0.1% wild-type;-,cross not done.

control mutants in S. pombe and in S. cerevisiae. Essen-tially two genetic screens have been employed:

1) Mutagenesis in a cdcl7.K42 background. In the firstscreen we selected initially for radiation sensitivity andalso for rapid cell death after a transient shift to therestrictive temperature in a cdcl7.K42 (ts DNA ligase)genetic background. The rationale for this was to at-tempt to identify, in addition to new checkpoint rad-like mutants, a further possible class of S. pombe mutantsthat might be defective in the radiation checkpoint butnot in the S phase feedback control (an equivalent phe-notype to the rad9 mutant of S. cerevisiae). UV-sensitive

mutants showing sensitivity to a transient temperatureshift (111 isolates) were backcrossed to wild-type. Mu-tants in which the UV sensitivity could be separatedfrom the transient temperature sensitivity by removalof the cdc17 background (and in which this sensitivitycould be restored by crossing cdc17 back in) were re-tained for further analysis (85 isolates).

2) Hydroxyurea sensitivity screen. A second screenwas designed to isolate checkpoint rad-like mutants andmutants that were defective specifically in the S phasefeedback controls but not in the radiation checkpoint.Wild-type cells were mutagenized, and hydroxyureasensitive clones were isolated.

Table 2. Phenotypes of selected mutants

Mutant Allele rad5 hu8 % Cut Morphology cl78 uvckPt

rad24 Ti ++ +/- 5 semi-wee + partialrad26 T12 ++ ++ 40 normal ++ normalrad27 T15 ++ - 3 normal +++ defectrad29 T45 +++ +/- 2 bi-nuc - NDrad30 TiN ++ ++ 28 normal ++ normalrad33 T9 ++ - 1 normal ++ normalrad34 T3 ++ - 0 normal +++ normalhus6 T21 +t ++ 50 normal + normalhus7 164 - + 35 normal + normalhus8 T39 - + 30 normal + normalhuwl T17 - + 11 wee ++ NDhusl T152 ++++ ++++ 58 normal +++ defecthus5 T62 ++ ++ 20 elongated + normalwildtype - - 0 normal - normal

rad' represents comparative radiation sensitivity; hu', comparative hydroxyurea sensitivity; and % cut, thepercentage of cut nuclei when an asynchronous culture is exposed to 10 mM hydroxyurea for 6 h. c178 representsthe severity of the rapid death phenotype after a transient shift to the restrictive temperature in a ts. cdcl 7.K42(DNA ligase) genetic background. uvck* represents the UV irradiation checkpoint status of the mutant cells asassayed by the cdc25 genetic synchronization method (Al-Khodairy and Carr, 1992). hus6 is temperature sensitivefor a moderate radiation sensitivity phenotype. rad29 cells are usually binucleate and large. It was not possibleto isolate cdc25 double mutants with rad29 for reasons which are unclear. huwi mapped close to cdc25, andthe double mutant with cdc25 could not be isolated with confidence.



60

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40

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60

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Figure 1. Ionizing radiation checkpoint analysis. The extent of thedelay to mitosis imposed on cells irradiated in G2 was assayed usingsynchronous cultures of wt, wild-type; r24, rad24.Tl; r26, rad26.T12;r27, rad27.T15; r33, rad33.T3; r34, rad34.T9; and h6, hus6.T21. Eachculture was divided in half, and one-half was irradiated with 50 Gyof ionizing radiation. The irradiated and unirradiated samples were

analyzed at 15-min intervals by DAPI and Calcofluor staining for thepercentage of cells that had passed through mitosis. Only the rad27mutant showed a complete loss of cell cycle delay after treatment. Apartial loss of the radiation checkpoint is seen with the rad24 mutant.This mutant is difficult to synchronize because of a morphology defect.

Linkage AnalysisA total of 561 mutants were isolated from the twoscreens. Each mutant was analyzed by spotting 103 cellsonto two plates: plate A, YE agar plus 10 mM hydroxy-urea; plate B, YE agar followed by 100 J/m2 UV radia-tion. After incubation overnight, plates were inspectedmicroscopically for growth and cell morphology. Onthe basis of these results 81 separate mutants were cho-sen for further analysis. These were assigned to threecategories: mutants with sensitivity to both UV and hy-droxyurea (73 isolates), mutants sensitive primarily toUV (5 isolates), and mutants sensitive primarily to hy-droxyurea (3 isolates). All mutants were crossed to theradl, rad3, rad9, and radi7 radiation and hydroxyurea-sensitive mutants of S. pombe (Al-Khodairy and Carr,1992; Rowley et al., 1992b). Remaining mutants were

crossed to the husl, hus2, hus3, hus4, and hus5 mutants(Enoch et al., 1992) and to the cdc2.3w mutant. In total,two alleles of radl, 49 alleles of rad3, nine alleles ofrad9, five alleles of radi7, one allele of husl, and one

allele of hus5 were identified. The remaining mutantswere then crossed to each other to assign them to com-plementation (linkage) groups. A total of 11 new com-

plementation groups were identified. With the exceptionof four alleles of rad3O and two alleles of rad34, eachnew complementation group contained a single mutant(see Table 1).

Primary CharacterizationThirteen mutants (11 new mutants plus the alleles ofhusl and hus5 isolated in this screen) were characterizedby measuring the radiation and hydroxyurea sensitivity.Growth rate and general morphology were assessed byeye, and the percentage of "cut" nuclei (Hirano et al.,1986) present after incubation for 6 h in the presence

of hydroxyurea was scored by DAPI staining. In addi-tion, the mutants were crossed to the ts. DNA ligase

40 - wt+hu - r24+hu

309

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Figure 2. S phase feedback control analysis. The extent of the delayto mitosis caused by exposing a synchronous culture of cells to 10mM hydroxyurea was assayed. wt, wild-type; r24, rad24.Tl; r26,rad26.T12; r27, rad27.T15; h6, hus6.T21; huw, huwl.T17. Each culturewas divided in half, and 10 mM of hydroxyurea was added to one-

half at time 30 min. The inhibition of S phase (which occurs at ap-

proximately the first septation peak) delays the subsequent mitosisfor the duration of the experiment in wild-type cells. A loss of the Sphase feedback control was only manifested in hus6.T21 cells. A con-

stant 10% of septating cells are seen with rad26.T12 and rad24.Tl inthe presence of hydroxyurea. This may signify a partial defect in theS phase control in these strains.

