cell cycle arrest of cdc mutants and specificity of the rad9 - genetics

Copyright 0 1993 by the Genetics Society of America

Cell Cycle Arrest of cdc Mutants and Specificity of the RAD9 Checkpoint

Ted A. Weinert*'+ and Leland H. Hartwell" *Department of Genetics, University of Washington, Seattle, Washington 98195, and 'Department of Molecular and Cellular

Biology, University of Arizona, Tucson, Arizona 85721 Manuscript received August 24, 1992

Accepted for publication January 15, 1993

ABSTRACT In eucaryotes a cell cycle control called a checkpoint ensures that mitosis occurs only after

chromosomes are completely replicated and any damage is repaired. The function of this checkpoint in budding yeast requires the RAD9 gene. Here we examine the role of the RAD9 gene in the arrest of the 12 cell division cycle (cdc) mutants, temperature-sensitive lethal mutants that arrest in specific phases of the cell cycle at a restrictive temperature. We found that in four cdc mutants the cdc rad9 cells failed to arrest after a shift to the restrictive temperature, rather they continued cell division and died rapidly, whereas the cdc RAD cells arrested and remained viable. The cell cycle and genetic phenotypes of the 12 cdc RAD mutants indicate the function of the RAD9 checkpoint is phase-specific and signal-specific. First, the four cdc RAD mutants that required RAD9 each arrested in the late S/ G2 phase after a shift to the restrictive temperature when DNA replication was complete or nearly complete, and second, each leaves DNA lesions when the CDC gene product is limiting for cell division. Three of the four CDC genes are known to encode DNA replication enzymes. We found that the RAD1 7 gene is also essential for the function of the RAD9 checkpoint because it is required for phase-specific arrest of the same four cdc mutants. We also show that both X- or UV-irradiated cells require the RAD9 and RAD17 genes for delay in the GP phase. Together, these results indicate that the RAD9 checkpoint is apparently activated only by DNA lesions and arrests cell division only in the late S/G2 phase.

"

T HE cell division cycle in eucaryotes proceeds by a defined sequence of events where late events

often depend upon completion of early events [reviewed in HARTWELL and WEINERT (1 989)]. The dependency of events appears critical for the high fidelity of chromosome transmission; an error in the order of events, if uncorrected, could lead to chromosome mutations (chromosome loss, gain, rearrangements) resulting in abnormal growth or cell death. Controls that enforce the dependency of events are called checkpoints and are defined by mutations that allow a late event to occur without completion of an early event. Checkpoints may order events in development as well as in the eucaryotic cell cycle (CUTTING et al. 1990). Distinct checkpoints in cell division appear to regulate the dependence of mitosis on DNA replication [see HARTWELL and WEINERT (1 989) and ENOCH and NURSE (1 99 l)] and the dependence of anaphase on spindle function (LI and MURRAY 1991 ; HOYT, TOTIS and ROBERTS 199 1). We anticipate that checkpoints may regulate the order of additional cell cycle events.

This study examines the checkpoint that enforces the dependence of mitosis on completion of DNA replication. Genes identified by mutations that allow mitosis without completion of DNA replication include the RAD9 gene in budding yeast (WEINERT and HARTWELL 1988), the RCCl/p imI+ gene in a hamster

Genetics 134 63-80 (May, 1993)

cell line and in fission yeast (NISHIMOTO, EILEEN and BASILICO 1978; MATUSOMOTO and BEACH 1991), the bimE gene in filamentous fungus (OSMANI et al. 1988), the cdc2+, cdc25+, weel+ and mikl+ genes in fission yeast (ENOCH and NURSE 1990; LUNGREN et al. 1991; ROWLEY, HUDSON and YOUNG 1992), and more recently five rad+ genes also in fission yeast (ROWLEY, SUBRAMANI and YOUNG 1992; AL-KHODAIRY and CARR 1992). Study of the mutants and corresponding wild-type gene products should provide insight into both the physiological role of the checkpoint controls and the molecular events that enforce the dependency of events. A proposed molecular mechanism for a checkpoint control involving the cdc2+ protein kinase is addressed in the DISCUSSION.

