MOLECULAR AND CELLULAR BIOLOGY,0270-7306/97/$04.0010

June 1997, p. 3356–3363 Vol. 17, No. 6

Copyright © 1997, American Society for Microbiology

Discrete Roles of the Spc1 Kinase and the Atf1 Transcription Factorin the UV Response of Schizosaccharomyces pombe

GENEVIEVE DEGOLS AND PAUL RUSSELL*

Departments of Molecular Biology and Cell Biology, The Scripps Research Institute, La Jolla, California 92037

Received 22 November 1996/Returned for modification 23 January 1997/Accepted 18 March 1997

Exposure of mammalian cells to UV irradiation or alkylating agents leads to the activation of the c-JunN-terminal kinase and p38 stress-activated protein kinase cascades, phosphorylation of c-Jun and ATF-2 bZIPtranscription factors, and finally to selective induction of gene expression. This UV response is believed to becrucially important for cell survival, although conclusive evidence is lacking. Here, we address this issue byinvestigating a homologous UV response pathway in the fission yeast Schizosaccharomyces pombe. In fissionyeast cells, UV irradiation induces activation of Spc1 stress-activated protein kinase, which in turn phosphor-ylates the Atf1 bZIP transcription factor. spc1 mutants are hypersensitive to killing by UV at a level equivalentto some checkpoint rad mutants. Whereas checkpoint rad mutants fail to arrest division in response to DNAdamage, spc1 mutants are defective at resuming cell division after UV exposure. Levels of basal and UV-induced transcription of ctt11, which encodes a catalase believed important for combating oxidative stresscaused by UV, are extremely low in spc1 mutants. Atf1 is required for UV-induced transcription of ctt11, butatf1 mutants are not hypersensitive to killing by UV. This surprising finding is explained by the observationthat ctt11 basal expression is unaffected in atf1 single mutant and spc1 atf1 double mutant cells, suggesting thatunphosphorylated Atf1 represses ctt11 expression in spc1 cells. In fact, the level of UV sensitivity of spc1 atf1double mutant cells is intermediate between those of the wild type and spc1 mutants. These findings suggestthe following. (i) Key properties of UV response mechanisms are remarkably similar in mammals and S. pombe.(ii) Activation of Spc1 kinase greatly enhances survival of UV-irradiated cells. (iii) Induction of gene expressionby activation of Atf1 may not be the most important mechanism by which stress-activated kinases function inthe UV response.

Mammalian cells exposed to shortwave UV irradiation oralkylating agents such as methyl methanesulfonate (MMS) re-spond by inducing the transcription of a specific set of genes bya process that is generically known as the UV response (8, 10,11, 13). Most of the details of the mammalian UV responseremain to be determined, but the emerging picture is that oftwo stress-activated protein kinase cascades activating multipletranscription factors that typically function in heterodimericcomplexes. One pathway involves a related family of c-JunN-terminal kinases (JNKs) that are activated by a variety offorms of stress and hence are also referred to as stress-acti-vated protein kinases (SAPKs) (8, 20). JNKs are related tomitogen-activated protein kinases (MAPKs) and thus followthe pattern of being activated by MAPK kinases (MAPKKs orMEKs) that are themselves activated by MAPKK kinases(MAPKKKs or MKKs) (39). A second pathway involved in theUV response leads to activation of a class of related proteinkinases that are most well known as p38/RK kinases. p38/RKkinases are also regulated by upstream protein kinase cascadesthat follow the MAPK format (9, 16, 22, 28, 30). Like JNKs,p38 kinases are activated in response to a variety of stresses,including UV and MMS.

The best-defined transcription factor targets of JNK and p38kinases belong to the family of bZIP transcription factors, socalled because they contain a region of basic amino acidsfollowed by leucine zipper sequences (42). Notable membersof the bZIP transcription factor family include Jun, Fos, andATF-2. A well-established target of JNKs is Jun, which to-

gether with Fos forms the AP-1 transcription factor complex(8, 17, 20). JNKs and p38 kinases are also capable of activatingATF-2 by direct phosphorylation (15, 37).

UV and MMS are well-known DNA-damaging agents, but itis believed that the mammalian UV response is not triggeredby DNA damage (10). Indeed, in enucleated HeLa cells, JNKbecomes highly activated in response to UV exposure, therebyruling out the possibility that damage of nuclear chromosomalDNA is required for the UV response (11). Instead, it is be-lieved that the UV response may be primarily induced at thecell membrane, possibly through the peroxidation of lipids.Likewise, alkylating agents may activate the UV responsethrough reactions with free SH groups. These reactions causea depletion of the intracellular pool of reduced glutathione(GSH), resulting in oxidative stress (21). A key finding insupport of this notion is that the AP-1-induced transcription ofc-jun is prevented by incubation of HeLa cells in mediumcontaining N-acetylcysteine (NAC) (10). NAC is rapidly con-verted to GSH, which in turn acts as an effective intracellularscavenger of free radicals.

The functional significance of the mammalian UV responseis not clearly understood. None of the AP-1-dependent-induc-ible genes are involved in DNA repair, whereas there are atleast two UV-inducible genes regulated by AP-1 for which arole in counteracting the adverse effects of free radicals mightbe inferred: genes encoding glutathione S-transferase and me-tallothioneins (2). Thus, it appears that the UV response islikely to be involved in combating oxidative damage, althoughit remains to be established whether the induction of oxidativestress genes actually has a protective effect against UV irradi-ation and exposure to alkylating agents. It is possible that inaddition to activating transcription factors, stress-activated ki-

* Corresponding author. Phone: (619) 784-8273. Fax: (619) 784-2265. E-mail: prussell@riscsm.scripps.edu.

3356

on April 8, 2018 by guest

http://mcb.asm

.org/D

ownloaded from

nases may have other functions that are important for protect-ing cells from UV damage.

