Proc. Nati. Acad. Sci. USAVol. 89, pp. 4952-4956, June 1992Genetics

The rad3+ gene of Schizosaccharomyces pombe is involved inmultiple checkpoint functions and in DNA repair

(G2 arres/COUPu of mitosis to DNA synthesis/moleculr checkpoints)

GRETCHEN JIMENEZ*, JENNIFER YUCEL*, RoY ROWLEYt, AND SURESH SUBRAMANI**Department of Biology, 0322 Bonner Hall, University of California, San Diego, La Jolla, CA 92093; and tDepartment of Radiology, University of UtahMedical Center, Salt Lake City, UT 84132

Communicated by Dan L. Lindsley, January 21, 1992

ABSTRACT A number of important molecular check-points are believed to control the orderly progression of cellcycle events. We have found that the radiation-sensitive Schizo-saccharomyces pombe mutant rad3-136 is deficient in twomolecular checkpoint functions. Unlike wild-type cells, themutant cells are unable to arrest in the G2 phase of the cell cycleafter DNA damage by -irradiation and are also incapable ofmaintaining the dependence of mitosis upon the completion ofDNA synthesis. An S. pombe genomic clone that complementsthe UV sensitivity of the rad3-136 mutant completely restoresthe missing checkpoint functions. The rad3+ gene is also likelyto play a role in DNA repair.

In recent years major advances have been made in ourunderstanding of the proteins involved in the entry intomitosis in eukaryotic cells (for reviews, see refs. 1-6).However, the regulatory circuits that modulate mitosis areless well-characterized. A variety of crucial molecular check-point functions ensure that entry into and exit from mitosisoccur only after the execution of an orderly progression ofevents (7). Among such checkpoints are functions that (i)delay mitosis or arrest cells in the G2 phase of the cell cyclein response to DNA damage (8-11), (ii) maintain the depen-dence of mitosis on the completion of chromosome replica-tion (12, 13), and (iii) make the exit from' mitosis dependentupon the completion of spindle assembly and proper chro-mosome segregation (14, 15).

Cells inflicted with DNA damage delay progression intomitosis by arresting the cell cycle temporarily in G2 so thatDNA repair can occur (7, 16, 17). In the budding yeastSaccharomyces cerevisiae (Sa. cerevisiae), the RAD9gene isinvolved in G2 arrest and the rad9 mutant is sensitive toDNAdamage because cells containing damaged DNA fail to arrest(9, 11). Several other mitotic entry checkpoint (mec) mutantshave been identified in Sa. cerevisiae (10).The checkpoint controlling the coupling of mitosis to DNA

synthesis is abolished in Schizosaccharomyces pombe (Sc.pombe) by overproduction of the phosphatase cdc25, whichdephosphorylates the cdc2 kinase and activates mitosis (18-21). Other genes involved in this checkpoint pathway includepimi (a homolog of the human RCCI gene), mutation ofwhich causes premature initiation of mitosis, and spil (asuppressor of pimi), which encodes a ras-like GTPase ho-mologous to human TC4 (22).The rad3-136 mutant of the fission yeast, Sc. pombe, is

sensitive to UV- and y-radiation, refractory to caffeine sen-sitization after exposure to UV-radiation, and exhibits areduced frequency of UV-induced forward mutation whencompared to wild-type cells (23, 24). We show in this paperthat the mutant rad3-136 is deficient in two checkpointfunctions, G2 arrest after y-irradiation and the coupling of

mitosis to DNA synthesis. We also show that a Sc. pombegenomic clone that restores resistance to UV- and -y-radiationcompletely restores the missing checkpoint functions whenreintroduced into the rad3-136 mutant. There also appears tobe a role for the rad3+ gene in DNA repair.

MATERIALS AND METHODSStains and Plasmids. The h- rad3-136 ura4 mutant was

obtained from Anwar Nasim (King Faisal Hospital, Riyadh,Saudi Arabia). h+cdc17-K42 was a gift of Peter Fantes(University of Edinburgh, Edinburgh, U.K.). The rad3Astrain (h- his3 leul-32 ura4D-18 rad3::pR3H1.0 leu2+), alsoreferred to as dR3-1, contains a disruption of the rad3 gene(B. L. Seaton, J.Y., P. Sunnerhagen, and S.S., unpublisheddata). The plasmid pSubl.41 contains a 9.8-kilobase Sc.pombe genomic DNA fragment encoding the rad3+ gene inthe BamHI site of the vector pFL20 (B. L. Seaton et al.,unpublished data).

