Copyright 0 1994 by the Genetics Society of America

Characterization of G, Checkpoint Control in the Yeast Saccharomyces cerevisiae Following Exposure to DNA-Damaging Agents

Wolfram Siede, Andrew S. Friedberg, Irina Dianova and Errol C. Friedberg

Laboratory of Molecular Pathology, Department of Pathology, University of Texas Southwestern Medical Center, Dallas, Texas 75235

Manuscript received February 24, 1994 Accepted for publication June 17, 1994

ABSTRACT The delay of Sphase following treatment of yeast cells with DNAdamaging agents is an actively regu-

lated response that requires functional RAD9 and RAD24 genes. An analysis of cell cycle arrest indicates the existence of (at least) two checkpoints for damaged DNA prior to Sphase; one at START (a G, checkpoint characterized by pheromone sensitivity of arrested cells) and one between the CDC4- and CDC7-mediated steps (termed the G,/S checkpoint). When a dnal-1 mutant (that affects early events of replicon initiation) also carries a rad9 deletion mutation, it manifests a failure to arrest in G,/S following incubation at the restrictive temperature. This failure to execute regulated G,/S arrest is correlated with enhanced thermosensitivity of colony-forming ability. In an attempt to characterize the signal for RAD9 genedependent G, and G,/S cell cycle arrest, we examined the influence of the continued presence of unexcised photoproducts. In mutants defective in nucleotide excision repair, cessation of Sphase was observed at much lower doses of UV radiation compared to excision-proficient cells. However, this re- sponse was not RADPdependent. We suggest that an intermediate of nucleotide excision repair, such as DNA strand breaks or single-stranded DNA tracts, is required to activate RADPdependent G, and G,/S checkpoint controls.

E UKARYOTIC cells can undergo transient arrest of cell cycle progression in the presence of damaged

or unreplicated DNA (MURRAY and HUNT 1993; LI and DESHAIES 1993; WEINERT and LYDALL 1993; MURRAY 1992). These regulated responses are part of the repertory of feedback controls which ensure the completion of meta- bolic steps pertinent to one cell cycle stage before ini- tiating the next. These controls appear to act at discrete transitional cell cycle stages and have been termed checkpoint controls (HARTWELL and WEINERT 1989; WEINERT and HARTWELL 1988).

Defective checkpoint responses have important implications for the process of neoplastic transforma- tion and for the phenotype of cancer cells (LANE 1992; HARTWELL 1992). For example, mammalian cells with a dysfunctional p53 gene fail to manifest G, arrest after treatment with ionizing irradiation (KUERBITZ et al. 1992; KASTAN et al. 1991, 1992). Additionally, p53 pro- tein levels are post-translationally enhanced after expo- sure of cells to X-rays, ultraviolet (W) radiation or al- kylating agents such as methyl methanesulfonate (MMS) (MALTZMAN and CZYZYK 1984; HALL et al. 1993; Z w e t al. 1993; Lu and LANE 1993). Reduced induction or altered kinetics of p53 activation has been reported in the human cancer-prone hereditary disorders Bloom’ syndrome and ataxia telangiectasia (AT) (-TAN et al. 1992; Lu and LANE 1993; KHANNA and LAWN 1993). AT cells are characterized by “radioresistant” DNA synthesis (PAINTER and YOUNG 1980; PAINTER 1986) that can be explained by defective GI checkpoint control. Genes

Genetics 138: 271-281 (October, 1994)

that are transcriptionally activated by p53 protein and induced in the presence of DNAdamaging agents in- clude MDM2 (PEmYet al. 1993), GADD45 (USTAN et al. 1992) and WAFl/CZPl (EL-DEIRY et al. 1993). WAFl/ CIPl protein (or p21) has been shown to bind to several human cyclindependent kinases known to be involved in the Gl/S transition and to inhibit their kinase activity (HARFJER et al. 1993). WAFl/CIPl protein is therefore an excellent candidate for an effector molecule medi- ating p53dependent cell cycle arrest. Indeed, GI arrest induced by DNAdamaging agents is correlated with en- hanced WAFl/CIPl protein levels (EL-DEIRY et al. 1994). Furthermore, diminished kinase activity of a Cdk/cyclin E complex has been observed in G,-arrested cells following treatment with ionizing radiation, and the WAFl/CIPl inhibitor has been found to be co- immunoprecipitated with Cdk/cyclin E complexes in G,-arrested cells (DULIC et al. 1994; EL-DEIRY et al. 1994).

The mechanism and physiological significance of G, arrest in the presence of DNA damage in mammalian cells is largely unknown. Loss of p53 activity in tumor cells is correlated with an enhanced probability of spon- taneous gene amplification (LMNGSTONE et al. 1992; YIN et al. 1992). However, cellular sensitivity to killing by ionizing irradiation is not enhanced by loss of p53 func- tion or GI arrest. Similarly, there is no direct correlation between the status of the p53 gene and X-ray sensitivity of tumor cell lines (BRACHMAN et al. 1993). In fact pri- mary cultures of human fibroblasts carrying a homozygous $153 deletion are more resistant to ionizing radiation than

272 W. Siede et al.

TABLE 1

List of yeast strains

Strain Genotype

SX46 A MATa RAD ade2 his3-532 trpl-289 ura3-52 SX46 A rad9A MATa rad9A::URA3 ade2 his3-532 trpl-289 ura3-52 SX46 A rad24A MATa rad24A::URA3 ade2 his3-532 trpl-289 ura3-52 SX46 A rad2A MATa rad2A::TRPI ade2 his3-532 trpl-289 ura3-52 SX46 A radl4A MATa radl4A::HISjr ade2 his3-532 trpl-289 ura3-52 SX46 A rad2A rad9A MATa rad2A::TRPI rad9A::URAjr ade2 his3-532 trpl-289 ura3-52 SX46 A radl4A rad9A MATa rad14A::HIS3 rad9A::URA3 ade2 his3-532 trpl-289 ura3-52

WS9110-3D rad9A MATa cdc4-I rad9A::URA3 ade2-l ura3-52

WS9120-1OA radYA MATa cdc7-l rad9A::URA3 ura3-52

WS9121-1B radYA MATa dnal-1 rad9A::URA3 ade ura3-52

L128-20 rad9A MATa dbf4-1 rad9A::URA3 a d d trpl ura3-52

WS9110-3D MATa cdc4-1 RAD ade2-1 ura3-52

WS9120-1OA MATa cdc7-1 RAD ura3-52

WS9121-1B MATa dnal-1 RAD ade ura3-52

~ 1 2 a - 2 0 MATa dbf4-l RAD ade5 trpl ura3-52

their normal counterparts (LEE and BERNS~EIN 1993; SLICHENMEYER et al. 1993).