Recent studies of the fission yeast Schizosaccharomycespombe have resulted in the identification of a protein kinase,Spc1, that closely resembles the stress-activated kinases of theJNK-p38 families in mammalian cells (19, 24, 34) and buddingyeast Hog1p kinase (4). Spc1, also known as Sty1 and Phh1, isactivated by a variety of stresses, including nitrogen limitation,high osmolarity, heat stress, and hydrogen peroxide treatment(7). The transmission of all of these stress signals requiresWis1, a MAPKK homolog (38). Spc1 activity is negativelyregulated by Pyp1 and Pyp2 tyrosine-specific phosphatases viadirect dephosphorylation of the tyrosine residue phosphory-lated by Wis1. Interestingly, the Wis1-Spc1 kinase cascade islinked to the G2-M cell cycle control mechanism (24, 34).Under optimum growth conditions, spc1 mutants exhibit amoderate delay of the onset of mitosis that is greatly exacer-bated under stressful conditions. Recent studies have providedstrong evidence that Spc1 directly regulates Atf1, a bZIP tran-scription factor (18, 35, 36, 41). Atf1 phosphorylation is di-rectly catalyzed by Spc1 in vivo. Moreover, osmotic stress-induced transcription of genes such as gpd11, which encodesan enzyme required for glycerol synthesis, is greatly reduced inspc1 and atf1 mutants. The discovery that Spc1 and Atf1 haveclose structural similarities to p38 and ATF-2, respectively, andthat Spc1 phosphorylates Atf1 in response to a range of stressstimuli suggests a remarkable degree of conservation betweenstress-activated signal transduction pathways in fission yeastsand mammals.

The discovery of similar stress response mechanisms in fis-sion yeasts and mammals presents an opportunity to investi-gate these signal transduction systems by means that were notpreviously possible, taking advantage of genetic methods toanswer questions that delve into causality and physiologicalsignificance. Accordingly, in this paper, we report experimentsthat have addressed a number of issues relevant to the re-sponse of fission yeast cells to genotoxic agents such as UV andMMS. We have found that the Wis1-Spc1 pathway is essentialfor a UV response that is triggered by oxidative stress. More-over, failure to activate Spc1 leads to a profound sensitivity tokilling by UV and MMS. We report that Atf1 is required forUV-induced elevation of stress response genes and explain theparadoxical finding that atf1 mutants are not abnormally sen-sitive to UV and MMS. These studies reveal discrete roles fora UV-responsive MAPK and its transcription factor substratein mediating the cellular response to UV irradiation.

MATERIALS AND METHODS

Yeast strains, media, and general methods. The S. pombe strains used in thisstudy are described in Table 1. YES and synthetic EMM2 media were used forgrowth media. Fission yeast experimental methods and media have been de-scribed previously (1, 25).

Gene expression analysis. Northern hybridization analyses were performed asdescribed previously (31). A 1-kb EcoRV fragment from pJK148 was used for thecontrol leu11 probe. A 2,461-bp fragment of rad81 was amplified by PCR withthe 59 oligonucleotide 59-TCGGATGCTTTGGTGTGGAGG-39 and the 39 oli-gonucleotide 59-GTCTCTAAAGCCGTCGACCTC-39. A 1,021-bp fragment ofctt11 was amplified by PCR with the 59 oligonucleotide 59-TCCTGAACGTGTCGTCCATGCAAAGG-39 and the 39 oligonucleotide 59-GACGGATTGAGGATGGATAGTTTG-39. A 2.3-kb BamHI fragment from the pREP3-pyp2 plas-mid was used to probe for pyp21 mRNA.

Detection of the Spc1Ha6H and Atf1Ha6H proteins. Immunoblot analysis ofSpc1 protein was performed with a strain having a chromosomal copy of spc1-Ha6H (34). This gene encodes Spc1 protein having a C-terminal tag containingtwo copies of the influenza virus hemagglutinin (HA) epitope followed by sixconsecutive histidine residues. Atf1 was also tagged at its C terminal end with twocopies of HA and six histidine residues, and this construct was expressed fromthe atf11 promoter (35). Spc1 and Atf1 proteins were purified by Ni21-nitrilo-triacetic acid (NTA)-agarose chromatography, resolved by sodium dodecyl sul-fate-polyacrylamide gel electrophoresis (SDS-PAGE), electroblotted to nitrocel-

lulose, and then detected with either anti-HA (12CA5) or anti-phosphotyrosine(4G10; Upstate Biotechnology) antibodies as described previously (34). Immu-noreactive bands were detected with horseradish peroxidase-conjugated second-ary antibodies and the ECL (enhanced chemiluminescence) Western blottingdetection system (Amersham). For the UV irradiation studies, cells were col-lected by filtration on 25-mm-diameter Metricel membrane filters (Gelman),irradiated with a Bio-Rad Genelinker, and then resuspended in liquid YESmedium.

UV and MMS survival studies. For the UV sensitivity experiments, 2,000 cellsof a log-phase culture were plated in duplicate for each dose and irradiated at254 nm with a Bio-Rad Genelinker. Survival curves represent the mean value ofthree experiments. For the MMS sensitivity experiments, exponentially growingcells were incubated for 1 h with the indicated concentrations of MMS. The drugwas inactivated with 5% sodium thiosulfate, and appropriate dilutions of cellswere plated on YES medium. Survival curves represent the mean value of threeexperiments.

RESULTS

UV irradiation and MMS treatment stimulate Spc1 tyrosinephosphorylation. The initial aim of this study was to determinewhether S. pombe has a UV response mechanism that is similarto the JNK and p38 kinase pathways in mammalian cells.MAPKKs activate MAPKs by phosphorylating threonine andtyrosine residues in the sequence Thr-X-Tyr; thus, a hallmarkof the mammalian UV response is the detection of tyrosine-phosphorylated JNK and p38 by the use of antiphosphotyro-sine antibodies. Therefore, the potential involvement of Spc1in a UV response in S. pombe was carried out by analysis ofSpc1 tyrosine phosphorylation. These studies used a strain thathas a single chromosomal copy of spc11 tagged with a se-quence encoding two copies of the HA epitope and six con-secutive histidine residues (34). This allowed us to purify Spc1by using Ni21-NTA beads and to detect it by using anti-HAantibodies.