The role of the RAD9 checkpoint in budding yeast has been examined by comparing cell cycle phenotypes of wild-type and rad9 null mutant cells. When unirradiated, rad9 null mutants are viable and have generation times and cell cycle kinetics indistinguish- able from wild-type cells but lose chromosomes spon- taneously at a higher rate than wild-type cells (WEI- NERT and HARTWELL 1990). Thus the RAD9 checkpoint is normally nonessential for cell viability though it does contribute to genomic stability. When cells contained DNA lesions due either to X-irradiation or to defects in DNA ligase, wild-type cells delayed o r arrested in the late S/G2 phase, whereas rad9 mutant

64 T. A. Weinert and L. H. Hartwell

Spindle pole initiation DNA RAD9 Nuclear

duplicatlon synthesis (mitosis) DNA synthests checkpoint division

a factor HU MBC

cdc28 cdc7 cd&,8,17 cdc16,20,23

cdc2.9.13.1 7 ( - 34)

cdcl5

I- G1 l- S -1- late S/G2/M 1-post 4 anaphase

FIGURE 1 .-Dependent events in the yeast cell cycle. The relationship between cell cycle events (top), arrest of cdc mutants after shift to the restrictive temperature, and phases of the cell cycle. For example cdcljr mutants after shift to the restrictive temperature nearly complete or complete DNA synthesis and arrest in the late S/G2/M phase. Origins and features of the model are discussed in the text. Independent or interdependent relationships are not shown. cdc mutants that cause an increase in mitotic recombination when limiting for cell division are underlined (HARTWELL and SMITH 1985). cdc mutants within the open box require intact RAD9 and RAD17 genes for cell cycle arrest at the restrictive temperature (this paper; HARTWELL and WEINERT 1989). CDC genes whose enzymatic functions are know include: CDC28 protein kinase (LORINCZ and REED 1984); CDC8 thymidylate kinase (SCLAFANI and FANGMAN 1984); CDC9 DNA ligase (JOHNSTON and NASMYTH 1978); CDC17 DNA polymerase I UOHNSON et al. 1985; CARSON 1987); and CDC2 DNA polymerase I11 (SITNEY, BUDD and CAMPBELL 1989; BOULET et al. 1989).

cells continued cell division without delay and died (WEINERT and HARTWELL 1988, 1990; HARTWELL and WEINERT 1989). The RAD9 gene is not essential for DNA repair per se because a delay in G2 imposed by incubation with a microtubule poison suppressed the radiation sensitivity of rad9 mutants (WEINERT and HARTWELL 1988). The RAD9 checkpoint provides a cell cycle delay to allow completion of DNA replication and repair before mitosis.

In this report we examine the function of the RAD9 checkpoint in cell division from the interactions of rad9 with cdc mutants, temperature-sensitive lethal mutants that arrest in specific phases of cell division after shift to the restrictive temperature. We antici- pated that interactions between rad9 and cdc mutants might reveal the specificity of the RAD9 checkpoint as well as the basis for cell cycle arrest in cdc mutants. For many CDC gene products and their corresponding mutants the biochemicaI and ceIl cycle functions have been characterized (see Figure 1) although the basis for the cell cycle arrest is unknown. In principle, the arrest of cdc mutants may be due directly to the primary lesion in the CDC gene product or a second- ary consequence of a checkpoint control. For example cdc28 mutants encode a temperature-sensitive protein kinase which at the restrictive temperature arrests cell division in G I (or in G2) (HARTWELL et al. 1973; PIGGOT, RAI and CARTER 1982; SURARA et al. 1991; REED and WITTENBERC 1990; reviewed in NURSE 1990). Presumably cell cycle progression requires the

activity of substrates of the CDC28 protein kinase that remain unphosphorylated and therefore inactive at the restrictive temperature; cdc28 mutants therefore arrest as a direct consequence of the CDC28 lesion. In contrast, cdc9 mutants encode a temperature-sensitive DNA ligase (JOHNSTON and NASMYTH 1978) which at restrictive temperature results in unligated Okazaki fragments and cell cycle arrest that requires the RAD9 gene (HARTWELL and WEINERT 1989); arrest in cdc9 mutants is a consequence of a checkpoint.

We find here that the cell cycle arrest of 4 cdc mutants (of 12 tested) at the restrictive temperature requires the RAD9 checkpoint. From the phenotypes of the cdc mutants examined, together with studies of irradiated and hydroxyurea-treated cells, we conclude that function of the RAD9 checkpoint is signal- and phase-specific; it is activated only by DNA lesions that arrest cell division specifically in the late S/G2 phase. Further support for these conclusions comes from our studies of cdc mutants together with a second checkpoint gene, RAD1 7.