y-Irradiation Experiments. Cells obtained from single col-onies were grown overnight in selective medium [0.67% yeastnitrogen base/2% (wt/vol) glucose plus supplements (aminoacids or uracil) at 75 mg/liter] or YPD [1% yeast extract/2%(wt/vol) Peptone/2% glucose], transferred to fresh selectivemedium or YPD, and irradiated using a 137Cs source at a rateof 2 krad/min (1 rad = 0.01 Gy) and a cell density of 107 cellsper ml. After y-irradiation, the cells were placed on icebriefly, diluted 1:10, and transferred to a 300C water bath(time = 0 min). Samples of the cells were removed at varioustimes for measurement of the septation index.Flow Cytometry. Nitrogen-starved cells were grown in

Edinburgh minimal medium (25) with 5 mM NH4Cl for 24 hr.Irradiated cells were treated as above. Cells were fixed in70o ice-cold EtOH, treated 1 hr at 370C with RNase at 250,ug/ml, stained with propidium iodide at 2.5 ;Lg/ml, soni-cated, and analyzed on a FACScan flow cytometer.Photomicrography of Cells. Exponentially growing cells

were fixed, stained with 4,6-diamidino-2-phenylindole(DAPI), and mounted on slides for fluorescence microscopyas described by Moreno and Nurse (25).Hydroxyurea (HU)-Sensitivity Experiments. Cells were

grown overnight in minimal medium [MM = 0.67% yeastnitrogen base plus supplements (uracil, leucine, or histidine)at 75 mg/liter] containing 0.5% glucose. Cells were diluted to106 cells per ml in this medium and brought 10 mM HU byadding HU from a 100 mM stock in water. At various timepoints after HU addition, cells were diluted into MM plus0.5% glucose and plated.Benomyl Induction of G2 Arrest. Single colonies were

grown in selective medium or YPD overnight at 300C and thentransferred to fresh medium at 107 cells per ml. Benomyl at20 jug/ml (from a stock at 5 mg/ml in EtOH) was autoclaved

Abbreviations: HU, hydroxyurea; DAPI, 4,6-diamidino-2-phenylin-dole.

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in MM. Cultures were incubated for 1.5 hr at 300C to arrestcells in G2 (26). The cells were irradiated (10 krad) with a 137CSsource at a rate of 2 krad/min. After irradiation, half of theculture was diluted 1:100 into fresh medium without drug.The other half of the irradiated culture was diluted 1:100 intofresh medium containing benomyl. Samples of the drug-freeculture were monitored for their septation index for up to 2hr after irradiation. The cultures were washed before the finalplating on YPD plates 2 hr after irradiation. The viability ofthe cells was not affected by benomyl alone but killingincreased when the cells were y-irradiated in the presence ofthe drug.

RESULTSThe rad3 Mutant Is Deficient in G2 Arrest and the Deficiency

Is Restored by pSubl.41. When exponentially growing 972 h-cells were irradiated with 60 krad of v-radiation, the cellsarrested in G2. Arrest was characterized by a rapid drop in theseptation index of irradiated cells as compared to unirradiatedcells (Fig. 1A), and the simultaneous elongation and accumu-lation of uninucleate cells. Flow cytometry of the irradiatedwild-type cells confirmed that they were arrested in G2 (Fig. 2

x

0

.

(I)

so 1o so

Time after irradiation, min240

FIG. 1. Complementation of the G2 arrest deficiency of the rad3mutant by pSubl.41 after DNA damage by virradiation. Cells wereirradiated with 60 krad of v-radiation and the septation index[(number of septated cells/total number of cells) x 100] was moni-tored as a function of time. (A) Behavior of wild-type cells. c,Unirradiated 972 h- cells; o, irradiated 972 h- cells. The drop in theseptation of irradiated cells is due to G2 arrest as shown in Fig. 2. (B)Septation indices of mutant cells. A, rad3-136 strain; c, rad3A strain;o, rad3-136 strain transformed with the plasmid pSubl.41, whichcarries the rad3+ gene. Note that the cloned rad3+ gene completelyrestores the G2 arrest deficiency of the rad3-136 mutant (see Fig. 2)as indicated by the drop in the septation index of the mutant carryingpSubl.41. The cells behave synchronously because they were dilutedfrom an overnight culture into fresh medium 2 hr prior to irradiation.

1

a 1

_ D

=:

i ......

jB 'Ci ~~~~10

2N

iIi

fi~~~ ~ ~~~~~~~~~~~~~~~..

DNA content

FIG. 2. Flow cytometric analysis ofDNA content of Sc. pombe.(A-C) 972h-. (D-F) rad3-136 ura4 h-. iN and 2N DNA content areindicated for wild-type cells. v-Irradiated cells were fixed 1.5 hr afterirradiation. Wild-type cells remain in G2 after -irradiation but therad3 mutant cells do not. The shoulders to the right of the G2 peaksin E and F result from the unequal segregation of DNA to daughtercells in the rad3-136 mutant such that an appreciable number of cellshave more than the 2N content of DNA. This observation isconsistent with the loss of the DNA synthesis checkpoint function inthe mutant. A and D, low nitrogen medium; B and E, exponentialcultures; C and F, v-irradiated cultures (60 krad).