Spc1 underwent rapid tyrosine phosphorylation in responseto UV irradiation (Fig. 1A). The response was quite transient,nearing peak levels within 10 min of irradiation and decreasingto preirradiation levels within 60 min posttreatment. To fur-ther confirm the activation of Spc1 by UV irradiation, we haveexamined the phosphorylation state of the Atf1 transcriptionfactor. Atf1 is a downstream component of the Wis1-Spc1pathway that becomes phosphorylated by Spc1 kinase duringstress (35). For this analysis, we used a strain carrying a chro-mosomal atf11 gene tagged with a sequence encoding the HAantigen and six consecutive histidine residues (35). Atf1 pro-tein purified from unstressed cells migrated with an apparentmolecular mass of ;85 kDa in SDS-PAGE (Fig. 1B). Fifteenminutes after UV irradiation, Atf1 migrated as a somewhatdiffuse band with a reduced mobility of ;90 kDa. This mobilityshift was previously shown to be caused by phosphorylation

TABLE 1. S. pombe strains used in this study

Straina Genotype Source or reference

PR109 h2 Laboratory stockKS1147 h1 spc1-M13 34KS1366 h2 spc1::ura41 34KS1376 h2 spc1HA6H(ura41) 34KS1455 h2 rad24::ura41 T. CarrKS1479 h2 atf1HA6H(ura41) 35KS1497 h2 atf1::ura41 35KS1533 h2 atf1::ura41 spc1::ura41 35KS1572 h2 spc1::ura41 atf1HA6H(ura41) 35JL1188 h1 rad1-1 S. SubramaniJM544 h2 wis1::ura41 Laboratory stockGD1520 h1 cdc17-K42 spc1HA6H(ura41) This studyGD1683 h1 wis1::ura41 spc1HA6H(ura41) This study

a All strains are leu1-32 ura4-D18.

VOL. 17, 1997 UV RESPONSE IN SCHIZOSACCHAROMYCES POMBE 3357

on April 8, 2018 by guest

http://mcb.asm

.org/D

ownloaded from

carried out by Spc1 kinase (35). In contrast, Atf1 protein pu-rified from a Dspc1 strain migrated with an apparent molecularmass of ;85 kDa even after UV irradiation. These data showthat Atf1 is phosphorylated in vivo in response to UV irradi-ation in an spc11-dependent manner.

Wis1 MAPKK is required for the transmission of high-tem-perature, oxidative stress and osmotic stress signals to Spc1 (7,24, 34). We therefore examined whether Wis1 was also re-quired for the UV-induced activation of Spc1 by comparingSpc1 tyrosine phosphorylation in wild-type and Dwis1 back-grounds. This analysis showed that UV-induced tyrosine phos-phorylation of Spc1 was abolished in Dwis1 cells (Fig. 1C).Similar experiments were carried out with cells treated withMMS. As shown in Fig. 1C, MMS treatment induced a highlevel of Spc1 tyrosine phosphorylation that was abolished in aDwis1 background. These studies establish that the Wis1-Spc1kinase cascade is activated in response to UV irradiation andan alkylating agent, findings that are highly reminiscent of themammalian UV response.

Mechanism of Spc1 activation by UV irradiation. The mam-malian UV response appears to be activated by damage tocomponents in the cell membrane, as opposed to damage ofnuclear DNA. Moreover, it is believed that the mammalianUV response occurs mainly as a reaction to oxidative stress,probably by the formation of free radicals in a process involv-ing lipid peroxidation (3, 10). To explore the situation in S.pombe, we examined the effect of incubating cells in NAC,which when taken up by cells is readily converted to GSH, ascavenger of free radicals (23). By itself, NAC had no effect onthe Spc1 tyrosine phosphorylation in unstressed cells (Fig. 2).

However, the UV-induced increase in Spc1 tyrosine phosphor-ylation was completely abrogated by preincubation of cells withNAC (Fig. 2). Similarly, MMS activation of Spc1 was pre-vented by incubation in medium containing NAC (data notshown).

To more directly address the possibility that DNA damageby itself leads to activation of Spc1, an experiment was carriedout to introduce DNA lesions into the genome without causingenvironmental stress. This experiment employed a strain car-rying the cdc17-K42 mutation, which encodes a temperature-sensitive version of DNA ligase (27). Incubation of cdc17-K42cells at the restrictive temperature of 35°C results in the accu-mulation of unligated DNA. As shown in Fig. 3A, incubationof cdc17-K42 cells at the restrictive temperature also leads tothe induction of expression of rad81, a gene believed to encodea DNA helicase that is involved in DNA repair (12). However,during the time course of this experiment, there was no changein the level of Spc1 tyrosine phosphorylation (Fig. 3B), evenafter 5 h of incubation at the restrictive temperature (data notshown). (Note that in this experiment the culture temperature

FIG. 1. Wis1-dependent activation of Spc1 in response to UV and MMS. (A)Wild-type (WT) cells (KS1376) were UV irradiated at 200 J/m2 (as described inMaterials and Methods) and incubated for the indicated time (minutes) in YESmedium. Epitope-tagged Spc1 was isolated by Ni21-NTA affinity precipitationand immunoblotted to detect phosphotyrosine (a-pTyr) or HA epitope tag(a-HA). (B) Spc1-dependent phosphorylation of Atf1 after UV irradiation.Wild-type (KS1479) and Dspc1 (KS1572) strains were grown in YES medium at30°C, and aliquots were harvested before or 15 min after UV irradiation (200J/m2). Epitope-tagged Atf1 was isolated by Ni21-NTA affinity precipitation andanalyzed by SDS-PAGE followed by immunoblotting with anti-HA antibodies.(C) UV and MMS activation of Spc1 is dependent on Wis1. Wild-type and Dwis1mutant cells were incubated under unstressful conditions (lanes 1 and 2), UVirradiated at 200 J/m2 followed by 15 min of incubation in YES medium (lanes3 and 4), or exposed to 0.05% MMS for 20 min (lanes 5 and 6). Spc1 wasanalyzed as described above.

FIG. 2. NAC prevents Spc1 tyrosine phosphorylation in response to UVirradiation. Wild-type cells were incubated in the presence or in the absence ofNAC (40 mM) for 1 h prior to exposure to UV irradiation (100 J/m2). Sampleswere collected 15 min later. Spc1 was isolated by Ni21-NTA affinity precipitationand probed for the presence of phosphotyrosine (a-pTyr) or HA epitope tag(a-HA).

FIG. 3. Spc1 tyrosine phosphorylation is not responsive to unligated DNA.Strain GD1514, having a temperature-sensitive mutation of the DNA ligase gene(cdc17-K42), was grown to mid-log phase at the permissive temperature of 30°C.The culture temperature was then gradually shifted to the restrictive tempera-ture of 35°C over a period of 30 min. Samples were collected at the indicatedtimes after the temperature had been shifted. (A) Northern analysis showedelevated expression of the rad81 gene, believed to encode a DNA helicaseinvolved in DNA repair. (B) Spc1 tyrosine phosphorylation was unchangedduring the time course of the experiment.

3358 DEGOLS AND RUSSELL MOL. CELL. BIOL.

on April 8, 2018 by guest

http://mcb.asm

.org/D

ownloaded from

was gradually raised from 30°C to 35°C to avoid causing heatstress.)