MATERIALS AND METHODS

Yeast strains: The yeast strains used in this study (Table 1) are isogenic with strain A364a except where noted. Strains were derived using standard genetic techniques (SHERMAN, FINK and HICKS 1986). The original strain containing the radl7-1 allele, X-55C (MATa RAD17-1 ade2-I obtained from the Berkeley Stock Center) was noncongenic with A364a, the strain background used in these studies. We therefore introduced the rad1 7-1 mutation into A364a

cdc Mutants and the RAD9 Checkpoint

TABLE 1

Yeast strains

65

Strainn Genotype

7859-10-2b MATa 7859-7-4b h2c2al 7867-2-1 h6clal

h7c4al 7843-3-1 h8clal 7844-1-3 598-3c 7851-3-2c 7845-7-4

hl6clal

h15c2al

hl7clal 7848-9-4 h20clal

h23clal 7850-2-4 h28clal

7868-13-3

7845-7-1

7848-2-1

7846-8-2

7849-1-3

7842-6-3 208-7-2 209-8-1 209-8-2 156-2-4 156-2-2 158-1-3 158-2-3 180-4-4 180-2-3 21 1-1-2 2 1 1-2-3 2 10-3-2 2 10-2-4 179-9-3 179-9- 1 155-3-2 155-7-3 228-1-3 228-2-3 208-4-1 208-2-3 159-3-2 159-7-1 7870-1-1 7870-1-4 7870

MATa MATa MA Ta MATa MATa MATa MA Ta MA Ta MATa MATa MA Ta MA Ta MA Ta MA Ta MA Ta MA Ta MATa MA Ta MA Ta MATa MA Ta MA Ta MA Ta MA Ta MATa MA T a MATa MATa MA Ta MATa MA T a MA Ta MATO MA T a MA Ta MATa MA Ta MA Ta MATa MA Ta MA T a MATa MA T a MATa MA Ta MATa MA Ta MATa MATa MATa MATa/MATa

his7 leu2 trpl ura3 his7 leu2 trpl ura3 rad9A::LEU2 cdc2-2 his7 u r a l cdc2-2 rad9A::LEU2 his7 ura l ura3 cyh2 canl cdc6-1 his7 ura l cdc6-1 rad9A::LEU2 cdc7-4 his7 u r a l cdc7-4 rad9A::LEU2 his7 ura l ura3 cdc8-1 his7 ura l cdc8-1 rad9A::LEU2 his3 trpl ural cyh2 cdc9-8 ade2 ade3 trpl leul-1 ura? SCE::URA3 canl cyh2 sap3 cdc9-8 rad9A::TRPl ade2 ade3 his3 leul leu2 cdcl3-1 his3 trpl ura3 canl cdcl3-1 rad9A::LEU2 his? trpl ural ura3 canl cyh2 cecl6-1 his7 ura l cdcl6-1 rad9A::LEU2 his3 trpl ura3 cyh2 canl cdc15-2 his7 ura l cdc15-2 rad9A::LEU2 his3 trpl canl cdcl7-1 his7 u r a l cdc17-I rad9A::LEU2 his3 trpl ural cyh2 cdc20-1 his7 ura l cdc20-1 rad9A::LEU2 his3 trpl ural canl cdc23-1 his7 u r a l cdc23-1 rad9A::LEUP his7 cyh2 cdc28-1 his7 ura l cdc28-I rad9A::LEU2 his3 trpl ural ura3 cyh2 rad17-1 ura? his7 cdc2-2 ura3 his7 canl cdc2-2 radl7-1 ura3 his7 canl Ade- cdc6-1 canl cdc6-1 radl7-1 ural trpl cdc7-4 ural ura3 his7 canl cdc7-4 radl7-1 ura l his7 cdc8-1 trpl ural ura3 his7 cdc8-1 radl7-1 trpl ura3 his7 cdc9-8 ade2 leul ura3 trpl canl cdc9-8 rad17-1 ade2 ade3 trpl ura3 his7 canl cdcl3-1 trpl ura3 his7 canl cdcl3-1 radl7-1 trpl ura? canl cdc15-2 his7 canl cdc15-2 radl7-1 trpl ural ura3 his7 canl cdcl6-1 ura3 trpl his7 his3 cdcl6-1 radl7-1 ural ura3 cdcl7-1 ura3 his3 leu2 trpl cdcl7-1 radl7-1 ura3 his3 leu2 cdc20-1 his7 cdc20-1 radl7-1 ural ura3 his7 canl cdc28-1 his7 trpl canl cdc28-I radl7-1 his7 ural ura3 trpl canl u r a l his7 radl7-1 ura l his7 ura3 radl7- l /+ ura3/+ mal /+ his7/+ t rp l /+ cad/+ cdc l3 /+