Vol. 5, February 1994

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rad24.Tl and rad26.T12; radiation-sensitive mutants,NdEB rad27.T15, rad33.T9, and rad34.T3; hydroxyurea-sensi-tive mutant, hus6.T21; hydroxyurea-sensitive mutant

B + with a wee phenotype, huwl.T17.Hp

Radiation and Replication Mitotic Checkpoint/Feedback Control Deficiencies in the SelectedMutants

ATOAvsl_ ~ ~ ~ ~~~I-A

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For each of the seven selected mutants, the timing ofmitosis after irradiation of synchronous cells was es-tablished (Figure 1). Only rad27.T15 displayed a totalloss of the radiation checkpoint, whereas rad24.TI ap-peared to be partially defective. For each mutant (withthe exception of rad33.T9 and rad34.T3) the septationindex of a synchronous population of cells was scored

P Nd after the addition of hydroxyurea to G2 cells (Figure 2).hus6.T21 cells did not arrest mitosis in this assay. Apercentage of rad24.Tl and rad26.T12 mutant cells sep-tated, but the bulk of the culture remained as singlemononucleate cells of variable length. The remainingmutants did not enter mitosis for the duration of theassay and elongated relatively uniformly.

PH P H Nd S N N B EP EB

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Figure 3. Cloning of rad26 and rad27 genes. (A) The restriction mapof the rad26 gene region. (B) The restriction map of the rad27 generegion. The organization of the ORFs is shown in the expandedl regions.U, introns; 0l, exons. The subclones used in the functional analysisare indicated by solid lines and the complementing activity is indicatedby + and -. 13, the region replaced by the ura4' gene. A, ApaI; B,BamHl; E, EcoRI; H, HindIII; Hp, HpaI; N, NcoI; Nd, NdeI; P, Pstl; S,Spel. Lines above and below the bar indicate sites derived from thelibrary vector.

mutant cdcl 7.K42. The rate of loss of viability in thedouble mutants was measured at various times of in-cubation at 35.50C and compared to a single cdc mutantcontrols. Each mutant (with the exception of huwl) wascrossed to cdc25.22 and tested for the G2 arrest phe-notype after UV radiation (Al-Khodairy and Carr, 1992)(see Table 2). On the basis of the information obtainedthe following seven mutants were selected for furtheranalysis: radiation and hydroxyurea-sensitive mutants,

Cloning of rad26 and rad27rad27.T15, which had lost the radiation checkpoint, dis-played considerably lower sensitivities to UV and ion-izing radiation when compared to the checkpoint radmutants radl, rad3, rad9, and radi 7. In contrast,rad26.T12 mutants had a normal G2 arrest deficiencybut remained sensitive to UV and ionizing radiation.We decided to clone the genes corresponding to thesetwo mutants to establish the phenotype of the null al-leles. The rad26 gene (Figure 3A) was cloned on a 3.8-kb fragment from the pURSP2 library, and the rad27gene (Figure 3B) was cloned on a 4.4-kb fragment fromthe pURSPl and the pURSP2 libraries described in Bar-bet et al. (1992). The regions corresponding to the com-plementing activity were determined by functionalanalysis, and the minimum region was cloned and se-

quenced.The rad26 complementing fragment could potentially

code for three exons, if two intervening sequences wereproposed on the basis of intron consensus sequences inS. pombe (Prabhala et al., 1992). PCR and functionalanalysis of cDNA indicated that the rad26 gene con-tained three exons separated by two introns (Figure 4)that could potentially code for an acidic protein of 615amino acids with a molecular weight of 69 kDa and apI of 4.92. Garnier-Robson analysis predicts a very longstretch of alpha helix at the N terminus of the protein.No homology was detected between the putative rad26protein and entries in the database. Two nuclear local-ization signals can be descemed from the sequence. PCRand functional analysis showed that rad27 was com-

posed of seven exons separated by six introns. The rad27gene could potentially code for a serine/threonine pro-

Molecular Biology of the Cell

APH

Nd

P EA

B

152


Figure 4. Sequence of the rad26gene. The DNA sequence and pre-dicted translation product of therad26 gene are shown. Two intronshave been identified by cDNAanalysis that bring together threeexons to create a 615 amino acidprotein. A further intron upstreamof. the ATG (with two differentsplice donor sites and a single ac-ceptor site, marked in bold) has alsobeen identified, but neither spliceevent extends the rad26 ORF (seeMATERIALS AND METHODS).Introns within the coding regionare shown in lower case, and donorand acceptor sequences are under-lined. Two potential nuclear loca-tion signals are underlined. EMBLaccession number X76558.