The highly conserved mechanisms of DNA repair and cell cycle regulation among eukaryotes (HOEIJMAKERS 1993; MURRAY and HUNT 1993) suggests the utility of the yeast Saccharomyces cerevisiae as a model for exploring the molecular mechanism(s) of G, arrest and its physi- ological significance in cells exposed to DNA damage. We have recently reported that the delayed entry into Sphase observed in yeast cells synchronized in G, and then treated with various DNA-damaging agents de- pends on the checkpoint control gene RAD9. This ob- servation established the regulated nature of G, arrest in the presence of DNA damage (SIEDE et al. 1993).

The RAD9, RAD1 7, RAD24, RAD53 (= MEC2), MECl and MEC3 genes have all been shown to regulate G, arrest after treatment of cells with ionizing or UV radiation (WEINERT et al. 1994; WEINERT and LYDALL 1993; WEINERT and HARTWELL 1988, 1993). RAD9 also regu- lates checkpoint controls during meiosis (THORNE and BYERS 1993; WEBER and BYES 1992). However, RAD9 is not required for the late S/G, arrest response to un- replicated DNA, i . e . , after hydroxyurea treatment (WEINERT et al. 1994). The function of RAD9must there- fore be considered specific for DNA damage-mediated checkpoint control. The only known phenotype of un- irradiated yeast cells deleted of RAD9 is an enhanced probability of chromosome loss (WEINERT and HARTWELL 1990). The RAD9 gene has been cloned and sequenced. The gene encodes a predicted polypeptide of 148.4 kD with no homology to other known proteins (WEINERT and HARTWELL 1990; SCHIESTL et al. 1989).

We have continued to characterize DNA damage- dependent G, arrest in yeast and have used the RAD9 dependency of the arrest phenomenon as a specific cri- terion of its regulated nature. We show here that in ad- dition to RAD9 the yeast RAD24 gene is involved in regulating G, arrest. Additionally, we present evidence for at least two damage-dependent cell cycle check-

points in UV- or y-irradiated cells. One is at START and another has been mapped between the CDC4- and CDC7-mediated steps of cell cycle progression. We have also observed that Gl/S arrest in the thermoconditional mutant dnal-1 (EBERLY et al. 1989) in the absence of exposure to exogenous DNA damaging agents is de- pendent on a functional RAD9 gene. Finally, in an at- tempt to characterize the specific signal(s) that triggers checkpoint arrest we have examined the G, arrest re- sponse to U V or ionizing radiation in mutants defective in nucleotide excision repair. Our results suggest that DNA strand breaks generated during this repair mode rather than base damage per se are important for regu- lated G, arrest after UV irradiation of cells.

MATERIALS AND METHODS

Yeast strains and construction of RAD gene deletions: The yeast strains used are listed in Table 1. The dbf4-1 strain L12&20 was kindly provided by L. H. JOHNSTON and used di- rectly. The original d n a l - 1 mutantusedfor strain construction was from A. SUGINO, and the cdc alleles were from strains pro- vided by the Yeast Genetic Stock Center, Berkeley, California. To construct strains of mating type MATa containing the a p propriate markers, spores from crosses of strains AS1001 ( d n u l - 1 ) and STX422-2A (cdc4-1) against our standard wild- type SX46 A (originally from J. RINE) were isolated (WS9121- lB, WS9110-3D; Table 1) . The cdc7-1 strain 6124 (originally from L. H. HARTWELL) was first crossed against BJ1991 (Yeast Genetic Stock Center, originally from E. W. JONES), and a spore from the resulting diploid strain was further crossed against YNN217 (R. W. DAVIS) to yield spore WS9120-1OA (Table 1). Plasmid manipulations were performed according to p u b lished protocols (SAMBROOK et al. 1989). Yeast strains were transformed by a modified lithium procedure (GIETZ et al. 1992). RAD gene deletion mutants were constructed by one-step

gene disruption (ROTHSTEIN 1983). A complete replacement of the RAD2 open reading frame (OW) with TRPl was created by transformation with the Sua-cut plasmid pWS52 I . This plas- mid contains regions upstream and downstream of the RAD2 gene on a SalI fragment in a pBR322derived vector. The RAD2 ORF was previously deleted by replacing a HpaI-NdeI fragmentwith TRPl on aEcoRI-BgllI fragment (from plasmid

S. cerevisiae G, Checkpoint Control 273

YRp7). The R A D 1 4 gene was first isolated on a 1.8kb fragment by the polymerase chain reaction (PCR) , and a deletion from amino acid residues 16-195 was created by replacing a HindIII-Sac1 fragment with HIS3 on a BamHI fragment. A SpeI-EcoRV digest was used for transplacement. The RAD24 gene was deleted using EcoRI-digested plasmid pRS406 rad24A (kindly supplied by T. WEINERT). This plasmid contains the RAD24 ORF deleted by URA3 replacing a XbaI-BgAI frag- ment. The construction of RAD9deletions has been described previously (SIEDE et al. 1993).

Cell synchronization and conditions of irradiation: a-Factor synchronization of early logarithmic phase cells and irradiation of suspensions with 254nm W light was per- formed as described by SIEDE et al. (1993), except that cdc mutants were synchronized at 25" for 3.5 hr. Synchronization of thermoconditional mutants was accomplished by incubat- ing cultures grown at 25" in YPD to early logarithmic phase (-2 X 106cells/ml) for 3 hr (for cdc7-1) or 2.25 hr (for cdc4-1, dbf4-1) at 36". Treatment with ionizing radiation was per- formed directly on a-factor- or temperature-arrested cultures in YPD with a '"Cs y-source u. L. Shepherd & Assoc.) at a dose rate of 470 rad/min. Cycloheximide, nocodazole and a-factor were from Sigma.

Flow cytometry: At various times after irradiation, samples (- 1 X lo7 cells) were removed, washed with water and fixed in 80% ethanol. Fixed cells were kept overnight at 4", washed with water, resuspended in 200 pl50 mM sodium citrate (pH 7.0) containing 25 pg/ml propidium iodide and 250 pg/ml RNase and incubated for 3 hr at 37". Stained cells were diluted 120 in 10 mM TrisHC1 (pH 9.0), briefly sonicated and ana- lyzed on a Becton-Dickinson FACScan flow-cytometer using the CELLFIT program (version 2.0) for analysis of DNA con- tent. A total of 5,000 cells were analyzed at each time point. In haploid yeast cells the peaks of propidium iodide-stained cells with G,, Sand G, DNAcontent are usually too close for reliable integration of histogram areas. Hence, we performed integra- tions [using the "sum of broadened rectangles" (SOBR) method] only in those cases where adequate resolution was obtained.

RESULTS

RADPdependent GI checkpoint arrest at START in W- and y-irradiated cells: We previously reported that cells synchronized at START with the mating phero- mone a-factor (Figure 1) undergo RABPdependent cell cycle arrest characterized by a G, DNA content when exposed to W light or "rays (SIEDE et al. 1993). In the present study we carried out a series of experiments de- signed to more precisely map the arrest stage(s) after treatment with DNAdamaging agents.