Strains with an “h” designation (e.g., h2c2al) from the collection from the HARTWELL laboratory. Strains were generated in this study

WEINERT and HARTWELL (1 990). HARTWELL and WEINERT (1989).

with the following exceptions, as noted below.

by repeated backcross (4-6 times) with A364a strains; we cell cycle delay after UV-irradiation and arrest and viability consider the resulting RAD+ and rad17 strains “nearly iso- in cdc9 and cdcl3 strains (data not shown). The radl7-1 genic” with A364a (the exceptions noted in the legend to mutation is the only allele available (COX and PARRY 1968) Figures 5 and 6 were backcrossed twice to A364a strains). and is uncharacterized as to whether it retains partial func- The cell cycle phenotypes were similar among the several tion. The rad9 mutation used in this study replaces 93% of rad17 and among the several RAD strains tested, including the RAD9 open reading frame with the LEU2 or TRPl gene;


we consider this a null mutation (WEINERT and HARTWELL

Media: Cells were grown either in complete or rich medium as noted (SHERMAN, FINK and HICKS 1986; HARTWELL 1967). For studies using radiation, X rays were delivered by a Machlett OEG60 tube and UV by a Stratalinker 1800 set at 40 J/m'. Hydroxyurea was obtained from Sigma, prepared immediately before use as a 2 M solution and used at a final concentration of 0.2 M at 30". Methylbenzimadazole- 2-yl-carbamate (MBC) was obtained as a gift from Du Pont, prepared as a 10 mg/ml stock solution in 100% dimethyl- sulfoxide (DMSO) and used at a final concentration of 100

Tests of cell cycle arrest in cdc mutants: Cells were grown to logarithmic phase (at 2-5 X lo6 cells/ml) in complete liquid medium at the permissive temperature (23") and shifted to the restrictive temperature (34", 36" or 38" as noted in Tables 2 and 5 ) for 4 hr. (We obtained similar results when cells were grown in rich or complete medium for the strains we tested; data not shown.) Four cell cycle parameters were analyzed: cell and nuclear morphology, first cycle arrest, cell viability and maximum permissive temperature for growth (each is discussed below). The fate of at least 100 cells was scored for each of the phenotypes. We also determined the DNA content for a subset of strains as indicated in Table 2.

Nuclear and cell morphology: Cells were fixed with 70% ethanol, stained with the DNA-binding dyes 4,6-diamino-2- phenylindole (DAPI) (PRINGLE et al. 1989) or with propidium iodide after RNase treatment for 2 hr (BAUM et al. 1988; WEINERT and HARTWELL 1990), and viewed using fluorescence and light microscopy.

First cycle arrest: A "microcolony assay" was used to determine whether cdc mutants arrested in the first cell cycle after a shift to the restrictive temperature. Cells grown in liquid medium at the permissive temperature were soni- cated to disrupt clumps and plated on prewarmed agar plates in complete medium. After 4 hr at the restrictive temperature random fields of microcolonies that were well dispersed were viewed by light microscopy to score cell cycle arrest. The number of buds in each of at least 100 microcolonies (each produced by a single cell) were recorded. Eleven of the cdc RAD mutant strains tested here arrest as large- budded cells at the restrictive temperature. Therefore in the microcolony assay any cell gives rise to either 2 buds (1 large-budded cell) or 4 buds (2 adjacent large-budded cells) depending on whether the cell was before or after (respectively) the execution point for the cdc mutant at the time of temperature shift. The cdc28 mutants studied here arrest as unbudded (GI) cells and therefore generate microcolonies that contain either 1 or 2 unbudded cells. We calculated the percentage of cells that show a first cycle arrest from the number of microcolonies that contained 2 or 4 buds (arrested) divided by the total number of microcolonies. Cells that gave rise to microcolonies that contained three or 2 5 buds we concluded did not undergo an efficient first cycle arrest. Typically an efficient first cell arrest is indicated if >80% microcolonies contain either 2 or 4 buds; however, comparison of mutants that arrest less efficiently also proves informative.