Patl 11/12 1/12 -241CTGCAGTCATATTCTGCTATTAGTGTTTC T1GrnTATAT TAGT

-240 12/12 -121

-120 EcoRl Apal -1ATTTATTATGAATTCAATTTTTTAGTaAAT&GACCTGMACAAACAAA1 120ATGATGGCTGATGAAAAOTTTGACT AGA OCAACAAGCTCAACCCOTCAAGTCAGTTCTCAM M A D E S F D L E S L O S D E I F E G V N L D E L E Q Q A Q T Q V Q A Q S S Q121 240GTAGTTGTACCGAGTGAGAAAAGC AAATAATTCATCATACACCAATT CGCGGTTCG TACCTCTAATGACV V V P S E K Q X Q N L N L P N S Y T N S S Q K V R E S T V N S Q A S L S S N D241 360CTTCGAACAGhACTTTTrlAATTAAGTCGGGAGAAAATGCATCTTGTACAACCTTCTAAAACAATC GACAAATCGCCCTAGAATCGTTAAATAATTCTATAAAGCAAAAL R T E LL I K S G E N A I L R A N L L K Q S E A N N A A L E S L N N S I K Q K361 480CAAGATGAAT _GAAAACTACCAAACGAATCCCT TGAQ D E Y Q R K L E E L K K E I E Y A K T K S L F H E R E A Q D A I E T M K K N E481 EcoRl 600AAGAATGTAAAGATCTCCGATTATGAAAAAATCCCATGAAGAAGATGGTGACATAAT_T _GCTGAAACAGCK R C K R N S P I M K K S H E E D G D N K L L S S S D Q L A K S T K H A A K N S601 720CCTTCT AA_GTAAAGCTGAAGC&AhTGCTCACAATTGAGTTAGTCATT GC CIOCCTGATGAAGATTTAP S K K K R K T S V A T A E D A S T D S V S S S I A I S D A S L S L S L MK D L721 840CaTCCAAGTTTCTATTCT aatttttaaactgtcgtgttaaattttattcccttttcttacttttc &ATTCAGAACTTTAO3CATACGTATTTGGTrGGL S L Q K R E D L Y F S R T L A Y V F G G841 960TTGTATGCATSCTT aA&GGAGaAOTOTTOTAATAACTTGAAAGCTCAATATACAGTCCCGATCTATCTATGGATTCCTCCAATTATGTACAATCC M H S L E T I E G E E E G E C L F N N L K A L I Y S P D L S N D S S N Y V Q S961 1080TGSCGTTCAAACACATC GTGTSAATI CTTTGCTATAACATCTTTGTTTAATGCATTATTAATTTTOGACCCTAAATCATCCACV V Q T S S S I L N Y S M K K L L Y N A S F A I T S L F N A L L I L D P K S S T1081 1200CTTTATCTTTCAGG3AAAATGcrrAA AGACAAATTTGA ATACTTF I F Q E N V V S L I S G F L L K E Y E K S N F L D S K F Y V L I D F L Y L Y L

1201 1320ATCTAT GAGGAGGCGTATTCAAACATTACTAAAGCGSTTGATCCGTCGTTATTCGAATCCTGCATTCGAGTrGCAAAATGCTCCTTCTGTATTGGTGS I A R E S A D D F A N I T K A V D P S L F E S C I R V QN A P S L I K C G V C

1321 Hpal 1440TTTAATTATTAGTTCCACCACTCCTT_ aTAAACCGGACTTAACAGCCTTTATGL I I S S T T P S F C A S V N L L N A D D K S Q E S L Q L F T T M A H I L V V1441 1560TACTACTAGGGAAAGGATTAAT?rTCCTTTCCTGAAgcfttacgaattgtgcttataatctttgcattgcttatttctaacaaat5gAATGAATGGATAACGCTTCATCGCTTTQTCAT T R E R I N F P E L N E W I T L H R F V I1561 1680TATCmAC =VCGTGA GGTTTAAAAGTATGCAATCCCT lM_G TATCATCAGCAACTCC

S F F T V F I Q M S G N I G K E I L K V C N P L I V C I G L A I T W Y H Q Q L L1681 1800TTTCGTCAATGTACCCTCAAAATGAATGCGTAGAGATTTTTAGTATCTCTTGTTCG&CTTCTGTACATTTTATCTTCCG^&mTACAAAGTGTAATCTA

S S M Y P Q N E C V E I L V S L V R L L Y I L S S E D L S S K F M L A E N A L Q1801 1920AACCTCGTTTCGTTATGCAATAGCATOCTGCGCTTCG&CGATACTGAAAAAAAAGCGTTTGGGCAATTTAmGCTGATACAAATIG

P R F V Y A I A C C A F G D T E Q K A F G N L G E E N Y F L T T E L L E V C V S1921 2040CTCCGAACGACGTTCCATTTGTAGACGTATTTT _ _ CGTC

P E E L E Q L Y T N F STOP2041 2160CCGCACCCACATCCTCGCAAATTAAATACAAAT(A cm CACAAAAACATCCCCATTCCGTTCTTT AGTAAAACTTACanaCTATATTACCTOCAA2161 2280TAAAAT _ ATATTAASlOICAAAGTACCACT CAATrCATTAAC7rST2281 2400CATAATAAAGAAAATCCGT CAAA A ACAACATCATAACATAACAGGAAAATCAGATAATTTCTCCAATT TCATTCTTTATCCATTGAA2401 2520TGCTTTTT SACAAAAlST TCCTTAAATGG&GAGiATCCATTCCCAGGTTGATGTGAACAGCTTTTOTTTGAGTATAATTGGTAAGCCGTAAGA2521 2640

2CCCAATTCGCGACCGATGCCACTCTCCTTATAACCACCAAAG641AGGATOTGNTAAGTTA CATCACAAAATCCTTC CTGACCTTGATGGC2641 N4del

tein kinase that shows no particular similarity to pre-viously reported protein kinases. The kinase domain islocated within the amino terminal half of the predictedprotein, which contains a total of 496 amino acid resi-dues and has a molecular weight of 56.5 kDa (Fig-ure 5).

Gene Deletion of rad26 and rad27For both genes a construct was generated in which asignificant region internal to the ORFs was deleted andreplaced with a functional ura4 gene. After integrationinto a diploid and subsequent genetic analysis, bothgenes were found to be inessential for growth. The DNAdamage survival and hydroxyurea survival phenotypesof the rad27 deletion mutant (rad27.d) were almost in-distinguishable from the rad27.T15 allele (Figure 6), in-dicating that this mutant is essentially also a null allele.

In contrast, the rad26 deletion mutant (rad26.d) wasconsiderably more sensitive to DNA-damaging agents(Figure 6) and showed a more acute defect after expo-sure to hydroxyurea than the rad26.T12 mutant (see alsoFigures 2, 9). This may indicate that rad26.T12 encodesa partially functional protein.