Cells of the haploid strain SX46 A were synchronized in GI with a-factor. Following synchronization the cells were UV-irradiated or not, released from a-factor arrest and incubated in fresh medium for 1 hr at 30". After this period of incubation the majority of the W-irradiated cells were arrested in G, as determined by DNA content, whereas a significant fraction of the unirradiated control culture had progressed beyond this phase of the cell cycle (Figure 2). In order to determine whether the GI- arrested cells were specifically arrested at START (a physiological stage in which cells are sensitive to a-factor), we divided the W-irradiated culture and

CDc28 cDc4 cDc7 DBF4 T I n d c p m d m a O f p r o t d n ~ # s i s """"""

wddiag """"""""

a-Factor

G1 S FIGURE 1.-Schematic representation of the genetic control

of cell cycle progression in yeast in relation to transcript induction.

treated one halfwith a-factor once again (Figure 2, third column). The dose of a-factor used (5.0 pg/ml) was independently shown to inhibit entry into Sphase of cells synchronized at START for -2 hr (data not shown).

The presence of a-factor prevented further cell cycle progression in the majority of cells previously exposed to UV radiation, suggesting that these cells were indeed arrested at START (Figure 2, third column). By 80 min after the addition of a-factor, cells that were insensitive to arrest had progressed in the cycle and yielded a dis- crete G, peak by flow cytometry analysis. We could there- fore determine that -63% of the W-irradiated cells re- mained sensitive to a factor (Figure 2, third column). The corresponding fraction in the unirradiated control was significantly smaller, but could not be precisely determined since the reentry of unirradiated cells into G, (Figure 2, first column) leads to an overestimate of the fraction of cells at START. Nevertheless, these results indicate that START constitutes a DNA damage checkpoint.

The remaining half of the cultures that were exposed to W radiation and post-irradiation incubation were treated with the protein synthesis inhibitor cyclohexim- ide (Figure 2, fourth column). It has been established that progression of the S phase is independent of de nouo protein synthesis downstream of the CDC7- mediated G,/S transition step in the yeast cell cycle (HEREFORD and HARTWELL 1973). Hence, the observation that cell cycle progression in W irradiated cells is sen- sitive to cycloheximide (Figure 2, fourth column) is con- sistent with the conclusion that W-irradiated cells un- dergo arrest at a stage earlier than the CDC7-dependent process. Alternatively, these results suggest that the re- sumption of cell cycle progression after W radiation- induced G, arrest requires de novo protein synthesis.

274 W. Siede et al.

0 min

60 min

80 min

100 min

120 min

140 min

0 J/m2 + a

RAD 30 J/m2 30 J/m2 30 J/m2

+ a +CYH

Addition of a-factor or cycloheximide

DNA Content

To investigate whether arrest at START is a regulated process dependent on specific checkpoint control genes we compared the response of the wild-type ( R A D 9 ) strain and an otherwise isogenic rad9A mutant. Figure 3 shows the results of a representative experiment in which cells were exposed to yirradiation.'Similar results were observed when cells were exposed to UV radiation (not shown). The DNA profiles observed 1 hr after ex- posure to y rays and release from a-factor arrest (Figure 3, A and B) confirm the defect in G, arrest in the rad9A mutant reported previously (SIEDE et al. 1993). Once again a-factor was added to part of the cultures following 1 hr of post-irradiation incubation to detect the fraction of cells arrested at START. In this experiment we also added the microtubuledestabilizing drug nocodazole (10 pg/ml) to all samples in order to block mitosis and prevent reentry of cycling cells into G,. Hence, in this case the majority of the unirradiated control cells were blocked in G, and the fraction of cells at START could be inferred from the G, peak detected in cultures in- cubated with nocodazole plus a-factor for the same length of time. This fraction was clearly greater in irra-

FIGURE 2.-Flow cytometric analysis of DNA content of a-factor-synchronized cells of strain SX46 A RAD after treatment with 0 or 30 J/m2 UV during incubation in YPD at 30". After 1 hr of incubation, a-factor (5.0 pg/ml, first and third column) or cycloheximide (200 pg/ml, fourth column) was added to part of the cul- tures. When plated out at 0 min, the survival of colony forming cells was 53% of the un- irradiated control. In this and in all other flow cytometric analyses shown the data are rep resentative of at least three independent experiments.

diated wild-type cells than in unirradiated controls (Fig- ure 3A). However, this difference was not observed in identically treated cultures of rad9A cells (Figure 3B).

The prolonged incubation of cultures in the presence of nocodazole results in distortion of the DNA profiles observed by flow cytometry. We therefore wished to con- firm the RADMependent arrest at START using an in- dependent experimental variable. Cells blocked in G, appear morphologically as large budded cells whereas cells blocked at START are unbudded. During the course of the experiment shown in Figure 3, A and B, we determined the fraction of unbudded cells by mi- croscopic examination (Figure 3, C and D) . The differ- ence between the fraction of unbudded cells in cultures with and without added a-factor after a further 120 min of incubation is a measure of cells at START at the time of addition (2 . e . , after 60 min of post-irradiation incu- bation). For wild-type cells these values were 19% cells at START in unirradiated cultures and 53% cells at START in irradiated controls (Figure 3C). The corre- sponding fractions in the rad9A cultures were 11 and IS%, respectively (Figure 3D). These results demon-

S. cerevisiae G, Checkpoint Control 275

A B RAD rad9A

0 Krad +a-factor

10 Krad 0 Krad +a-factor

10 Krad +a-factor

c- noaxlazole Addition of

+I- a-factor

150 mino

DNA Content

C

3b €b do 120 150 A0 Time after Irradiation (min)

0 I 0 30 50 90 120 4 5 0 180

Time after Irradiation (min)

FIGURE 3.-Flow cytometric analysis of DNA content (A, B) and microscopic determination of the fraction of unbudded cells (C, D) in a-factor-synchronized cultures of strains SX46 A RAD (A, C) and SX46 A rad9A (B, D) after treatment with 0 ( 0 , O ) or 10 krad ?rays (0, m). As indicated by the arrows, nocodazole (10 pg/ml) and a-factor (5.0 pg/ml) were added after 60 min of post-irradiation incubation, the latter only to part of the cultures (second and fourth column in A, B; filled symbols in C, D).

strate that DNA damage-induced arrest at START is de- pendent on a functional RAD9 gene.

RADMependent GI checkpoint arrest can also occur between the CDC4- and CDC7-mediated steps of cell cycle progression: We next addressed whether RAD9 dependent arrest can occur downstream of START dur- ing late GI (G,/S transition). In these experiments we used cells synchronized at the CDCCmediated step in the cell cycle (Figure 1 ) by incubating cultures of cdc4-1 cells at the restrictive temperature. As expected, cycle progression of cells arrested at the CDC4mediated step was insensitive to a-factor (data not shown). The syn- chronized cultures were then UV-irradiated and incu- bated at the permissive temperature. Samples were re- moved at various times and analyzed by flow cytometry. In RAD9 cdc4 mutants cell cycle arrest was observed at a stage characterized by a G, DNA content (Figure 4). However, in the otherwise isogenic cdc4 rad9A mutant the extent of this arrest was considerably reduced. Simi- lar results were observed after treatment with 7-rays (Fig- ure 5). Thus, in addition to a checkpoint at START, RADPdependent checkpoint arrest can occur down- stream of the CDC4-mediated step.