The microcolony assay may in some instances over- estimate the first cycle arrest; for example cells that are before the execution point at the time of temperature shift and generate four buds (instead of 2 buds) during the 4-hr incubation would be scored as "arrested." (Time-lapse photomicroscopy, though laborious, avoids this particular prob- lem. We have used both the microcolony and time-lapse photomicroscopy assays to test for first cycle arrest in cdc9

1990).

pg/ml.

and cdc9 rad9 strains and found that results from the two assays are quantitatively similar; 79% and 80% of cdc9 cells and 36% and 43% of cdc9 rad9 strains, respectively showed a first cycle arrest [HARTWELL and WEINERT (1989) and Table 2 in this study.]) The microcolony assay does not reveal the nuclear morphology of the cells. Therefore, we find results from both the microcolony assay, the cell and nuclear morphology assay, and the cell viability assays (see below) are required to assess whether mutants arrest cell division synchronously at the restrictive temperature.

Cell viability: Cell viability was determined for cells incubated at the permissive temperature of 23" and after 4 hr at the restrictive temperature in liquid culture. Cells were plated on solid agar medium and colony formation determined by light microscopy usually after 1 day of growth at the permissive temperature; viable cells give rise to "macro- colonies" that contain typically > 50 cell bodies (buds) and inviable cells to microcolonies of < l o cell bodies. In some instances viability was determined from visible colony formation after 3 days of incubation. At least three determi- nations were made for each strain, and the mean values and standard deviations are shown (Tables 2, 3 and 5 ) . We also determined the cell viability for many cdc RAD and cdc rad9 strains after incubation for up to 8 hr at the restrictive temperature [see HARTWELL and WEINERT (1 989); data not shown]. We found that the differences in viability after 4 hr at the restrictive temperature was representative of the differences in viabilities from later times and permits cor- relation with cell cycle arrest phenotypes which were also determined after 4 hr. We do not consider viabilities that differ by less than twofold between the cdc RAD and cdc rad strains as significantly different especially if other phenotypes are similar.

DNA content: Flow cytometry was performed on cells that were fixed with 70% ethanol, digested with RNase for 2 hr at 37", stained with 5 0 pg/ml propidium iodide (PI), and diluted into 5 pg/ml PI just prior to analysis by flow cytometry (BAUM et al. 1988). At least 5000 cells were counted in each analysis. The position of the 1C and 2C peaks were confirmed where tested with a factor- or MBC- arrested cultures (arrested in GI and GP, respectively; data not shown).

Maximum permissive temperature for growth Cells were streaked for single colonies onto agar plates containing complete medium and incubated at 23 O , 2 5 O , 28 O , 30°, 32" or 36" for 2-4 days. The maximum permissive temperature is the highest temperature tested at which visible colony formation was comparable to that seen at the permissive temperature of 23". At temperatures higher than the maximum permissive temperature typically no or a few visible colonies are formed. cdc6 rad9 strains consistently formed a few colonies (papillation) at one temperature interval higher than the maximum permissive temperature.

The role of RAD17 in cell cycle control: tests for cell cycle arrest after X-irradiation: The cell cycle arrest in RAD and radl 7 cells after X-irradiation was determined by time-lapse photomicroscopy (WEINERT and HARTWELL 1988). Logarithmically growing cells were plated on solid agar slabs (in rich medium), X-irradiated ( 2 krad) and pho- tographed at the time of plating and after incubation for 8- 10 hr at 23 O . Pairs of photographic negatives were projected and the fate of individual cells determined.