The Radiation Checkpoint in rad26.d andrad27.d CellsAfter exposure to ionizing radiation, synchronous cul-tures of rad26.d and rad27.d cells displayed no significantdelay in cell cycle progression (Figure 7). In commonwith the radl, rad3, rad9, and radl7 null alleles, rad26.dmutant cells exhibited a high sensitivity to irradiation.Like the radiation sensitivity of the checkpoint rad mu-tants (Al-Khodairy and Carr, 1992; Rowley et al., 1992),only a small fraction of this sensitivity could be reversed



HindIII -361AAGCTTATTACAAACCATTTAATATA TCAAGATGGCAACGTTTTTACTTGAACGCTATG

-360 -241ACCGTCAACAGCCATGC CCGATTGATATTATTAGAATCCATTCTAlG GCS ATGASraG-240 -121TAGGAGGTAAGTATAACACAAACAACTGTOCTATGTAATAAAGACOGTAC CATTTTATATCCAATTATCATTAAGmA A

-120 NdeI -1TTACCTTA-rATTTAATGc _ CTTTCCCTTTACACCAAAGA

1 120ATGGCTCAAAAATTAGALTAACTTT __otoattttgatttgaaaaatactatatactaacttaagcaaoCOTCCGTM A Q K L D N F P Y H I G R E I G T A F A S V R121 SpeI 240TTATGTTACGATGATAATGCTAAAATATATGCT TAATocAAAAcTcAcTATTGTATGAATGCAGGOGTAToGGCTAGAAGGATGGCTAGTGAAATACAACTTCACL C Y D D N A K I Y A V K F V N K K H A T S C M N A G V W A R R M A S E I Q L H241 360AAACTATGCAATGGACATAAAAATATCATTCATTTTTmACAGCAGAAAATCC AT GC AGT GGTGACTTA TTGACAAAATAGO tosoK L C N G H K N I I H F Y N T A E N P Q W R W V V L E F A Q G G D L F D K I E361_-s_--- s

480

P D V G I D E D V A Q F Y F A Q L M E G I S F M H S600

K G V A H R D L K P E N I L L D Y N G N L K I S D F G F A S L F S Y K G K S R L601 720TTGAATAGTCCAGTGGGTAGTCCACCATACOCTGCT CCAGAAATTACACAGCAGTATGATOG TTCAAAAOTTGATGTCTO= CTGCTTAT AGAAL N S P V G S P P Y A A P E I T Q Q Y D G S K V D V W S C G I I L F A L L L G N

721 NcoI 840CACACCTTGGGATGAAGCAATTAGCAACACTGGTGACTACTTGTTTATAAAAAACAATGTGAACOCCCGTCTTACCACCCATGATGTTTCTCCAGGAGCCTATTaMtatCCtgT P W D E A I S N T G D Y L L Y K K Q C E R P S Y H P W N L L S P G A Y S841 NcoI 960actgaaattcctttaaactaatgatS§agCGATTATCACCGGAATGCTTCGAGGTCATAGGCATTTCAAACATGTTGTACAGCACCCATGrCTACCTCAAGTACACCA

I I T G M L R S D P F K R Y S V K H V V Q H P W L T S S T P961 1080TTCGAACTAAAAATGGGAATTGTGCTACaTA _ T TGCTCATCTAGAG CATCTCAAAAataaF R T K N G N C A D P V A L A S R L M L K L R I D L D K P R L A S R A S O N1081 1200gatcttgcacagttgtttcatatttctaactct ttcaatCG CGATACGGrmTTTTT GTTTAAATACAAG;GTGACTTAAGTA

D S G F S M T Q P A F XK N D Q K E L D R V E V Y G1201 1320CGCCTTATCCCAGeCAGTACAATTAATAAAAATATTGACGTTACTGaAATCCTTGAAAMGACCCCTCATTCwrrGGTG^AGVOtattattA L S Q P V Q L N R N I D V T E I L E R D P S L S Q F C E N E G F I K R L1321 1440tatttaaacctaaat tttttctaactatatgaaaG IGCAAAGCTAAGAATTTCTACGAAATTTGTCCTCCTGAAGCCACTAGGTTTTATTCTAGGGCGTCGAGGACCAT

A K I A K N F Y E I C P P E R L T R F Y S R A S R E T I1441 1560TATAGATCATCTTTATGACAGTCTACGACTACTTGCAATCTCAGTGACTATGAAATATGTT GATGCACTTTTTAAAGTACGATTCTCTTGCAAGGI D H L Y D S L R L L A I S V T M K Y V R N Q T I L Y V N L H D R R K C L L Q G

1561 BamHI 1680TGTTATTGAACTAACTAATTAGGCCATAACTTAGAGsCTTATAAACTTTATAAAGCGTAATGGOGATCCTCTTGhATTGAGAeAAATTTTTTAAAgtatgtctttatcatagggatttcatV I E L T N L G H N L E L I N F I K R N G D P L E W R K F F S1681 1800atactaatccatgtgaaagAACGTTGTCAGTTCAATAGGGAAGCCGATTGTTCTTACAGATGTTTCACAAAATTAATTGCACATCTTTGAAGGTCGTGCCAGTTCGTTTGTAAAAGAA

N V V S S I G K P I V L T D V S Q N STOP1801 1920ACCTGTTGTTTGACTTCGTT GTGAATT ATTTTCAATTTTTGTAGTACAT CTAGAAT GATT CAAGCAAATATCAATGGTTGCGAATGCGAG1921 2040ATATATTATTAGCATAAGGATAGTTCTTCCGACTGCATGCCGTTTAATACAATGAcAATTmrCMr_TAGTGAGATATCTGA2041 2160C CATTTTCTTGGTCGGTTCAAbGOSISGTGATAAAAACTCATCTTCTTTACAAAATAGTAAAAG2161 EcoRI 2280GTTCTTTTTTGTGAATTCTTGTCTCTCTTTGGCT CAGGTAAGCATGCAAAGG AAAAT AGTAC ATAAAATATCGTCTGGAGTCTCPstITOCAG

by an artificial arrest of the cell cycle (unpublished data),indicating a further defect in the response to radiationin this group of mutants. In contrast, rad27.d cells weresignificantly less radiation sensitive, and the majorityof this sensitivity could be rescued by an artificial blockto mitosis after irradiation. This is similar to the situationseen in S. cerevisiae rad9 mutant cells (Weinert andHartwell, 1988) and indicates that the defect in rad27.dcells after irradiation can be fully accounted for by itsinability to arrest mitosis.UV irradiation did not delay mitosis significantly in

rad26.d or rad27.d cells when the cells were releasedfrom G2 into mitosis immediately after irradiation (wild-type cells manifest the radiation checkpoint in thesecircumstances, see Figure 8A). If cells were held in G2for a brief period of time after irradiation (15 min), ashort delay in the timing of mitosis could be detectedwith rad27.d but not rad26.d mutants (Figure 8B). It ispossible that a rad27 independent radiation checkpointpathway is functioning in rad27.d cells under these cir-cumstances. However, as no delay was detected afterr-irradiation and as repair synthesis is more extensive