To test whether RADWependent G,/S checkpoint arrest can occur at an even later step of G,/S transition we synchronized cells at the CDC7-mediated and the closely related DBF4mediated steps (CHAPMAN and JOHNSTON 1989; KITADA et al. 1993) (Figure I) , by incu-

bation of cdc7-1 or dbf4-I mutant cells under restrictive conditions. Once again a significant dose-dependent de- lay of !+phase was evident after UV irradiation. However, in contrast to the results obtained with the cdc4 mutant, this effect was not significantly influenced by the status of the RAD9 gene in the cdc7-1 and dbf4-1 mutants (Figure 6 and data not shown). We independently dem- onstrated that checkpoint arrest at START is intact in these mutants by irradiating cdc 7 and cdc 7 rad 9A strains synchronized with a-factor under permissive conditions. G, arrest was readily detected and was dependent on a functional RAD9 gene (Figure 7) .

G,/S arrest of the dnal-1 mutant is also RAD9 dependent: A temperature-sensitive yeast mutant des- ignated d n a l - 1 has been reported to undergo cell cycle arrest at elevated temperatures (EBERLY et al. 1989). This and other studies have led to the suggestion that the DNA1 gene functions during early events in replicon initiation and is required throughout Sphase (EBERLY et al. 1989). Surprisingly, we found that cell cycle arrest in the dna l -1 mutant requires a functional RAD9gene, as evidenced by a failure to observe an accumulation of cells with a G, DNA content in a dnal-1 rad9A mutant after shifting the temperature of an asynchronously di- viding culture from 25" to >35" (Figure 8). This mutant was also characterized by enhanced thermosensitivity of colony formation. Both dna l -1 RAD9 and dna l -1 rad9A mutant cells were viable at 23" or 30°, but not at

276 W. Siede et al.

cdc7 RAD cdc7 rad9A

0 min

60 min

cdcl W cdcl rad9A 0 Jim' 30 Jim'

90min 3 4n 120 min

150 min

M) J/m2 0 Jim' 30 I/m2

A DNA Content

FIGURE 4,"Flow cytometric analysis of DNA content of cells synchronized at the CDC4 step of strains WS9110-3D cdc4-1 RAD and WS9110-3D cdc4-1 rad9A after treatment with 30 or 60 J/m2 UV during incubation in YPD at 25". Survival: 67% (cdc4 RAD, 30 J/m2), 12% (cdc4 R A D , 60 J/m2) us. 1.2% (cdc4 rad9A, 30 J/m2), 0.0025% (cdc4 rad9A, 60 J/m2).

cdcl RAD cdcl rad9A LkLk 0 min

120 min

DNA Content

FIGURE 5."Flow cytometric analysis of DNA content of cells synchronized at the CDC4 step of strains WS9110-3D cdc4-1 RAD and WS9110-3D cdc4-1 rad9A after treatment with 10 h a d y-rays during incubation in YPD at 25". Survival: 2% (cdc4-1 RAD) us. 1% ( c d c 4 - l r a d 9 A ) .

36" (Figure 9A). At 33" the double mutant was inviable, whereas the dnal -1 RAD9 strain maintained colony growth. Colony formation of the double mutant was also more sensitive to transient incubation at restrictive tem- peratures (Figure 9B).

RAD24 is necessary for DNA damageinduced G, ar- rest: A mutant defective in the RAD24 gene is charac- terized by a moderate sensitivity to UV and ionizing ra- diation (ECKARDT-SCHUPP et al. 1987). The rad24-1

IS0 min

OJ/m2 MJ/m2 601/m2 Ol/m2 301/m2 60J/m2

120 min

DNA Content

FIGURE 6.-Flow cytometric analysis of DNA content of cells synchronized at the CDC7 step of strains WS9120-10A cdc7-1 RAD and WS9120-1OA cdc7-1 rad9A after treatment with 30 or 60 J/m2 UV during incubation in YPD at 25". Survival: 29% (cdc7-1 RAD, 30 J/m2), 7% (cdc7- l RAD, 60 J/m2) us. 10% (cdc7-1 rad9A, 30 J/m2), 1% (cdc7-1 rad9A, 60 J/m2).

cdc7 RAD cdc7 rad9A

0 mi"

60 mm

DNA Contcnl

FIGURE 7.-Flow cytometric analysis of DNA content of a-factor-synchronized cells of strains WS9120-1OA cdc7-1 RAD and WS9120-1OA cdc7-1 rad9A after treatment with 30 or 60 J/m2 UV during incubation in WD at 25". Survival: 39% (cdc7-1 RAD, 30 J/m2), 13% (cdc7-1 RAD, 60 J/m2) us. 12.7% (cdc7-l rad9A, 30 J/m2), 0.35% (cdc7-l rad9A, 60 J/m2).

mutant shows an epistatic interaction with radY-1 in UV- irradiated cells (ECKARDT-SCHUPP et al. 1987). We con- firmed this observation using congenic strains bearing a rad9 deletion mutation (W. SIEDE and E. C. FRIEDBERG, unpublished observations). These results support the view that RAD9and RAD24 are involved in a linear path- way which determines resistance to W radiation in yeast.

277

0 min 35°C d 3 3 2 -

150 rnin 35°C

DNA Content

FIGVRE 8.-Flow cytometric analysis of DNA content in cells of strains M'S9121-1B d n n l R A D and WS0121-1B dnnl m d 9 A after shifting the incubation temperature of an asynchro- nously growing culture from 25" to 35".

A 36°C 33°C 30°C 23°C

dnalRAD

B

L

t

4 dnalrad9A

dnalRAD

dnalrad9A

FIGLIRE 9.-Thermosensitivity of colony forming ability of strains WS9121-1B dnnl RAD and M'S0121-1R d n n l m d 9 A . Samples of stationary-phase cultures grown at 25" were streakcd out on W D plates and incubated at 23". 30", 33" and 36" (A) or incubated for 40 hr at 36", then at 23" (R) .

It has recently been demonstrated that, in addition to RAD9, RAD24 is required for G, arrest after treatment of cells with ionizing radiation (WEINERT et al. 1994). We used the cloned RAD24 gene (T. WEINERT, personal communication) to generate a rad24 deletion strain. A comparison of the mutant with an otherwise isogenic wild-type strain indicated a requirement for a functional RAD24 gene for G, arrest after exposure of cells syn- chronized with a-factor to UVradiation (Figure 10). The same result was obtained by measuring budding delay (data not shown). A direct comparison with results ob- tained using a rnd9A mutant did not reveal significant quantitative differences in cell cycle progression after W radiation (data not shown). Additionally both mu- tants have comparable levels of UV radiation sensitivity.