Genetic linkage of cell-cycle arrest defect and radiation sensitivity in radl 7 mutants: We tested for cosegregation of cell cycle arrest-defect and radiation sensitivity in strains produced from a r a d l 7 / + heterozygote (strain 7870). Log- arithmically growing cells were plated on solid agar media containing rich medium and X-irradiated with 2 krad. Cell


TABLE 2

Requirement for RAD9 in cell cycle arrest of cdc mutants

67

Arrest Viabilityf

morphologyd Nuclear

Maximum Strain

genotypea Restrictive

temperatureb Cell cycle permissive

phase' 23" R T First cyclee 23" RT temperaturd

cdc l3 cdcl3 rad9

cdc2 cdc2 rad9

cdc9 cdc9 rad9

cdcl7 cdcl7 rad9

cdcl7 cdcl7 rad9

cdc8 cdc8 rad9

cdc6 cdc6 rad9

cdc28 cdc28 rad9

cdc7 cdc7 rad9

cdcl6 cdcl6 rad9

cdc20 cdc20 rad9

cdc23 cdc23 rad9

cdcl5 cdcl5 rad9

CDC+ CDC+ rad9

36 36

36 36

36 36

34 34

38 38

36 36

36 36

36 36

36 36

36 36

36 36

36 36

36 36

36 36

GP* Asyn*

G2* As y n

G2* Asyn*

GP * Asyn * S* S*

S* S*

Late S* Asyn

GI G1

GI-S G,-S

GP GP

GP GP

GP G2

Postanaphase Postanaphase

Asyn Asyn

20 98 12 16

15 95 14 64

21 95 25 42

13 91 14 71

19 99 14 81

18 96 27 83

24 88 29 69

33 88 53 97

37 71 29 76

18 79 19 83

36 91 34 99

48 94 31 88

30 83 27 94

14 17 13 27

94 1 1

85 29

80 43

72 28

73 71

88 81

85 74

78 88

81 75

58 57

90 89

81 70

69 69

10 14

93 f 2.1 86 f 1.2

94 f 0.8 72 f 7.1

94 f 2.3 72 ? 7.8

97 f 1.7 87 f 6.0

97 f 1.7 87 f 6.0

95 f 3.0 83 f 4.5

96 f 2.6 82 f 5.5

96 f 1.6 93 f 2.3

93 f 3.6 93 f 2.3

97 f 0.7 92 f 1.6

93 f 3.1 90 f 5.0

97 f 2.4 95 f 3.1

95 f 3. I 84 f 7.9

94 f 1.2 93 f 2.4

69 f 2.6 1 1 f 4.7

32 f 7.8 0.2 f 0.8

8.6 f 1.2 0.5 f 0.3

88 f 1.6 12 f 3.3

46 f 6.7 17 f 3.1

28 f 1.4 29 f 3.2

28 f 5.4 15 f 5.0

69 f 5.9 61 * 3.4

25 f 2.9 22 f 1.2

76 f 4.0 59 f 3.8

65 f 13.5 63 f 2.9

78 f 4.8 66 f 4.7

52 f 3.3 44 f 2.5

92 f 2.1 72 ? 1.9

25 28

30 25

32 25

32 28

32 28

30 30

30 28

32 32

28 28

30 30

30 30

30 30

30 30

36 36

a Strains are shown in Table 1. Restrictive temperature (RT) for cdc mutation. Conclusions from these and previously published observations (and see Figure 1). * Indicates strains that were analyzed by flow cytometry. Percentage of cells with a large bud and undivided nucleus, except cdc28 (unbudded). Percentage of microcolonies that contain either 2 or 4 buds except for cdc28 that contain 1 or 2 buds (microcolony assay).

f Mean value from 3-6 cultures; standard deviation shown. g Maximum permissive temperature of colony formation.

cycle arrest was determined from the percentage of microcolonies that contained either 2 (1 large-budded cell) or 4 buds (2 large-budded cells) 10 hr after plating and X- irradiation. Cell lethality was determined from macrocolony formation 24 hr. after X-irradiation at the same dose. Ra- diation sensitivity was determined by exposing freshly replicated patches of cells to 8 krad of X rays twice at 8-hr intervals and scoring growth of survivors after incubation for 2-3 days at 23". The cell cycle arrest defect was quan- tified using a convenient metric calculated from the percentage of X-irradiated cells that arrest divided by cell inviability at the same dose (WEINERT and HARTWELL 1988, 1990). Typically the ratio of cell cycle arrest to cell inviability is >0.8 for arrest-proficient strains (e.g., RAD) and (0.3 for arrest-deficient strains (e.g., rad1 7).