Figure 5. Sequence of the rad27gene. The DNA sequence and pre-dicted translation product of therad27 gene are shown. Six intronshave been identified by cDNAanalysis that bring together sevenexons to create a 496 amino acidprotein with the seven domainscommon to serine/threonine pro-tein kinases. Introns are shown inlowercase, and the kinase homol-ogy is underlined. EMBL accessionnumber L13742.

after UV rather that T-irradiation, it is possible that thisbrief delay to mitosis is the result of the DNA synthesisassociated with DNA repair activating the replicationfeedback pathway (which is still operational in rad27.dcells, see below).

Hydroxyurea Feedback Controls in rad26.d andrad27.d Cellsrad26.d mutants are completely defective in the feedbackcontrol that prevents mitosis when DNA synthesis isinhibited by hydroxyurea (Figure 9A). rad26.d cells arealso highly sensitive to exposure to hydroxyurea, show-ing a much greater loss of viability than the cdc2.3wmutant, which is similarly unable to prevent mitosisafter the inhibition of DNA synthesis by hydroxyurea(Enoch and Nurse, 1990). A similar phenotype has beenreported for husi mutant cells. This is of interest as En-och et al. (1992) demonstrated that synchronous culturesof husi cells and cdc2.3w cells entered mitosis with thesame kinetics when exposed to hydroxyurea but thatthe husi cells died more rapidly than the cdc2.3w cells.

Molecular Biology of the Cell

481

..gcatgaacctttcgtttttactaacggacttacaattLa_qAGCCTGATOTAGGTATTGAT CATGCACTC

154


0 100 200 300 0 1000uv dose J/m2 y dose Gy

wild-type (Figure 7A). This observation indicates that_ this mutant separates a sector of the radiation sensitivity

(A in Figure 6B) from that caused by failure to arrest inG2 (B in Figure 6B).The radiation sensitivity of the rad26.d (null) allele is

thus composed of two parts: the first because of a G212 arrest deficiency and the second because of a G2 arrest-

independent defect. The rad26.T12 mutant is defectiveonly in this second (G2 arrest-independent) damageresponse. Combining the rad26.T12 mutation with amutation that (on its own) causes only a G2 arrest defectshould, therefore, result in a double mutant with a ra-

7d diation sensitivity similar to the rad26.d (null) mutant.7T15 We have been able to recreate such a phenotype by2 creating a double mutant strain with the rad26.T12 (ar-2000

Figure 6. Radiation survival of rad26 and rad27 strains. The UV (A)and gamma (B) survival of wild-type (wt), rad26.T12 (r26T12), rad26.d(r26.d), rad27.T15 (r27T15), and rad27.d (r27.d) cells are shown. Littledifference is evident between the two rad27 alleles. The rad26 nullallele is considerably more sensitive than the rad26.T12 allele.

The authors concluded that the increased lethality ofthe husl mutant, when compared to the cdc2.3w mutant,could not be simply because of entry into mitosis. Thisimplied that a second defect was present in husl cellsthat occurs earlier in the cell cycle (possibly during Sphase) and that this defect could cause cell death evenif the delay to mitosis was intact.We have shown that a similar early lethality is evident

in rad26.d (null) mutant cells and that the time at whichexposure to hydroxyurea causes cell death coincideswith the expected time of S phase (Figure 9B). Thesedata indicate that rad26.d cells, in common with huslcells, have a defect in the reversibility of arrest of DNAsynthesis after exposure to hydroxyurea, which can bedistinguished from the defect in preventing mitosis aftersuch treatment.

rad27.d cells retained the replication feedback controland showed no evidence of entering mitosis after ex-posure to hydroxyurea (Figure 9A). rad27.d cells werenot sensitive to exposure to hydroxyurea for <10 h,longer than the period for which bulk DNA synthesisis inhibited by hydroxyurea in wild-type S. pombe cells(Sazer and Nurse, 1994).

Separation of the Radiation Sensitivity from a G2Arrest Defect in rad26.T12rad26.T12 mutant cells are significantly less sensitive toirradiation than rad26.d mutant cells (Figure 6). If thiswas because of a partially active protein, we would ex-pect to see a partial defect in G2 arrest after ionizingradiation. When tested, no difference could be seen be-tween the timing of mitosis after T-irradiation of a syn-chronous culture of rad26.T12 cells when compared to

A100

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- r26dOGv r26d 5GYr26d1 OO;yr26d250Gy

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- r27d1G

yr27d 250Gy

i.6.

O 100 100 200

w-* t OGywt 5OGy

* wt 1 OOGy-- wt 25OGy

wt 500Gyn wt 75OGy

r26p OGyr26p SOGy

--r26p IlOOGyr26p 250gy

r26p 500Gy

Ir26p 75OGyI

300Time (min)

B

10 20 30 0 500 1 0uv dose J/m2 Gamma dose Gy

Figure 7. (A) Gamma irradiation checkpoint analysis of rad26 andrad27. wt, wild-type; the null mutants of rad26 (r26.d) and rad27 (r27.d)and the rad26.T12 (r26T12) mutant have been analyzed for the abilityto arrest mitosis after treatment with increasing doses of ionizing ra-diation. Synchronous cultures prepared from lactose gradients wereirradiated in G2, and the percentage of cells passing mitosis was scoredby DAPI and Calcofluor staining. (B) Radiation sensitivity of therad26.T12 rad27.d double mutant. The UV and gamma radiation sen-sitivities of the rad26.T12 (r26T12), rad26.d (r26d), and rad27.d (r27d)single mutants and the rad27.d/rad26.T12 (r27d-r26T12) double mu-tant were tested at low doses of radiation. The phenotype of therad26.T12/rad27.d double mutant is similar to the rad26.d single mu-tant, supporting the suggestion that rad26.T12 is defective in a G2arrest-independent radiation response (see Table 4).