Signal for W radiation-induced G , arrest: If repair- able DNA damage per se (such as cyclobutane pyrimi- dine dimers or 6 4 photoproducts produced by W ra- diation), constitutes a signal for cell cycle arrest we would expect to detect RADMependent arrest at sig- nificantly lower doses of UV radiation in excision repair- defective strains than in repaircompetent cells. The RAD14 and RAD2 genes are indispensable for nucle-

0 min

€4 min

2 I

e - - 90 rnin

1 2 0 min

RAD 0 J/m2 ,30 !/m2

rad24A

DNA Conlcnt

FIGURE 10.-Flow cytometric analysis of DNA content of a-factor-synchronized cells of strains SX46 A RAD and SX46 A m d 2 4 A after treatment with 30 J/m' UV during incubation in YPD at 30". Survival: 70% ( R A D ) , 8% ( m d 2 4 A ) .

otide excision repair. Hence, mutants deleted of these genes are highly sensitive to UV radiation (CUZDER et nl. 1993; FRIEDRERC et nl. 1991; FRIEDRERC 1985). We there- fore examined the cell cycle response in a rad14 dele- tion mutant. Delayed entry into Sphase and a prolon- gation of Sphase was observed when excisiondefective cells synchronized with a-factor were treated with a dose of only 3 J/m2 (Figure 11). However, this phenomenon was not dependent on the RAD9gene and hence cannot be considered to be determined by regulated check- point controls (Figure 11). This conclusion is strength- ened by the failure to observe preferential arrest at START in W-irradiated excisiondeficient cells (Figure 12, A-D). Once again a-factor was added to portions of irradiated and unirradiated cultures after 1 hr of post- irradiation incubation, and incubation was continued in the presence of nocodazole. Neither the DNA profiles (Figure 12, A and B) nor the fraction of unbudded cells (Figure 12, C and D) provided evidence for a greater fraction of cells at START in irradiated compared to unirradiated cultures. Identical results were observed in the rnd9A strain (Figure 12B). In contrast, when these cells were exposed to ionizing radiation (which pre- dominantly produces DNA damage that is not repaired by nucleotide excision repair), typical RADMependent GI checkpoint arrest was observed (data not shown). Identical results were found in strains deleted for either the RAD14 or RAD2 nucleotide excision repair genes (data not shown).

DISCUSSION

As is the case in mammalian cells, GI arrest in yeast after treatment with DNAdamaging agents such as UV light or ionizing radiation is an actively regulated re- sponse. In yeast this phenomenon depends on a func- tional RAD9gene (SIEnE et nl. 1993) and, as shown here,

278 W. Siede et al.

radl4ARALl radl4A rad9A O h z 31/m2 SUm*

90 min ii B

180 min

DNA Content

FIGURE 11.-Flow cytometric analysis of DNA content of a-factor-synchronized cells of strains SX46 A rad1 4A RAD and SX46 A radl4A rad9A after treatment with 3 or 5 J/m’ UV during incubation in WD at 30”. Survival: 16% ( radl4A RAD, 3J/m2),0.85% (rud14ARAD,5J/m2) vs.996 (rudl4A rad9A, 3 J/m2), 0.7% (radl4A rad9A, 5 J / m 2 ) .

on the RAD24 gene. Both genes are also required for G2 arrest after treatment with DNAdamaging agents, but not for cell cycle arrest triggered by the presence of unreplicated DNA (WEINERT et al. 1994; WEINERT and LYDALL 1993; WEINERT and HARTWELL 1988).

We attempted to define the precise stage(s) of G, checkpoint arrest in cells exposed to DNA damage. Pro- gression beyond START in yeast commits the cell to en- ter Sphase and coincides with a loss of sensitivity to a-factor and to nutritional signals (MURRAY and HUNT 1993; NASMY~H 1993). When cells were synchronized at START with a-factor and then irradiated with W light or y rays a greater fraction of cells retained sensitivity to a-factor ( i. e., remained at START) than in unirradiated controls. This arrest phenomenon was dependent on a functional RAD9 gene. Hence, checkpoint arrest in the presence of damaged DNA can apparently occur at START.

Mutants defective in the CDC4 gene arrest as budded cells (WITTENBERG and REED 1991) (Figure 1 ) . Hence, bud emergence (a frequently used indicator of GI ar- rest) should not be influenced if a checkpoint defect located downstream of CDC4-mediated events was the exclusive or primary checkpoint in a-factor arrested cells exposed to DNA damage. However, previous stud- ies have shown that RADPdependent G, arrest of a-factor synchronized cells exposed to W or ”irradiation is indeed characterized by delayed bud emergence (SIEDE et aZ. 1993). These observations are

consistent with an additional G, checkpoint that is ear- lier than CDCBmediated events (i. e . , at START).

We found evidence for at least one additional check- point in yeast which is downstream of START and maps between the CDC4- and CDC7-mediated events (Figure 1 ) . Our results also suggest that RADPdependent GI checkpoint controls do not operate downstream of the CDC7-mediated step. While we cannot formally elimi- nate the existence of RADPindependent damage- responsive checkpoint controls in yeast, the cessation of cell cycle progression observed in cdc7 rad9 mutants (Figure 6) more likely reflects the passive interference of DNA replication and/or transcription by DNA damage (BERGER and EDENBERG 1986; SAUERBIER and HERCULES 1978; SETLOW et al. 1963).

Checkpoints for DNA damage at both START and G,/S transition might facilitate DNA repair by comple- mentary mechanisms. Exit from cell cycle progression associated with arrest at START is expected to increase the kinetic window for nucleotide excision repair. In- deed, substantial repair of W radiation damage has been observed in stationary phase cells under non- growth conditions; so called “liquid holding recovery” (HAWES and KUNZ 1981). Arrest of cells between the CDC4- and CDC7-mediated steps is characterized by el- evated levels of a number of transcripts ( e . g . , CDC9, CDC8, CDC21) whose polypeptide products are directly involved in DNA replication (WITTENBERG and REED

1991; WHITE et al. 1987) (Figure 1) . Several transcripts in this group are also inducible by DNA-damaging agents (ELLEDGE and DAVIS 1989; PETERSON et al. 1985). Hence, up-regulation of genes involved in DNA synthe- sis by both of these mechanisms may facilitate repair synthesis during nucleotide excision repair. The phe- notype of mutants defective in the RAD53 checkpoint gene (= MEC2, S A D l ) suggests that DNA damage- dependent gene induction and cell cycle arrest may ac- tually be coregulated to some extent u. B. ALLEN, W. SIEDE, E. C. FRIEDBERG and S. J. ELLEDGE, submitted for publication).