A-C). The G2 arrest was also observed at 5, 10, 25, or 50 kradbut the duration of arrest was shorter (data not shown).

In contrast to the wild-type cells, the rad3-136 and therad3A (rad3 gene disruption) strains did not exhibit G2 arrestand continued to septate after t-irradiation (Fig. 1B). Celldivision in the irradiated mutant cells was accompanied by adecrease in DNA content of a fraction of cells, which isconsistent with entry into the G1 and S phases (Fig. 2 D-F).Unlike the wild-type cells, which had a viability of 35% at 60krad of y-irradiation and arrested without cell division (Fig.3 A and B), the mutant cells exhibited reduced survival uponplating (0.017% at 60 krad) and did not elongate, and dividingcells accumulated chromosomal abnormalities (Fig. 3 C andD). Many daughter cells contained noDNA or only fragmentsof DNA. The proportion of mutant cells with chromosomalabnormalities increased to 35% during the 2 hr after irradi-ation. Similar effects were observed at lower doses (10 krad)of v-radiation (data not shown).

In rad3-136 cells transformed with the pSubl.41 plasmidand irradiated with 60 krad of -y-radiation, the G2 arrestresponse was similar to that of the wild-type cells (Fig. 1B),as was the radiosensitivity for killing (26% survival at 60krad). Furthermore, the irradiated cells elongated dramati-cally (Fig. 3 E and F), consistent with the incidence of a cellcycle block. Interestingly, the pSubl.41-containing cellswere longer than wild type even before irradiation.The rad3 Mutant Is Deficient in Coupling DNA Synthesis and

Mitosis and This Deficiency Is Restored by pSubl.41. Todetermine whether the rad3 mutant was also deficient in thecheckpoint pathway responsive to DNA synthesis, we testedthe sensitivities of the rad3-136 and rad3A strains to the DNAsynthesis inhibitor HU. Both mutant strains were extremelysensitive to HU under conditions where wild-type cells wereessentially resistant (Fig. 4A). Wild-type cells ceased divisionas judged by the drop in the septation index, whereas themutant cells continued to septate (Fig. 4B). Staining of theHU-treated cells with DAPI also confirmed that while therewas an accumulation of unseptated elongated uninucleatecells in the culture of wild-type cells as observed by Enochand Nurse (12) (Fig. SA), the mutant strains continued to

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FIG. 3. Photomicrographs of DAPI-stained Sc. pombe cells. (A) Unirradiated 972 h- cells. (B) 972 h- cells 2 hr after v-irradiation. (C)rad3-136 cells 1.5 hr after v-irradiation. (D) rad3A cells 2 hr after virradiation. (E) rad3-136 cells transformed with pSubl.41 and fixed 2 hr aftervirradiation. (F) rad3-136 cells transformed with pSubl.41 but without irradiation. The dose of y-radiation used was 60 krad.

divide. Premature division of the mutant strains was accom-panied by the accumulation of cells with chromosomal ab-normalities (Fig. 5 B and C) and by lower viability (Fig. 4A).During the 4.5 hr after HU treatment, the proportion of cellswith chromosomal abnormalities increased to 40% for rad3-136 and to 70% for rad3A. The high septation index observedfor the rad3A strain (Fig. 4B) reflects this high proportion ofabnormally dividing cells.The resistance to HU was restored to wild-type levels in

rad3-136 cells transformed with pSubl.41 (Fig. 4A). Theseptation index of the rad3-136 strain carrying pSubl.41 wasalso similar to that of the wild-type strain (Fig. 4B). Further-more, the cells arrested normally (Fig. SD). These resultssuggest that the rad3+ gene has an additional role in couplingevents in the S phase to the onset of mitosis.The rad3 Mutation Enhances the Lethality of Ligase Inac-

tivation in the cdc17 Mutant. The cdcl7-K42 mutant of Sc.pombe is deficient in DNA ligase at the nonpermissivetemperature (27) and is known to arrest in late S phase. Acomparison of the viabilities of the cdcl7-K42 and the rad3-136 cdcl7-K42 double mutant at 350C shows clearly that thedouble mutant is less viable (Fig. 6). Increased loss ofviability results from the inability of the double mutant toexercise the rad3-mediated checkpoint function involved incoupling mitosis to DNA synthesis.The rad3 Mutant Is also Deficient in DNA Repair. Since the