I/ j


0 100 200 300Time (min)

100 20CTime (min)

Figure 8. UV irradiation checkpoint analysis of rad26 and rad27.The delay to mitosis caused by increasing doses of UV radiation ismeasured in wild-type (wt) and the null alleles of rad26 (r26d) andrad27 (r27d) by synchronizing the cells using the cdc25.22 allele. (A)Wild-type cells show a dose-dependent delay to mitosis. No delay isseen after irradiation in either null mutant when cells are releasedinto mitosis immediately after irradiation. (B) When cells are held inG2 for 15 min after irradiation, wild-type cells delay mitosis, a shortdelay is seen in rad27 null cells, and no delay is seen in rad26 nullcells. The possible reasons for the delay in rad27 but not rad26 nullmutants in this experiment are discussed in the text.

rest proficient) mutation and the rad27.d (arrest deficient)mutation (see Figure 7B). The double mutant shows asimilar sensitivity to both UV and T-radiation as therad26.d mutant.

In the rad27.d (null) mutant, maximum radiation sen-sitivity occurred immediately preceding mitosis, corre-lating with the loss of G2 arrest in this strain. In therad9.d and rad26.d mutants, the maximum radiationsensitivity was also seen in late G2, but the cells weremore radiation sensitive than the rad27.d mutant at allstages of the cell cycle. In contrast, the rad26.T12 mutantwas maximally sensitive in S phase, indicating that thecomponent of radiation sensitivity that is independentof G2 arrest and is defective in the rad26.T12 mutant iscaused by a defect in an S phase-related process. Giventhe increased sensitivity of rad26.T12 during S phase(indeed at the dose tested there was essentially no hy-persensitivity in such cells irradiated during G2), it might

A B

0 200 400Time (min)

120 1202 -~2.-w-sur.wva

13100,1 --- ---2.3w-hu -100u -. 2.3w+huX 80 \80,

60 60 Q*40 . / 40w

e 20 / 20

0o 100 200 300 40Time (min)

600

Radiation Sensitivity Through the Cell CycleAs discussed above, the sensitivity of the null allele ofthe rad26.d checkpoint rad mutant is composed of atleast two elements. One is because of a defect in pre-venting mitosis after DNA damage, whereas the otheris a consequence of as yet uncharacterized defects inthe response to DNA damage. A similar bipartite sen-sitivity appears to apply to the response to hydroxyurea(Figure 9B and Enoch et al., 1992), where viability islost both because of an aberrant mitosis (equivalent tothe loss of viability in cdc2.3w cells) and because of adefect in the reversible arrest of DNA synthesis. To testif the G2 arrest-independent sensitivities to hydroxy-urea and to DNA damage were functionally linked, wetested the sensitivity to UV and ionizing radiation ofwild-type, rad27.d, rad9.d, rad26.d, and rad26.T12 mu-tants through the cell cycle (Figure 10).

Figure 9. (A) S phase feedback controls in rad26 and rad27 nullalleles. The ability of wild-type (wt), rad26 (r26d), and rad27 (r27d)null alleles to arrest mitosis after inhibition of S phase by hydroxyureahas been assayed by exposing one-half of a synchronous culture to10 mM hydroxyurea. The percentage of septated cells was estimatedby DAPI and Calcofluor staining. Nuclear morphology indicated mi-tosis preceded septation in all cases. For the duration of the experiment,no evidence of septation (mitosis) is detected in wild-type and rad27.dcells. rad26.d cells entered a catastrophic mitosis at a time slightlypreceding mitosis in the untreated control. This phenomenon of ad-vanced attempted mitosis and septation is reproducible and is alsoevident in synchronous cultures of rad9 and radi7 deletion mutantsafter exposure to hydroxyurea. (B) Hydroxyurea sensitivity throughthe cell cycle. The sensitivity of rad26.d cells to hydroxyurea exposurecorrelates to S phase of the cell cycle, whereas the sensitivity of thecdc2.3w mutant correlates to mitosis. Survival was measured by ex-posing one-half of a synchronous culture to hydroxyurea and removingsamples at regular intervals for survival assays. In addition, treatedand untreated samples were fixed in methanol at 30-min intervals.Septation index of each sample was determined by fluorescent mi-croscopy after DAPI and Calcofluor staining.



Time (min) Time (min) Time (min)

L2200 300Time (min) Time (min)

Figure 10. UV and T survival through the cell cycle. rad27.d (r27), rad9.d (r9), rad26.d (r26), and rad26.T12 (r26T12) mutants are compared towild-type (wt) cells for survival profile through the cell cycle. The top panels show the septation index, the bottom panels show the mitoticindex, and the middle panel shows the survival profile. All three rad deletion (null) strains appeared to show similar profiles, being mostsensitive immediately before mitosis and recovering to maximum resistance rapidly after mitosis. The increased radiation sensitivity in rad9and rad26 strains, when compared to the rad27 strain, appears to be evident through the cell cycle. The maximum sensitivity of the rad26.T12mutant occurs in S phase for both UV and gamma irradiation. rad9.d 15 J/m2 and 75 Gy, rad26.d 15 J/m2 and 75 Gy, rad27.d 30 J/m2 and 75Gy, rad26.T12 15 J/m2 and 50 Gy, wild-type 30 J/m2 and 100 Gy. Note that the survival axis for rad26.T12 and wild-type are 10-100%, andthe survival axis for rad9.d, rad26.d, and rad27.d are 1-100%.

be anticipated that the rad9.d and rad26.d mutants mightalso have shown additional sensitivity (compared torad27.d) during S phase, rather than manifesting thisextra sensitivity throughout the cycle. This discrepancycould be explained if the potentially lethal DNA damagecaused by the irradiation persisted for a period of timeapproaching that taken for a single cell cycle. If thiswere so, irradiated rad9.d and rad26.d mutant cells wouldenter S phase with damaged DNA irrespective of thetime during the cycle at which they had been irradiated.In contrast, rad26.T12 cells (which have a normal G2arrest response) irradiated in G2 (the majority of thecell cycle for S. pombe) would not enter mitosis or thesubsequent S phase until a period of time had elapsedthat allowed all the damage to be repaired.