The observed dependency on RAD9 of cell cycle ar- rest mediated by a defect in the DNAl gene is remark- able. RADMependent cell cycle arrest and accelerated cell death have been observed in late S/G2 in mutants such as cdc2, cdc9, cdcl3and cdcl7 (SCHIESTL et al. 1989; WEINERT and HARTWELL 1993; HARTWELL and WEINERT 1989). Arrest in GI or G,/S has been observed with cdc 7, dbf4, cdc4 and cdc28 mutants under restrictive condi- tions, but this arrest has been shown to be R A D 9 independent (WEINERT and HARTWELL 1993; this study). Thus, dnal-1 is the first known conditional-lethal mu- tant that manifests RADPdependent cell cycle arrest in G,. This observation suggests that a late G, (G,/S) check- point might be utilized when untreated dnal mutant cells are incubated under restrictive conditions. D N A l is believed to encode a function necessary for replicon

S. cerevisiae G, Checkpoint Control 279

A B radl4ARAD radl4Arad9A

0 Jlm' 3 J/m2 +a-factor +a-factor +a-factor

- nocodazole Addiiion of

+I- a-factor

DNA Content

I 0 30 en 90 120 15a 180

Time after Irradiation (min)

FIGURE 12.-Flow cytometric analysis of DNA content (A, B) and microscopic determination of the fraction of unbudded cells (C, D) in a-factor-synchronized cultures of strains SX46A r a d l 4 8 RAD (A, C) and SX46A radl 4Arad9A (B, D) after treatment with 0 (0, 0) or 3 J/m2 UV (0, W). As indicated by the arrows, nocodazole (10 pg/ml) and a-factor (5.0 pg/ml) were added after 60 min of post-irradiation incubation, the latter only to part of the cultures (second and fourth column in A, B; filled symbols in C, D).

initiation (EBERLY et al. 1989). After synchronization of d n a l - 1 RAD9 cells by incubation at restrictive tempera- tures followed by a shift to permissive conditions, further cell cycle progression is not sensitive to a-factor but is still sensitive to the DNA synthesis inhibitor hydroxyurea (data not shown). During prolonged incubation at re- strictive temperatures cells are able to perform a limited number of very slow cell divisions (data not shown). Ex- periments with cell-free extracts indicate a direct in- volvement of the DNA1 gene product in the initiation of DNA replication rather than a regulatory role (EBERLY et al. 1989). We suggest that the failure to carry out normal initiation of replication in d n a l - 1 at restrictive temperatures results in structural alterations in the ge- nome that mimic those associated with DNA damage (or its processing), and hence activate the RADMependent arrest pathway.

We addressed the nature of the inducing signal asso- ciated with G, and/or G,/S checkpoint arrest. Our re- sults suggest that unrepaired substrates for nucleotide excision repair or their persistence in the genome do not per se constitute a signal for checkpoint arrest in yeast. In rad2 and rad14 mutants that are completely deficient in the incision step of nucleotide excision re- pair (GUZDER et al. 1993; FRIEDBERG et al. 1991) we de- tected a significant Sphase delay at about 10-fold lower doses of W radiation than in the excision-proficient strain (SIEDE et al. 1993). However, at the UV doses used

we found no evidence for G, arrest at START, or indeed for regulated checkpoint arrest at all. This is not due to a specific requirement for the RAD2 or RAD14 gene products since RADPdependent GI arrest after treat- ment with ionizing radiation was intact in the absence of these genes. We cannot formally exclude that the o b served delay in cell cycle progression in repairdefective mutants reflects the operation of a RADPindependent checkpoint mechanism(s). However, as argued above, this phenomenon more likely results from the passive interference with replication and/or transcription by the high concentration of unrepaired photoproducts in the genome. By this argument, the independence of RAD9 function also implies that the passive stalling of replication and/or transcription does not elicit a signal that triggers the RADPdependent checkpoint pathway. The persistence of W radiation damage during several rounds of replication in excisiondeficient yeast (JAMES et al. 1978) is also indicative of the inability of such dam- age to trigger G, checkpoint controls, or the ability of cells to overcome the effects of such damage by adaptive mechanisms (KADYK and HARTWELL 1993).

What then constitutes the signal for cell cycle arrest in UV-irradiated cells? One possibility is that photoprod- ucts that are not subject to nucleotide excision repair (and in fact may not even be DNA products) are im- portant, and that the dose of UV radiation used in these experiments was too small to detect their contribution

280 W. Siede et al.

to checkpoint arrest. DNAstrand breaks have been iden- tified as important determinants for G, checkpoint ar- rest (WEINERT and LYDALL 1993) and for induction of the p53 gene in mammalian cells (NELSON and -TAN 1994; I,u and LANE 1993). Thus, a second, and in our view more likely, possibility is that a checkpoint signal for G, arrest is generated during nucleotide excision repair. Besides DNA single-strand nicks or single-stranded DNA tracts that result during this process, double strand breaks (induced by incision events at closely spaced le- sions on different strands) may act as a triggering signal.

Recent data on the DNAdamage induced increase in p53 levels in XP-A cells (defective in the incision step of nucleotide excision repair) suggest that a similar mecha- nism might operate in human cells (NELSON and KASTAN

1994). In contrast to wild-type cells, UV-irradiation did not increase p53 levels in XP-A cells if replication was inhibited, however, the increase was normal in replicat- ing cells and, under both conditions, following ionizing radiation. Comparable to our studies, these results sug- gest that p53 induction and p53-regulated cell cycle ar- rest depend on DNA strand breaks which can be in- duced directly ( e . g . , by ionizing radiation) or indirectly during replication of a damaged template or through the action of nucleotide excision repair. Induction of p53 in replicating cells defective in nucleotideexcision repair most likely occurs downstream of the p53mediated GI checkpoint(s) and hence does not result in arrest in G,.

Characterization of the GI arrest associated with ex- posure of yeast cells to DNAdamaging agents has re- vealed interesting common features between this check- point response and that observed in the G, phase of the cell cycle. Both involve common gene products such as RAD9 (WEINERT and HARTWELL 1988; SIEDE et al. 1993), RAD24 (WEINERT et al. 1994; this study) and RAD53 (= MEC2, SADl) (WEINERT et al. 1994; J. B. ALLEN, W. SIEDE, E. C. FRIEDBERG and S. J. ELLEDGE, sub- mitted for publication). There are also indications that both processes utilize common signals (i. e. , DNA strand breaks). These observations, together with the finding that GI arrest can occur in at least two different stages during GI, are consistent with the operation of a sur- veillance system for DNA damage that is not cell cycle stage specific, but rather elicits a signal which stops cell cycle progression at the soonest possible physiological arrest stage. Such a model is also consistent with the induction of inhibitors of Cdk activity as proposed for mammalian cells (EL-DEIRY et al. 1994; DULIC et al. 1994). Defective arrest at multiple checkpoints in rad9 and rad24 cells (i. e . , at START, in Gl/S and G,) is also reminiscent of the situation in AT cells, in which a de- fective ability to arrest at the restriction point, during replicon initiation and in G, has been observed after treatment with ionizing radiation (RUDOLPH and LATI 1989; NAGASAWA et al. 1987; PAINTER 1986; LITTLE and NAGASAWA 1985; ZAMBETTI-BOSSELER and SCOTT 1981; PAINTER and YOUNG 1980; LITTLE 1968).