These findings strongly suggest that treatments such as UVirradiation and exposure to alkylating agents activate Spc1kinase by causing oxidative stress. DNA lesions do not appearto be involved in activating the Spc1 pathway.

Survival of spc1-M13 mutants after exposure to UV andMMS. Although the existence of a UV response in mammaliancells has been established for a number of years, a clear un-derstanding of the physiological importance of the UV re-sponse has been lacking, mainly due to the inability to specif-ically ablate the UV response. Therefore, the existence of spc1mutants of fission yeast presented a valuable opportunity toaddress the question of whether the UV response is importantfor combating the cytotoxic effects of UV irradiation. Wild-type and spc1-M13 mutant cells were grown to mid-log phase,exposed to increasing doses of UV, and assayed for viability bycolony-forming ability. This analysis revealed that spc1-M13 orDspc1 cells were highly sensitive to UV irradiation (Fig. 4 anddata not shown). For example, at a dose of 300 J/m2, at which;30% of the wild-type cells survived, only ;0.2% of the spc1-M13 cells were able to form colonies. Dwis1 cells were alsoprofoundly sensitive to UV irradiation, at a level equivalent tothat of spc1-M13 cells (data not shown), in agreement with theobservation that Wis1 is required for the tyrosine phosphory-lation of Spc1 that is induced by UV.

An experiment was carried out to gain a sense of the impor-tance of the Spc1-regulated UV response relative to the DNAdamage repair and checkpoint mechanisms. Fission yeast radmutants can be broadly divided into two groups: those that aredefective both for DNA repair and the G2-M checkpoint, suchas rad1 mutants, and those that are defective only in the check-point itself, such as rad24 mutants (5). We therefore comparedthe UV sensitivities of spc1-M13, rad1-1, and rad24::ura41

mutants (Fig. 4). This analysis showed that the UV sensitivityof spc1-M13 cells was approximately the same as that ofrad24::ura41 cells. In contrast, rad1-1 cells were much moreUV sensitive than either spc1-M13 or rad24::ura41 cells.

The ability of spc1-M13 cells to survive exposure to MMSwas also investigated. Consistent with the studies of UV sen-sitivity, we found that the ability of spc1-M13 cells to surviveMMS treatment was dramatically decreased relative to that ofwild-type cells (data not shown and see Fig. 6B). As was the

case for UV treatment, the MMS survival properties of spc1-M13 cells were equivalent to those of Dwis1 and rad24 strains(data not shown and Fig. 6B). These findings show that acti-vation of Spc1 is very important for survival of UV and MMStreatment.

Cell cycle defect of spc1 mutants exposed to UV irradiation.In S. pombe, a number of UV-sensitive rad mutants have beenclassified as checkpoint-defective mutants because they fail toarrest cell cycle progression in G2 in response to DNA-dam-aging agents (33). In this sense, checkpoint proteins such asRad24 are involved in inhibiting cell cycle progression. In con-trast, Spc1 kinase has a positive role in promoting the onset ofmitosis (24, 34). The discovery that Spc1 is involved in con-trolling the response to agents that have both cytotoxic andgenotoxic consequences prompted an examination of the effectof UV irradiation on the cell division properties of spc1 mutantcells. As was the case for wild-type cells, UV exposure (200J/m2) caused the cell division (septation) index of spc1-M13cells to drop to 0 within 90 min after irradiation. However,UV-irradiated spc1-M13 cells were highly defective in resum-ing cell division. At 6 h postirradiation, spc1-M13 cells wereelongated and very few cells had division septa, whereas wild-type cells had resumed cell division at this time (Fig. 5). Dwis1cells behaved exactly like spc1-M13 mutants (data not shown).These findings show that activation of Spc1 kinase as part ofthe UV response is required to resume cell division after ex-posure to UV irradiation.

Datf1 cells are not UV sensitive. A hallmark of the mamma-lian UV response is the transcriptional induction of stressresponse genes. In S. pombe, Spc1 kinase promotes the induc-tion of expression of stress response genes such as gpd11 andpyp21 by a mechanism that requires Atf1, a bZIP transcriptionfactor (35, 41). In fact, both basal transcription and stress-induced transcription of gpd11 and pyp21 are extremely low inDatf1 mutants. Consistent with this finding, Datf1 mutants growvery poorly on medium supplemented with 1 M KCl, althoughthe defect is less severe than that of spc1 mutants. As shown inFig. 1B, Atf1 is phosphorylated after UV irradiation in anSpc1-dependent manner. To further explore the role of Atf1 inthe UV response, we examined the UV sensitivity of Datf1mutants. Much to our surprise, we found that the UV sensi-tivity of Datf1 mutants was not enhanced relative to that ofwild-type cells (Fig. 6A). This pattern was replicated when

FIG. 4. Spc1 is important for survival of UV irradiation. Wild-type (WT)(PR109), spc1-M13 (KS1147), rad24::ura41 (KS1455), and rad1-1 (JL1188)strains were exposed to the indicated doses of UV irradiation, and survival wasmeasured as described in Materials and Methods. The spc1-M13 cells werehypersensitive to killing by UV at a level equivalent to that of the Drad24 cells,whereas rad1-1 cells were several orders of magnitude more sensitive to UV.

FIG. 5. spc1-M13 cells exhibit a cell cycle arrest phenotype when exposed toUV irradiation. Exponentially growing wild-type (WT) and spc1-M13 cells wereirradiated at 200 J/m2. Cells were then incubated in YES medium at 30°C for 6 h,fixed with ethanol, and stained with the DNA dye 49,6-diamidino-2-phenylindole.

VOL. 17, 1997 UV RESPONSE IN SCHIZOSACCHAROMYCES POMBE 3359

on April 8, 2018 by guest

http://mcb.asm

.org/D

ownloaded from

Datf1 cells were tested for MMS sensitivity: Datf1 and wild-typecells survived equally well following exposure to MMS, where-as spc1-M13 cells were profoundly sensitive to MMS treatment(Fig. 6B).

These results might be explained if Atf1 is not required fortransmitting the Spc1-dependent transcription induction signalthat is activated following exposure to UV irradiation. Perhapsthere is a transcription factor target other than Atf1 that isimportant for the UV response. We addressed this question bymeasuring pyp21 expression in Datf1 cells following UV expo-sure. UV-induced expression of pyp21 was abolished in Datf1cells and Dspc1 cells (Fig. 7). These findings show that an intactSpc1-Atf1 signaling pathway is required for the UV-inducedexpression of pyp21.