Summary of the Phenotype of the RemainingMutantsIn addition to mutants in the rad26 and chkl (rad27)genes, our study has also identified a number of otherelements either directly or indirectly involved in thefeedback control and checkpoint pathways. The mainproperties of these are summarized below. The exampleof the rad26.T12 and rad26.d phenotypes serves to il-lustrate that the molecular analysis of these genes, andthe subsequent creation and analysis of null alleles, isessential for a full understanding of their function.huwl. huwl is mildly sensitive to hydroxyurea exposurebut not demonstrating a feedback control deficiency.Cells are phenotypically wee, dividing at a size equiv-alent to the weel and cdc2.3w mutants. Genetic analysismaps this mutation close to the cdc25 locus, but it ap-pears to be distinct from this gene (P. Russell, personalcommunication). Although not directly involved in the

replication feedback control, the wee phenotype suggestsa role in the regulation of p34cdc2 activity.rad24. rad24 is partially defective in the radiationcheckpoint with an additional semi-wee phenotype andmild sensitivity to hydroxyurea exposure with a possiblepartial defect in the replication feedback control. Thismutant is difficult to work with as it also appears tomanifest a defect in cell morphology.rad29. rad29 displays large binucleate cells sensitive toradiation. Although a complementing gene has beencloned, no further analysis has been performed as themutant cells have proved difficult to synchronize.rad3O. The rad3O phenotype is reminiscent of rad26.T12.No obvious defect in the replication feedback controlor the radiation checkpoint has been detected.rad33 and rad34. Lethality of a temperature shift forcdcl7.K42 is substantially increased in rad33 and rad34mutant backgrounds but does not obviously correlatewith attempted mitosis. Both strains are relatively UVsensitive but they appear to retain the radiation check-point.hus6. hus6 is defective in the replication checkpoint andmildly sensitive to DNA damage. No radiation check-point deficiency was detected.hus7 and hus8. hus7 and hus8 are hydroxyurea sensitiveonly. The feedback control phenotype has not been es-tablished.There has been some recent confusion as to the

checkpoint/feedback control phenotype of the radia-tion and hydroxyurea-sensitive mutants of S. pombe.Whereas it is desirable to report the existence and pre-liminary characterization of the new mutants describedabove, we are concerned not to increase the confusion.We have therefore tabulated the potential checkpointand feedback control genes and mutants of S. pombe



Table 3. Summary of potential checkpoint/feedback control mutants of S. pombe

Strain ck hu cdc17 weel.50 Comment Refs.'

radl.d absent absent rapid d. inviable cpr cloned 1 2rad3.d absent absent rapid d. inviable cpr cloned 1 2rad9.d absent absent rapid d. inviable cpr cloned 1 2radl7.d absent absent rapid d. inviable cpr cloned 1 2rad2l partial' present rapid d. viable essential cloned 1 3rad24.d partial partial rapid d. inviable clonedrad25.d partial present ND ND clonedrad26.d absent absent rapid d. inviable cpr clonedrad29 present present normal viable clonedrad3O present present normal inviablerad33 present present rapid d2 viable interaction cdcl 7rad34 present present rapid d2 viable interaction cdc17chkl.d absent present rapid d. inviable3 kinase clonedhusl absent absent rapid d. inviable cpr cloned 4hus2 ND absent ND inviable cloned 4hus3 ND ND4 ND inviable 4hus4 ND ND4 ND viable5 4hus5 present ND4 ND inviable 4hus6 present absent normal partialhus7 present ND ND NDhus8 present ND ND NDhuwl present present rapid d. inviable wee phenotypecdc2.3w present absent rapid d. inviable allele of cdc2 5 6weel.d present present partial cdc2-Y15 kinase 5 7mikl.d present partial normal inviable cdc2-Y15 kinase 8cdc18 ND absent - required for S 9

ck, presence or absence of the radiation checkpoint; hu, presence or absence of replication feedback control;cdc1 7, rapid death in cdc17 genetic background at restrictive temperature linked to premature mitosis; weel .50,inviability and mitotic catastrophe in weel.50 genetic background at restrictive temperature; ND, not done;partial, partial phenotype; cpr, member of the checkpoint rad group of mutants.1 A partial checkpoint defect has been reported (Al-Khodairy and Carr, 1992), but not confirmed (Birkenbihland Subramani, 1992). Rad2l is essential, making analysis difficult. Birkenbihl and Subramani (1992) did reportan increased frequency of cut nuclei after irradiation.2 Rapid death in rad33 and rad34 cdcl 7 double mutants does not appear to be linked to premature mitosis.3 Mitotic catastrophe not dramatic compared to checkpoint rad weel.50 double mutants.4 Late or constant cut phenotype (Enoch et al., 1992) is not necessarily evidence of loss of feedback control.5 hus phenotype suppressed at 35°C. Notation such as radl.d indicates a deletion/null allele.'References: 1) Al-Khodairy and Carr, 1992; 2) Rowley et al., 1992b; 3) Birkenbihl and Subramani, 1992; 4)Enoch et al., 1992; 5) Enoch and Nurse, 1990; 6) Sheldrick and Carr, 1993; 7) Barbet and Caf, 1993; 8) Rowleyet al., 1992a; 9) Kelly et al., 1993.

that we are aware of, and listed their known involve-ment in the feedback control and checkpoint pathways(Table 3).