We thank LEE JOHNSTON and AKIO SUGINO for dbf4 and d n a l mutant strains, respectively, and TED WEINERT for providing RAD9 and RADZkontaining plasmids prior to publication. These studies were supported by research grant CA12428 from the U.S. Public Health Service and Institutional grant W-142 from the American Cancer Society.

LITERATURE CITED

BERGER, C. A, and H. J. EDENBERG, 1986 Pyrimidine dimers block simian virus 40 replication forks. Mol. Cell. Biol. 6 3443-3450.

BRACHMAN, D. G., M. BECKETT, D. GRAVES, D. HARAF, E. VOKES et al., 1993 p53 mutation does not correlate with radiosensitivity in 24 head and neck cancer cell lines. Cancer Res. 5 3 3667-3669.

CHAPMAN, J. W., and L. H. JOHNSTON, 1989 The yeast gene, DBF4, essential for e n q into S phase is cell cycle regulated. Exp. Cell Res. 180: 419-428.

DULIC, v., W. K. K A U ~ N , S. J. WILSON, T. D. T u n , E. LEES et al., 1994 p53dependent inhibition of cyclindependent kinase ac- tivities in human fibroblasts during radiation-induced G , arrest. Cell 76: 1013-1023.

EBERLY, S. L., A. S m and A. SUGINO, 1989 Mapping and character- ization of a new DNA replication mutant in Saccharomyces cer- evisiae. Yeast 5: 117-129.

ECKARDT-SCHUPP, F., W. SIEDE and J. C. GAME, 1987 The RAD24 (= P I ) gene product of Saccharomyces cerevisiae participates in two different pathways of DNA repair. Genetics 115: 83-90.

EL-DEIRY, W. S., T. TOKINO, V. E. VELCULESCU, D. B. LEVY, R. PARSONS et al., 1993 WAFI, a potential mediator of p53 tumor suppres- sion. Cell 75: 817-825.

EL-DEIRY, W. S., J. W. HARPER, P. M. O’CONNOR, V. E. VELCULESCU, C. E. CANMAN et at., 1994 WAFI/CIPI is induced in p53-mediated G, arrest and apoptosis. Cancer Res. 5 4 1169-1174.

ELLEDGE, S. J., and R. W. DAVIS, 1989 DNA damage induction of ri- bonucleotide reductase. Mol. Cell. Biol. 9 4932-4940.

FRIEDBERG, E. C., 1985 DNA Repair. W. H. Freeman, New York. FRIEDBERG, E. C., W. SIEDE and A. J. COOPER, 1991 Cellular responses

to DNA damage in yeast, pp. 147-192 in The Molecular Biology of the Yeast Saccharomyces: Genome Dynamics, Protein Synthesis, and Energetics, edited by J. R. BROACH, J. R. PRINCLE and E. W. JONES. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.

GIETZ, D., A. ST. JEAN, R. A. WOODS and R. H. SCHIESTL, 1992 Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res. 20: 1425.

GUZDER, S. N., P. SUNG, L. PRAKMH and S. PRAKASH, 1993 Yeast DNA- repair gene RAD14 encodes a zinc metalloprotein with affinity for ultravioletdamaged DNA. Proc. Natl. Acad. Sci. USA 90: 5433-5437.

HALL, P. E., P. H. MCKEE, H. D. MENAGE, R. DOVER and D. P. LANE, 1993 High levels ofp5? protein in UV-irradiated normal human skin. Oncogene 8 203-207.

HARPER, J. W., G. R. ADAMI, N. WEI, K. KEYOMARSI and S. J. ELLEDGE, 1993 The p21 Cdk-interacting protein Cipl is a potent inhibitor of G, cyclindependent kinases. Cell 75: 805-816.

HARTWELL, L., 1992 Defects in a cell cycle checkpoint may be re- sponsible for the genetic instability of cancer cells. Cell 71: 543-546.

HARTWELL, L. H., and T. A. WEINERT, 1989 Checkpoints: controls that ensure the order of cell cycle events. Science 246 629-634.

HAYNES, R. H., and B. A. KUNZ, 1981 DNA repair and mutagenesis in yeast, pp. 371-414 in The Molecular Biology of the Yeast Sac- charomyces, edited by J. N. STRATHERN and J. R. BROACH. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.

HEREFORD, L. M., and L. H. HARTWELL, 1973 Role of protein synthesis in the replication of yeast DNA. Nat. New Biol. 244: 129-131.

HOEIJMAKERS, J. H. J., 1993 Nucleotide excision repair: 11. From yeast

JAMES, A. P., B. J. fiLBEYand G. J. PREFONTAINE, 1978 The timing o f w to mammals. Trends Genet. 9: 211-217.

mutagenesis in yeast: continuing mutation in an excision- defective ( r a d l - I ) strain. Mol. Gen. Genet. 165: 207-212.

mkx, L. C., and L. H. HARTWELL, 1993 Replicationdependent sister chromatid recombination in rad1 mutants of Saccharomyces cer- evisiae. Genetics 133 469-487.

TAN, M. B., 0. ONYEKWERE, D. SIDRANSKY, B. VOGELSTEIN and R. W. CRAIG, 1991 Participation ofp53protein in the cellular response to DNA damage. Cancer Res. 51: 6304-6311.

S. cerevisiae G, Checkpoint Control 28 1

&TAN, M. B., Q. ZHAN, W. S. EL-DEIRY, F. CARRIER, T. JACKS et al., 1992 A mammalian cell cycle checkpoint pathway utilizing p53 and GADD45 is defective in ataxia-telangiectasia. Cell 71:

KHANNA, K R, and M. F. LAWN, 1993 Ionizing radiation and W i n - duction of p53 protein by different pathways in ataxia- telangiectasia cells. Oncogene 8: 341 1-3416.

KITADA, K., A. L. JOHNSON, L. H. JOHNSTON and A. SUGINO, 1993 A multicopy suppressor gene of the Saccharomyces cerevisiae G, cell cycle mutant gene dbf4 encodes a protein kinase and is identified as CDC5. Mol. Cell. Biol. 1 3 4445-4457.

KUERBITZ, S. J., B. S. PLUNKETT, W. V. WAISH and M. B. KASTAN, 1992 Wild-type p53 is a cell cycle checkpoint determinant fol- lowing irradiation. Proc. Natl. Acad. Sci. USA 8 9 7491-7495.

LANE, D. P., 1992 p53, guardian of the genome. Nature 358: 15-16. LEE, J. M., and A. BERNSTEIN, 1993 p53 mutations increase resistance

to ionizing radiation. Proc. Natl. Acad. Sci. USA 90: 5742-5756. LI, J. J., and R. J. DESHAIES, 1993 Exercising self-restraint: discourag-

ing illicit acts of S and M in eukaryotes. Cell 7 4 223-226. LITTLE, H., and H. NAGASAWA, 1985 Effects of confluent holding on

potentially lethal damage repair, cell cycle progression, and chro- mosomal aberrations in human normal and ataxia-telangiectasia fibroblasts. Radiat. Res. 101: 81-93.