Atf1 is required for UV-induced transcription of ctt11, agene encoding catalase. These results called into question thenotion that Spc1-dependent induction of stress response genesis important for survival of UV and MMS treatment. It is likelythat Spc1 has important functions that do not involve Atf1;indeed the cell cycle defects of spc1-M13 mutants are notreplicated in Datf1 strains (35, 41). Therefore, it is possible thatSpc1 carries out functions required for UV survival that do not

involve transcriptional induction. However, a deficiency in thisargument was that genes such as pyp21 have no obvious rolesin combating oxidative stress; thus, a defect in expressingpyp21 is in itself not incompatible with the fact that Datf1 cellsare not abnormally sensitive to UV and MMS. Therefore, itbecame important to identify an Atf1-regulated gene that waslikely to be directly involved in protecting cells from oxidativestress. One such protein is catalase, which rapidly catalyzes thedecomposition of hydrogen peroxide to water and oxygen.Quite recently, the gene encoding cytosolic catalase, ctt11, wasidentified in S. pombe (26). Both oxidative stress and UVirradiation cause a large increase in the level of ctt11 mRNA,indicating that induction of ctt11 transcription may be an im-portant part of the UV response in S. pombe. In Saccharomycescerevisiae, catalase T is important for survival of oxidativestress, heat shock, and osmotic stress (40). Moreover, theHOG1 pathway regulates the osmotic induction of the CTT1gene (32).

We found that transcription of ctt11 was regulated by Spc1.UV irradiation caused a large increase in the level of ctt11

mRNA by a mechanism that was abolished in a Dspc1 orspc1-M13 background (Fig. 8A and data not shown). More-over, the basal level of ctt11 expression was also reduced inDspc1 or spc1-M13 cells. These observations suggested that thereduced level of ctt11 basal expression in Dspc1 cells may, inpart, account for their high sensitivity to killing by UV andMMS.

Interestingly, there was a very different pattern of ctt11

expression in Datf1 mutants relative to Dspc1 cells (Fig. 8A).The basal amount of ctt11 mRNA was not decreased in Datf1mutants relative to that in the wild type, but there was noincrease in the level of ctt11 mRNA after UV irradiation.Thus, Atf1 was not required for the basal level of ctt11 expres-sion but was necessary for the increased level of ctt11 expres-sion that is part of the UV response. These differences betweenDspc1 and Datf1 mutants may explain why Dspc1 cells aresensitive to UV and MMS, whereas Datf1 cells behave like thewild type.

Atf1 represses ctt11 mRNA expression and enhances UVsensitivity in Dspc1 cells. Two explanations came to mind toexplain how ctt11 expression could be highly dependent onSpc1 for both basal and UV-induced expression but requireAtf1 only for the increased level of expression which occurs inresponse to UV stress. Perhaps the simplest possibility was thatSpc1 regulated two transcription factors: one was sufficient forthe basal level of ctt11 expression as observed in Datf1 mu-tants, whereas the second factor, Atf1, was required for ex-pression that is induced by UV irradiation. In this scheme, thefirst transcription factor would not be sufficient for UV-in-

FIG. 6. Datf1 cells are not hypersensitive to killing by UV irradiation orMMS treatment. (A) Cell survival assays were performed with wild-type (WT)(PR109), atf1::ura41 (KS1497), and spc1-M13 (KS1147) cells exposed to theindicated doses of UV irradiation. (B) Cell survival assays were performed withwild-type (PR109), Datf1 (KS1497), spc1-M13 (KS1147), and rad24::ura41

(KS1455) cells exposed to the indicated doses of MMS.

FIG. 7. Induction of pyp21 mRNA transcription in response to UV is de-pendent on Atf1 and Spc1. RNA samples were collected at the indicated times(minutes) from wild-type (WT), Dspc1, and Datf1 cells after the cells had receiveda 200-J/m2 dose of UV irradiation. Northern analysis was performed with pyp21

and leu11 probes.

3360 DEGOLS AND RUSSELL MOL. CELL. BIOL.

on April 8, 2018 by guest

http://mcb.asm

.org/D

ownloaded from

duced transcription. The second model proposed that Atf1 hasboth transcriptional activator and repressor properties. In theabsence of Spc1, Atf1 acts as a transcriptional repressor. Onthe other hand, in the absence of Atf1 a transcriptional mech-anism that is independent of both Spc1 and Atf1 provides thebasal level of ctt11 expression. In this model, Spc1 convertsAtf1 from a transcriptional repressor to a transcriptional acti-vator.

The two possibilities, which we named the two-factor andAtf1 activator-repressor models, made contrary predictionsabout the pattern of ctt11 expression in Dspc1 Datf1 doublemutants. The two-factor model proposed that ctt11 mRNAexpression should be very low in the double mutant, equivalentto that observed in Dspc1 single mutant cells. In contrast, theAtf1 activator-repressor model made the opposite prediction,namely that there should be significant basal expression ofctt11 in the double mutant, equivalent to that seen in Datf1single mutant. Analysis of the Dspc1 Datf1 double mutantstrongly supported the activator-repressor model: the patternof ctt11 expression in the double mutant was indistinguishablefrom that of the Datf1 single mutant (Fig. 8A). This observa-tion strongly suggests that Spc1 activity converts Atf1 from atranscriptional repressor to a transcriptional activator.

The question arose as to whether the basal expression ofgenes such as ctt11 contributed to cell survival following ex-

posure to UV. This question was addressed by comparing theUV survival properties of wild-type, Dspc1 single mutant, andDspc1 Datf1 double mutant cells. Interestingly, the UV survivalphenotype of the Dspc1 Datf1 cells was intermediate betweenthose of the wild-type and Dspc1 cells (Fig. 8B). Thus, thetranscriptional repressor activity of Atf1 enhances UV killingin cells that lack Spc1 activity, although defects that cannot beascribed to the repressor activity of Atf1 also contribute to theUV sensitivity of Dspc1 cells.