DISCUSSION

We have identified a number of new mutants deficientin the feedback controls and radiation checkpoints ofS. pombe. We have not tested these against all possiblereported mutants that could conceivably affect thefeedback controls or the radiation checkpoint as thiswould be rather a daunting task. We have, however,tested for allelism with the known checkpoint rad mu-tants (Al-Khodairy and Carr, 1992; Rowley et al.,1992b), the hus group of mutants reported in Enoch etal. (1992), and the cdc2 locus. In addition to alleles of

all the checkpoint rad mutants and of husl, we havenow identified another member of this class of mutants,rad26. The deletion mutant of the corresponding rad26gene shares the same phenotype as deletions in othermembers of this class.The rad27 gene defines a new class of gene product

required for the radiation checkpoint but not the rep-lication feedback controls that maintain the dependencyof mitosis on the completion of DNA synthesis. Inter-estingly, while this manuscript was in preparation, thesame gene has been reported by Walworth et al. (1993)as a multi-copy suppressor of a specific cold-sensitiveallele of the cdc2 gene, cdc2.r4. These authors havenamed this gene chkl. To avoid confusion, we will referto it from now on as chkl/rad27. To distinguish ourdeletion construct from the one reported by Walworth



Table 4. Summary of radiation sensitivities in rad mutants

wt r26.d r27.d r26.T12 1, 3, 9, 17

G2 checkpointdependent sensitivity absent present present absent present

S phase checkpointsensitivity absent present absent present present

G2 dependent sensitivity is that because of an inability to prevent mitosis after DNA damage. S phase checkpointsensitivity is the sensitivity to DNA damage that correlates to S phase and is independent of the defect inpremitotic arrest.

et al. (1993) we will call our deletion chkl.27d. The chkl/rad27 gene has been localized to chromosome III byhybridization to a cosmid library array and has beenreported in Hoheisel et al. (1993) as locus rad27.The multicopy suppression of cdc2.r4 implies that the

chkl gene product may interact with the mitotic appa-ratus and places it close to the end of the checkpointpathway. However, as pointed out by Walworth et al.(1993), we should remain cautious of interpreting thisas evidence that chkl acts directly or indirectly throughp34cdc2, because it is not clear why an inessential geneproduct that is required for mitotic arrest under specificcircumstances should relieve a cell cycle block whenoverproduced.Our work has demonstrated the loss of the radiation

checkpoint response in chkl.r27d cells. Walworth et al.(1993) reported a defect in the radiation checkpoint intheir deletion allele of chkl after UV irradiation. How-ever, they suggested that this defect was only partialand that the reduced radiation sensitivity of the chklnull mutant when compared to the radl. I mutation wasbecause of a partial arrest via a chkl independent mech-anism. Although we observed a brief arrest after UVirradiation under specific circumstances (which we at-tribute to the activation of the replication feedback con-trol by repair specific DNA synthesis), we have not de-tected any arrest after treatment with ionizingirradiation. The brief arrest observed after treatmentwith UV is not sufficient to account for the differencein phenotype observed between rad26 and chkl deletionstrains. We have therefore concluded that chkl.r27dlacks a radiation checkpoint and that the additionalsensitivity observed in rad26.d and other checkpoint radmutant cells is a consequence of the loss of a separateresponse to DNA damage.The original allele of rad26 (rad26.T12) is radiation

sensitive but not defective for G2 arrest after irradiation.This, and the phenotype of chkl/rad27 null mutants,has enabled us to separate the cause of the radiationsensitivity in the rad26.d (null) mutant into two parts.The first is because of an inability to arrest mitosis afterDNA damage and is seen in the rad26.d and chkl.r27dnull mutants, but not in the rad26.T12 mutant. The sec-

ond defect appears to be in a DNA damage responsespecific to S phase and is seen in the rad26.d andrad26.T12 mutants, but not the chkl.r27d mutant (SeeTable 4). The precise nature of the second defect seenin rad26.d and, by extension, the other checkpoint radmutants, is unknown. However, it seems likely that anS phase-specific radiation checkpoint is lost and thisresults in an inability to control DNA synthesis in re-sponse to DNA damage. This explanation is consistentwith the observation that excision repair is still func-tional in the radl 7 (checkpoint rad) deletion mutant(Barbet et al., 1994).The phenotype of the rad26.T12 mutant (which re-

mains significantly radiation sensitive but displays noapparent G2 arrest defect) provides a possible tool withwhich the cause of the S phase-specific radiation sen-sitivity of the checkpoint rad mutants can be investi-gated. It is also of interest when attempting to under-stand the multiple feedback control and checkpointdeficiencies evident in the checkpoint rad group of mu-tants. We propose that the checkpoint rad mutants forma "guardian complex" that is required for correct func-tioning of the feedback controls (which ensure the de-pendency relationships between cell cycle events) andof the radiation checkpoints (Barbet et al., 1994). In thismodel we suggest that all of the individual proteinscorresponding to the checkpoint rad mutants are re-quired for the integrity of the complex and that loss ofa single gene product results in a totally nonfunctionalcomplex. Individual proteins in the complex could,however, be responsible for a single one of the differentfeedback controls and checkpoints that are mediated bythe complex. For example, Radl 7, which has limitedhomology to subunits of RF-C (a DNA polymerase ac-cessory factor), could interact directly with the repli-cation complexes and initiate the replication feedbackcontrol signal (Barbet et al., 1994). Although the com-plete loss of Radl7 protein results in concomitant lossof the radiation checkpoint, the model allows that Radi 7does not necessarily have to be directly involved in theradiation checkpoint response.One consequence of such a model is the prediction

that mutations in the checkpoint rad genes could be



generated that result in loss of a specific function, whilenot destroying the integrity of the complex. In this con-text the S phase-associated radiation sensitivity ofrad26.T12 mutant cells, which retain a normal cell cycledelay in response to irradiation, presumably reflects afunction for this protein in the S phase-specific DNAdamage response that is separate from the mitotic defect(see Table 4). Only the loss of the protein (which wouldstructurally perturb the guardian complex) leads to lossof all the feedback controls and checkpoint functions.

In summary, our work has identified a number ofnovel mutants and genes involved in the feedback con-trols and checkpoint pathways in fission yeast and hasfacilitated a preliminary dissection of the defects in thecheckpoint rad group of mutants that lead to radiationsensitivity and an inability to tolerate cell cycle pertur-bations.

ACKNOWLEDGMENTSWe thank Wendy Muriel for technical assistance, Tamar Enoch fordiscussion and confirming the genetics of hus5, and N. Walworth, S.Davey, and D. Beach for communicating their chkl cDNA sequencedata before publication. This generosity helped us to identify intron6 and exon 7 as part of the chkl/rad27 gene. This work was supported,in part, by Commission of the European Communities contract F13P-CT920007. F. Al-K. is supported by the Saudi Arabian Government.

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