LIITLE, J. B., 1968 Delayed initiation of DNA synthesis in irradiated human diploid cells. Nature 218: 1064-1065.

LMNGSTONE, L. R., A. WHITE, J. SPROUSE, E. LWANOS, T. JACKS et al., 1992 Altered cell cycle arrest and gene amplification potential accompany loss of wild-type p53. Cell 7 0 923-935.

Lu, X., and D. P. LANE, 1993 Differential induction of transcription- ally active p53 following UV or ionizing radiation: defects in chro- mosome instability syndromes. Cell 7 5 765-778.

IMALTzMAN, W., and L. Czlzlx, 1984 UV irradiation stimulates levels of p53 cellular tumor antigen in nontransformed mouse cells. Mol. Cell. Biol. 4 1689-1694.

MURRAY, A. W., 1992 Creative blocks: cell-cycle checkpoints and feed- back controls. Nature 359: 599-604.

MURRAY, A,, and T. HUNT, 1993 The Cell Cycle. Oxford University Press, New York.

NAGASAWA, H., K. H. KKAEMER, Y. SHILOH and J. B. L r m , 1987 De- tection of ataxia telangiectasia heterozygous cell lines by postir- radiation cumulative labeling index: measurements with coded samples. Cancer Res. 47: 398-402.

NASMYTH, K., 1993 Control of the yeast cell cycle by the Cdc28 protein kinase. Curr. Opin. Cell Biol. 5 166-179.

NELSON, W. G., and M. B. KASTAN, 1994 DNA strand breaks: the DNA template alterations that trigger p53dependent DNA damage re- sponse pathways. Mol. Cell. Biol. 1 4 1815-1823.

PAINTER, R. B., 1986 Inhibition of mammalian cell DNA synthesis by ionizing radiation. Int. J. Radiat. Biol. 49: 771-781.

PAINTER, R. B., and B. R. YOUNG, 1980 Radiosensitivity in ataxia telangiectasia: a new explanation. Proc. Natl. Acad. Sci. USA 77:

PERRY, M. E., J. PIETTE, J. A. ZAWADZKI, D. HARVEY and A. J. LEVINE, 1993 The mdm-2 gene is induced in response to W light in a p53dependent manner. Proc. Natl. Acad. Sci. USA 90: 11623-11627.

PETERSON, T. A., L. PRAKASH, S. PRAKASH, M. A. OSLEY and S. I. REED, 1985 Regulation of CDC9, the Saccharomyces cerevisiae gene that encodes DNA ligase. Mol. Cell. Biol. 5: 226-235.

ROTHSTEIN, R. J., 1983 One-step gene disruption in yeast. Methods Enzymol. 101: 202-211.

587-597.

7315-7317.

RUDOLPH, N. S., and S. A. LATT, 1989 Flow cytometric analysis of X-ray sensitivity in ataxia telangiectasia. Mutat. Res. 211: 31-41.

SAMBROOK, J., E. F. FRITSCH and T. MANIATIS, 1989 Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.

SAUERBIER, W., and K. HERCULES, 1978 Gene and transcription unit mapping by radiation effects. Annu. Rev. Genet. 12: 329-363.

SCHIESTL, R. H., P. REWOLDS, S. F'RAKASH and L. PRAKASH, 1989 Cloning and sequence analysis of the Saccharomyces cerevisiaeRAD9gene and further evidence that its product is required for cell cycle arrest induced by DNA damage. Mol. Cell. Biol. 9 1882-1896.

SEnow, R. B., P. A. SWENSON and W. L. CARRIER, 1963 Thymidine dimers and inhibition of DNA synthesis by ultraviolet irradiation of cells. Mutat. Res. 142: 1464-1466.

SIEDE, W.,A. S. F R I E D B E R G ~ ~ ~ E . C. FRIEDBERG, 1993 RADMependent G , arrest defines a second checkpoint for damaged DNA in the cell cycle of Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 90: 7985-7989.

SLICHENMEIER, W. J., W. G. NELSON, R. J. SLEBOS and M. B. &TAN, 1993 Loss of a p53-associated G, checkpoint does not decrease cell survival following DNA damage. Cancer Res. 5 5 4164-4168.

THORNE, L. W., and B. BIERS, 1993 Stage-specific effects of X-irradiation on yeast meiosis. Genetics 134: 29-42.

WEBER, L., and B. BIERS, 1992 A RADMependent checkpoint blocks meiosis of cdcl? yeast cells. Genetics 131: 55-63.

WEINERT, T. A,, and L. H. HARTWELL, 1988 The RAD9gene controls the cell cycle response to DNA damage in Saccharomyces cerevi- siae. Science 241: 317-322.

WEINERT, T. A,, and L. H. HARTWELL, 1990 Characterization of the RAD9 gene of Saccharomyces cerevisiae and evidence that it acts posttranslationally in cell cycle arrest after DNA damage. Mol. Cell. Biol. 10: 6554-6564.

WEINERT, T. A., and L. H. HARTWELL, 1993 Cell cycle arrest of cdc mutants and specificity of the RAD9 checkpoint. Genetics 134 63-80.

WEINERT, T., and D. LYDALL, 1993 Cell cycle checkpoints, genetic in- stability and cancer. Semin. Cancer Biol. 4: 129-140.

WEINERT, T. A., G. L. KISER and L. H. HARTWELL, 1994 Mitotic check- point genes in budding yeast and the dependence of mitosis on DNA replication and repair. Genes Dev. 8: 652-665.

WHITE, J. H. M., S. R. GREEN, D. G. BARKER, L. B. DUMAS and L. H. JOHNSTON, 1987 The CDCB transcript is cell cycle regulated in yeast and is expressed coordinately with CDC9 and CDC2I at a point preceding histone transcription. Exp. Cell Res. 171:

WITTENBERG, C., and S. I. REED, 1991 Control of gene expression and the yeast cell cycle. Crit. Rev. Eukaryotic Gene Expression 1:

YIN, Y., M. A. TAINSKY, F. Z. BISCHOFF, L. C. STRONG and G. M. WAHL, 1992 Wild-type p53 restores cell cycle control and inhibits gene amplification in cells with mutant p53 alleles. Cell 7 0 937-948.

ZAMBETTI-BOSSELER, F., and D. SCOTT, 1981 Cell death, chromosome damage and mitotic delay in normal human, ataxia telangiectasia, and retinoblastoma fibroblasts after X-irradiation. Int. J. Radiat. Biol. 3 9 547-558.

ZHAN, Q., F. CARRIER and A. J. FOKNACE, JR., 1993 Induction of cellular P53 activity by DNAdamaging agents and growth arrest. Mol. Cell. Biol. 13: 4242-4250.

223-231.

189-205.

Communicating editor: E. W. JONES