DISCUSSION

The major aim of our studies was to use fission yeast toinvestigate how eukaryotic cells respond to cytotoxic damagecaused by agents such as UV and MMS. The first goal of thestudies was to determine whether S. pombe has a UV responsemechanism that is broadly analogous to the mammalian sys-tem. As described above, the mammalian UV response is be-lieved to involve the activation of two classes of MAPK ho-mologs, JNKs and p38 kinases. Thus, we initially askedwhether Spc1 kinase, which is most closely related to p38kinase, is activated in response to UV and MMS treatment.This analysis showed that Spc1 undergoes a large increase inactivating tyrosine phosphorylation in response to both typesof genotoxic agents. Importantly, the increase in Spc1 tyrosinephosphorylation was quite rapid and transient, typically ap-proaching maximal levels within 10 min of exposure to UV anddecreasing to basal levels within 60 min. Wis1 kinase wasrequired for the increase of Spc1 tyrosine phosphorylation inresponse to UV and MMS. We also found that UV and MMSexposure resulted in the induced expression of the pyp21 andctt11 genes, and this response was dependent on Atf1 tran-scription factor. Moreover, we observed that Atf1 was phos-phorylated in vivo after UV irradiation in an Spc1-dependentmanner. Thus, our findings show that fission yeast has a UVresponse mechanism that is transmitted through the Wis1-Spc1kinase cascade via Atf1 transcription factor.

A second aim of our studies was to identify the nature of thestress or damage that is sensed by the Spc1 UV responsepathway. As noted above, UV and MMS are well-known mu-tagens; thus, in theory these genotoxic agents could trigger theSpc1 UV response by damaging DNA. However, our findingsstrongly suggest that the Spc1 pathway is not responsive toDNA damage, or at least not to the presence of unligatedDNA as occurs in cdc17-K42 mutant cells. More importantly,our studies have shown that incubation of cells in NAC abol-ishes the UV-induced activation of Spc1. Intracellular NAC isconverted to GSH, which in turn reacts with oxygen free rad-icals. Thus, the ability of NAC to suppress UV-induced acti-vation of Spc1 kinase strongly suggests that Spc1 kinase isresponding to oxidative stress. Indeed, previous studies haveshown that the Spc1 kinase pathway becomes highly activatedwhen cells are exposed to hydrogen peroxide, which causesacute oxidative stress (7).

The third important aim of our studies was to evaluatewhether the UV response is important for survival of UVexposure. This is a disarmingly straightforward question thatdoes not have a simple answer. First, it is clear that activationof Spc1 via Wis1 kinase is quite important for survival of UVirradiation and MMS treatment. For example, at a UV dose of300 J/m2, the survival of spc1-M13 or Dspc1 cells is decreased;100-fold relative to wild-type cells. The UV sensitivity ofspc1-M13 cells is comparable to that of Drad24 mutants, whichare defective in the G2-M checkpoint (14). Likewise, spc1-M13cells exhibit a hypersensitivity to MMS treatment that is com-parable to that exhibited by Drad24 cells.

FIG. 8. Induction of expression of the ctt11 catalase gene in response to UVis regulated by Spc1 and Atf1. (A) RNA was isolated from wild-type (WT)(PR109), Dspc1 (KS1366), Datf1 (KS1497), and Dspc1 Datf1 (KS1533) cells afterthe cells had received a 200-J/m2 dose of UV irradiation. Northern analysis wasperformed with ctt11 and leu11 probes. In wild-type cells, there was a largeincrease of ctt11 mRNA in response to UV. This response was absent in thethree mutant strains. However, basal transcription of ctt11 was reduced only inthe Dspc1 strain, suggesting that Atf1 may act as a repressor of ctt11 expressionin Dspc1 cells. (B) UV irradiation survival properties of wild-type, Dspc1, andDspc1 Datf1 cells. The Dspc1 Datf1 double mutant exhibits a UV survival phe-notype intermediate between that of wild-type and Dspc1 mutant cells.

VOL. 17, 1997 UV RESPONSE IN SCHIZOSACCHAROMYCES POMBE 3361

on April 8, 2018 by guest

http://mcb.asm

.org/D

ownloaded from

Questions relating to the physiological importance of theUV response become complicated when one considers the roleof Atf1 transcription factor. Surprisingly, Datf1 mutants are nothypersensitive to killing by UV or MMS; in fact, Datf1 mutantsand wild-type cells cannot be distinguished in this assay. Thisresult could be easily understood if Atf1 was not involved inthe UV response or if there were other Atf1-like transcriptionfactors that provided redundant activities, but our studies showthat Atf1 is required for the transcriptional induction of severalUV-responsive genes. Of particular significance is the ctt11

gene, which encodes catalase, because the role of catalase incombating oxidative stress is well established. Expression ofctt11 mRNA is highly elevated in response to UV; this re-sponse is abolished in a Datf1 mutant. Why then are Datf1 andDspc1 mutants not similarly sensitive to killing by exposure toUV or MMS? Our studies suggest the difference between Datf1and Dspc1 mutants in UV and MMS sensitivity may be partlyexplained by differences in basal expression of important stressresponse genes. In Dspc1 cells, the basal expression of ctt11 isvery low, much less than is observed in wild-type cells grownunder unstressful conditions. In contrast, basal ctt11 expres-sion in Datf1 mutants is not decreased relative to that of thewild type. Basal expression of ctt11 is also approximately nor-mal in Dspc1 Datf1 double mutant cells, indicating that Atf1acts as a repressor of ctt11 expression in Dspc1 cells. The basalexpression of ctt11 and perhaps other genes appears to bephysiologically important, because UV survival is enhanced inthe Dspc1 Datf1 double mutant cells relative to that in theDspc1 single mutant.

These observations lead to the following additional conclu-sions. (i) The increased expression of ctt11 that occurs follow-ing exposure is not important for survival of exposure to UV orMMS, at least not as assayed by the methods described here.The same can be said for other stress response genes regulatedby Atf1. (ii) The fact that Dspc1 Datf1 double mutant cells aremore sensitive to UV killing than Datf1 single mutant cells,even though both strains exhibit the same patterns and levelsof ctt11 expression, shows that Spc1 contributes to UV survivalby a mechanism that does not involve Atf1. In theory this couldoccur by a transcriptional induction mechanism that does notrequire Atf1 or regulate ctt11 expression, or it may be thatSpc1 has important substrates that are not involved in tran-scriptional regulation. Resolution of this question will comewith the identification of additional in vivo substrates of Spc1.

An ancillary discovery arising from our studies is that Atf1transcription factor appears to act as a repressor of ctt11 ex-pression in Dspc1 cells. As mentioned above, this conclusion isbased on the observation that basal expression of ctt11 isenhanced in a Dspc1 Datf1 double mutant relative to that in aDspc1 single mutant. The simplest interpretation of this obser-vation is that Atf1 is negatively influencing ctt11 expression inthe absence of Spc1 activity. To our knowledge, this is the firstindication that a bZIP transcription factor is converted from atranscriptional repressor to activator by the action of a MAPKhomolog. We do not yet know exactly how Spc1 regulates Atf1activity, other than that it involves direct phosphorylation ofAtf1 by Spc1 in vivo (35).