RAD9 gene of Sa. cerevisiae is involved in G2 arrest but notin DNA repair per se (10), we asked whether the impositionofan artificial G2 arrest would provide rad3 cells with enoughtime to repair the DNA after damage. The rad3-136 cells weresynchronized in G2 by a 1.5-hr pretreatment with benomyl

(26) and then irradiated with 10 krad of y-radiation. Half thecells were grown without benomyl and the other half weremaintained in benomyl for another 2 hr. Wild-type cellsdisplayed the expected G2 delay when they were irradiatedand released from the benomyl block after the 1.5-hr pre-treatment with the drug (Table 1). This indicates that theDNA-damage-induced arrest occurs after the benomyl-sensitive step. The rad3-136 mutant showed no G2 arrestwhen released from benomyl after irradiation. However,when benomyl was present in the cultures after irradiation,both the wild-type and rad3-136 strains remained arrested, asexpected. The viabilities of the irradiated cells in the pres-ence or absence of benomyl were found to be similar.Survival of h-972 cells 2 hr after irradiation at 10 krad was61.7% in the absence of benomyl and 47.9o in the presenceof benomyl. Survival of h- rad3-136 ura4 cells 2 hr afterirradiation was 2.1% in the absence of benomyl and 6.7% inthe presence of benomyl. In several experiments with therad3 mutant cells, there was never more than a 2- to 3-folddifference in viability between the cultures maintained in thepresence or the absence ofthe drug after y-irradiation. Theseresults suggest that the rad3+ gene must also be involveddirectly or indirectly in DNA repair since viability was notsignificantly increased by holding the rad3 strain in G2.

DISCUSSIONRole of rad3 in Checkpoint Functions. The inability of the

rad3-136 and the rad3A strains to arrest in G2 after y-irradi-ation shows that mutations in the rad3+ gene of Sc. pombeimpair G2 arrest. A similar deficiency has been noted in the

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Table 1. Induction of G2 arrest by benomyl does not enhancethe survival of rad3-136 cells significantly after y-irradiation

Septation index, %Time afterTirradiatin h-972 h- rad3-136 ura4irradiation,

min - benomyl + benomyl - benomyl + benomyl

0 0.4 0.4 0.4 0.430 1.0 0.9 1.5 0.960 0.8 1.4 19.3 3.590 4.3 0 5.8 0.8120 6.2 1.3 3.8 1.1

Data represent the average of two experiments. Cells were irra-diated with 10 krad.

DNA repair because the viability of the mutant cells was notrestored by the artificial imposition of a period of G2 arrestwith benomyl. If the repair pathway controlled by rad3 is anerror-prone one, then the relative immutability (40-fold lowerthan wild-type) of the rad3 mutant (24, 30), after UV irradi-ation, could be explained by cell death resulting from a lossof multiple checkpoint and DNA repair functions. Finally,unlike the null allele of the Sa. cerevisiae RAD9 gene, therad3 mutant exhibits a higher level of spontaneous intragenicrecombination relative to wild-type (24). The expression ofthe rad3+ gene from a multicopy plasmid results in elongatedcells even in the absence of DNA damage or unreplicatedDNA (Fig. 3 A and F). Since DNA damage or unreplicatedDNA is not essential for rad3-mediated arrest, we speculatethat the rad3 protein may function in relaying informationregarding the presence of unreplicated or damaged DNArather than in its detection.

Properties of the rad3 Mutant. The sensitivity of the rad3mutant to UV and ionizing radiation probably results fromthe dual defect in G2 arrest and DNA repair. The prematureentry into mitosis without repair of DNA lesions leads tochromosome segregation problems (Fig. 3) that eventuallyresult in cell death (31). A similar problem accounts forsensitivity to HU (Figs. 4 and 5) and is likely to explain theincreased sensitivity of the rad3 cdcl7 mutant at the nonper-missive temperature (Fig. 6).

Caffeine is known to abolish G2 arrest in mammalian cells(32-34). A similar effect in Sc. pombe would explain thelower survival of caffeine-treated cells after DNA damage(24). Since G2 arrest is impaired in the rad3 mutant, it is notsurprising that, unlike 972 h- cells, the mutant cells areinsensitive to further sensitization by caffeine (24).

In summary, the rad3 mutant of Sc. pombe is deficient intwo molecular checkpoint functions and in DNA repair.These deficiencies provide an explanation for the knownphenotype of the rad3-136 mutant.

We thank Joe Trotter and Merl Hoekstra for assistance with the

FACS analyses. This work was supported by grants from theNational Institutes ofHealth (GM31253) and the Council for TobaccoResearch to S.S. and Grant CA40245 to R.R. G.J. was supported bya National Institutes of Health Training Grant.

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