The mammalian UV response, first identified as a charac-teristic pattern of altered gene expression induced by briefexposure to UV, has been recognized for more than a decade.Recent investigations have resulted in the identification ofproteins that are believed to have important signal transduc-tion functions in the UV response, such as p38/RK kinase andATF-2 transcription factor. Other recent advances have alsoled to the availability of ATF-2-deficient mice and compoundsthat specifically inhibit p38/RK kinases (6, 22, 29), but as of yet,

the effect of these mutations and kinase inhibitors on thesurvival of cells following UV exposure has not been described.Thus, the actual physiological significance of the mammalianUV response is uncertain. Our findings may be instructive inseveral regards. First, our studies of Spc1 kinase in fission yeastcells provide direct evidence that UV-activated kinases areimportant for survival of UV irradiation; thus, we may expectthat JNK and p38 kinase will have similarly important func-tions in mammalian cells. Second, although our studies haveclearly shown that Spc1 kinase is required for the transcrip-tional induction of a gene encoding a protein that has a wellknown role in combating oxidative stress, namely catalase, itcannot be assumed that transcriptional induction of stress re-sponse genes is important for survival of UV stress. Datf1mutants fail to induce ctt11 expression in response to UV andyet exhibit no defect in surviving UV irradiation. There hasbeen an implicit assumption that the increased expression ofAP-1-regulated genes that is the hallmark of the mammalianUV response is critically important for survival of UV damage,but our findings clearly indicate that for fission yeast cells, thisassumption may be incorrect. Our findings suggest that stress-activated kinases such as p38 and JNK may have substratesother than transcription factors that have primary importancein promoting survival of cells in the face of UV irradiation. Achallenge of future studies will be to identify these substrates;we anticipate that fission yeast cells will prove useful for ac-complishing these goals.

ACKNOWLEDGMENTS

We thank the TSRI Cell Cycle Groups for their support, with par-ticular thanks going to Kazuhiro Shiozaki, Frederique Gaits, andJunko Kanoh. Suresh Subramani, Tony Carr, and Susan Forsburgprovided rad strains and the cdc17-K42 strain.

G.D. was supported by an American Cancer Society InternationalCancer Research Fellowship. This work was funded by an NIH grantawarded to P.R.

REFERENCES

1. Alfa, C., P. Fantes, J. Hyams, M. McLeod, and E. Warbrick. 1993. Experi-ments with fission yeast. A laboratory course manual. Cold Spring HarborPress, Cold Spring Harbor, N.Y.

2. Angel, P. 1995. The role and regulation of the jun protein in response tophorbol ester and UV light, p. 62–92. In P. Baeuerle (ed.), Inducible geneexpression and cytoplasmic/nuclear signal transduction. Birkhauser Verlag,Boston, Mass.

3. Black, H. S. 1987. Potential involvement of free radical reactions in ultravi-olet light-mediated cutaneous damage. Photochem. Photobiol. 46:213–221.

4. Brewster, J. L., T. de Valoir, N. D. Dwyer, E. Winter, and M. C. Gustin. 1993.An osmosensing signal transduction pathway in yeast. Science 259:1760–1763.

5. Carr, A. M. 1995. DNA structure checkpoints in fission yeast. Semin. CellBiol. 6:65–72.

6. Cuenda, A., J. Rouse, Y. N. Doza, R. Meier, P. Cohen, T. F. Gallagher, P. R.Young, and J. C. Lee. 1995. SB 203580 is a specific inhibitor of a MAP kinasehomologue which is stimulated by cellular stresses and interleukin-1. FEBSLett. 364:229–233.

7. Degols, G., K. Shiozaki, and P. Russell. 1996. Activation and regulation ofthe Spc1 mitogen-activated protein kinase in Schizosaccharomyces pombe.Mol. Cell. Biol. 16:2870–2877.

8. Derijard, B., M. Hibi, H. Wu, T. Barret, B. Su, T. Deng, M. Karin, and R. J.Davis. 1994. JNK1: a protein kinase stimulated by UV light and Ha-Ras thatbinds and phosphorylates the c-jun activation domain. Cell 76:1025–1037.

9. Derijard, B., J. Raingeaud, T. Barrett, I. W. Wu, J. Han, R. J. Ulevitch, andR. J. Davis. 1995. Independent human MAP kinase signal transductionpathways defined by MEK and MKK isoforms. Science 267:682–685.

10. Devary, Y., R. A. Gottlieb, T. Smeal, and M. Karin. 1992. The mammalianultraviolet response is triggered by activation of Src tyrosine kinase. Cell71:1081–1091.

11. Devary, Y., C. Rosette, J. A. DiDonato, and M. Karin. 1993. NF-KB activa-tion by ultraviolet light is not dependent on a nuclear signal. Science 261:1442–1445.

12. Doe, C. L., J. M. Murray, M. Shayeghi, M. Hoskins, A. R. Lehmann, A. M.Carr, and F. Z. Watt. 1993. Cloning and characterization of the Schizosac-

3362 DEGOLS AND RUSSELL MOL. CELL. BIOL.

on April 8, 2018 by guest

http://mcb.asm

.org/D

ownloaded from

charomyces pombe rad8 gene, a member of the SNF2 helicase family. NucleicAcids Res. 21:5964–5971.

13. Engelberg, D., C. Klein, H. Martinetto, K. Struhl, and M. Karin. 1994. TheUV response involving the ras signaling pathway and AP-1 transcriptionfactors is conserved between yeast and mammals. Cell 77:381–390.

14. Ford, J. C., F. al-Khodairy, E. Fotou, K. S. Sheldrick, D. J. Griffiths, andA. M. Carr. 1994. 14-3-3 protein homologs required for the DNA damagecheckpoint in fission yeast. Science 265:533–535.

15. Gupta, S., D. Campbell, B. Derijard, and R. J. Davis. 1995. Transcriptionfactor ATF2 regulation by the JNK signal transduction pathway. Science267:389–393.

16. Han, J., J. D. Lee, L. Bibbs, and R. J. Ulevitch. 1994. A MAP kinase targetedby endotoxin and hyperosmolarity in mammalian cells. Science 265:808–811.

17. Hibi, M., A. Lin, T. Smeal, A. Minden, and M. Karin. 1993. Identification ofan oncoprotein- and UV-responsive protein kinase that binds and potenti-ates the c-Jun activation domain. Genes Dev. 7:2135–2148.

18. Kanoh, J., Y. Watanabe, M. Ohsugi, Y. Iino, and M. Yamamoto. 1996.Schizosaccharomyces pombe gad71 encodes a phosphoprotein with bZIPdomain, which is required for proper G1 arrest and gene expression undernitrogen starvation. Genes Cells 1:391–408.

19. Kato, T., K. Okazaki, H. Murakami, S. Stetter, P. A. Fantes, and H.Okayama. 1996. Stress signal, mediated by a Hog1-like MAP kinase, controlssexual development in fission yeast. FEBS Lett. 378:207–212.

20. Kyriakis, J., P. Banerjee, E. Nikolakaki, T. Dai, E. Rubie, M. Ahmad, J.Avruch, and J. Woodgett. 1994. The stress-activated protein kinase subfamilyof c-Jun kinases. Nature 369:156–160.

21. Lauthier, D., P. Luscher, and R. M. Tyrrell. 1992. Endogenous glutathionelevels modulate both constitutive and UV-A radiation/hydrogen peroxideinducible expression of the human heme oxygenase gene. Carcinogenesis 13:227–232.

22. Lee, J., J. Laydon, P. McDonnell, R. T. Gallaghe, S. Kumar, D. Green, D.McNulty, M. Blumenthal, J. Heys, and S. Landvatter. 1994. A protein kinaseinvolved in the regulation of inflammatory cytokine biosynthesis. Nature372:739–746.

23. Meister, A. 1991. Glutathione deficiency produced by inhibition of its syn-thesis and its reversal, applications in research and therapy. Pharmacol.Ther. 51:155–194.

24. Millar, J. B. A., V. Buck, and M. G. Wilkinson. 1995. Pyp1 and Pyp2 PTPasesdephosphorylate an osmosensing MAP kinase controlling cell size at divisionin fission yeast. Genes Dev. 9:2117–2130.

25. Moreno, S., A. Klar, and P. Nurse. 1991. Molecular genetic analysis of fissionyeast Schizosaccharomyces pombe. Methods Enzymol. 194:795–823.

26. Nakagawa, C. W., N. Mutoh, and Y. Hayashi. 1995. Transcriptional regula-tion of catalase gene in the fission yeast Schizosaccharomyces pombe: mo-lecular cloning of the catalase gene and northern blot analyses of the tran-script. J. Biochem. 118:109–116.

27. Nasmyth, K. A. 1977. Temperature-sensitive lethal mutants in the structuralgene for DNA ligase in the yeast Schizosaccharomyces pombe. Cell 12:1109–1120.

28. Raingeaud, J., S. Gupta, J. Rogers, M. Dickens, J. Han, R. Ulevitch, and R.

Davis. 1995. Pro-inflammatory cytokines and environmental stress cause p38mitogen-activated protein kinase activation by dual phosphorylation on ty-rosine and threonine. J. Biol. Chem. 270:7420–7426.

29. Reimold, A., M. Grusby, B. Kosaras, J. Fries, R. Mori, S. Maniwa, I. Clauss,T. Collins, R. Sidman, M. Glimcher, and L. Glimcher. 1996. Chondrodys-plasia and neurological abnormalities in ATF-2-deficient mice. Nature 379:262–265.

30. Rouse, J., P. Cohen, S. Trigon, M. A. L. Morange, D. Zamanillo, T. Hunt,and A. Nebreda. 1994. A novel kinase cascade triggered by stress and heatshock that stimulates MAPKAP kinase-2 and phosphorylation of the smallheat shock proteins. Cell 78:1027–1037.

31. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: alaboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.

32. Schuller, C., J. L. Brewster, M. R. Alexander, M. C. Gustin, and H. Ruis.1994. The HOG pathway controls osmotic regulation of transcription via thestress response element (STRE) of the Saccharomyces cerevisiae CTT1 gene.EMBO J. 13:4382–4389.

33. Sheldrick, K. S., and A. M. Carr. 1993. Feedback controls and G2 check-points: fission yeast as a model system. Bioessays 15:775–782.

34. Shiozaki, K., and P. Russell. 1995. Cell-cycle control linked to extracellularenvironment by MAP kinase pathway in fission yeast. Nature 377:739–743.

35. Shiozaki, K., and P. Russell. 1996. Conjugation, meiosis and the osmoticstress response are regulated by Spc1 kinase via Atf1 transcription factor infission yeast. Genes Dev. 10:2276–2288.

36. Takeda, T., T. Toda, K. Kominami, A. Kohnosu, M. Yanagida, and N. Jones.1995. Schizosaccharomyces pombe atf11 encodes a transcription factor re-quired for sexual development and entry into stationary phase. EMBO J.14:6193–6208.

37. van Dam, H., D. Wilhelm, I. Herr, A. Steffen, P. Herrlich, and P. Angel. 1995.ATF-2 is preferentially activated by stress-activated protein kinases to me-diate c-jun induction in response to genotoxic agents. EMBO J. 14:1798–1811.

38. Warbrick, E., and P. A. Fantes. 1991. The wis1 protein kinase is a dosage-dependent regulator of mitosis in Schizosaccharomyces pombe. EMBO J.10:4291–4299.

39. Waskiewicz, A. J., and J. A. Cooper. 1995. Mitogen and stress responsepathways: MAP kinase cascades and phosphatase regulation in mammalsand yeast. Curr. Opin. Cell Biol. 7:798–805.

40. Wieser, R., G. Adam, A. Wagner, C. Schuller, G. Marchler, H. Ruis, Z.Krawiec, and T. Bilinski. 1991. Heat shock factor-independent heat controlof transcription of the CTT1 gene encoding the cytosolic catalase T ofSaccharomyces cerevisiae. J. Biol. Chem. 266:12406–12411.

41. Wilkinson, M. G., M. Samuels, T. Takeda, W. M. Toone, J. C. Shieh, T.Toda, J. B. A. Millar, and N. Jones. 1996. The Atf1 transcription factor is atarget for the Sty1 stress-activated MAP kinase pathway in fission yeast.Genes Dev. 18:2289–2301.

42. Ziff, E. B. 1990. Transcription factors: a new family gathers at the cAMPresponse site. Trends Genet. 6:69–72.

VOL. 17, 1997 UV RESPONSE IN SCHIZOSACCHAROMYCES POMBE 3363

on April 8, 2018 by guest

http://mcb.asm

.org/D

ownloaded from