Delay of nuclear division (mitosis) by irradiation: To

quantify the delay in nuclear division by DNA damage, cells growing logarithmically in rich liquid medium were first synchronized in the GP phase by plating on agar plates containing the microtubule inhibitor MBC and incubated for 3 hr at 23". DNA damage was induced in the MBC- blocked GP cells by irradiation (X rays, 4 krad; or UV, 40 J/m') and cells were immediately resuspended in medium, washed twice to remove the MBC and resuspended in fresh medium. The kinetics of progression through mitosis was determined by anal sis of nuclear morphology. Aliquots of cells were fixed wit K 70% ethanol at specific times, stained with DAPI and nuclear morphology determined by fluorescence microscopy.

RESULTS Rational-cdc mutants and the RAD9 checkpoint:

We tested the role of the RAD9 checkpoint in cell


cycle arrest in 12 cdc mutants which have been characterized physiologically and genetically, including some for which the biochemical functions are known (see legend to Figure 1). We reasoned that interaction between the R A D 9 checkpoint and any of these cdc mutants would provide information on the basis for cell cycle arrest in the cdc mutants and on the phase and signal specificity of the R A D 9 checkpoint. Figure 1 shows the dependent events of the cell cycle defined by these cdc mutants (PRINGLE and HARTWELL 1981) as modified by the results presented here (see DISCUS- SION). The phase of cell division where cdc mutants arrest at their restrictive temperatures is in some cases ambiguous. We can not at present distinguish by cytological or biochemical criteria late S phase (as defined below), the G2 phase, and metaphase (see NURSE 1985). These distinctions are particular rele- vant because the R A D 9 checkpoint acts in this part of the cell cycle, and because better resolution would allow comparisons to checkpoint controls in other organisms where the three phases are better distin- guished. Formally we must describe the function of the RAD9 checkpoint as acting in the late S/G2/M phase (Figure l), but we will refer to its function in the text as in “late S/G2 phase” reflecting our bias that it acts near the end or immediately after DNA replication. The arrest of cdclb, cdc20, cdc23 and MBC- treated cells is referred as in “G*/M” because arrested cells in each case appear to contain fully replicated, intact sister chromosomes (KOSHLAND and HARTWELL 1987). The order of function of cdc mutants that are required in late S/G2 phase and those required in G2/ M phase has not been formally established beyond their order with respect to the block imposed by the microtubule inhibitor, benomyl (WOOD and HART- WELL 1982).

We used two sets of criteria to test the cell cycle arrest of cdc R A D and cdc rad9 mutants after shift to a restrictive temperature. A direct measure of cell cycle arrest comes from determination of the nuclear and cell morphology, and from whether the cells arrested in the first cell cycle (scored by observing microcolonies on solid medium) 4 hr after shift to the restrictive temperature. A second criterion was the effect of a rad9 mutation on cell viability when cells were limited for the cdc function. CDC function was limited either by incubation for 4 hr at the fully restrictive temperature (36”) or by continuous incubation at an intermediate temperature. For some CDC mutants, we predicted that limiting the CDC function generates errors that can be repaired provided cell division is delayed by the R A D 9 checkpoint. For these CDC mutants we expected the cdc rad9 mutant would have a lower viability compared to the cdc R A D mutant because cell division with uncorrected errors would be lethal. We measured synthetic lethality at both the

fully restrictive temperature where cdc R A D mutants arrest because they have insufficient CDC function to complete cell division as well as at an intermediate temperature where cdc R A D mutants delay because the residual CDC function may be limiting but sufficient for cell division (see for example HARTWELL and SMITH 1985; SCLAFANI and FANGMAN 1986). For example, cdc9 R A D mutants require the R A D 9 gene both for arrest and viability at the fully restrictive temperature (see Table 2; HARTWELL and WEINERT 1989) as well as for viability, and presumably for delay of cell division, at intermediate temperatures (CDC9 apparently is limiting for cell division at intermediate temperatures because the percent of large-budded cells with an undivided nucleus increases with increas- ing temperature of incubation; data not shown). In sum, the two criteria of morphological cell cycle arrest and cell viability provide a comprehensive test for the role of the R A D 9 checkpoint in arrest of cells limited for a CDC gene product.

Arrest in the late S/G2 phase of four cdc mutants requires the RAD9 gene, We found that the cell cycle arrest of cdc2, cdc9, cdcl3 and cdcl7 after shift to the restrictive temperature required an intact R A D 9 gene. Results of tests of morphological arrest and viability of cdcl? and cdcl7 mutants are presented in detail below.

c d c l 3 R A D mutants shifted to the restrictive temperature for 4 hr arrested in the first cell cycle (Figure 2A) as large-budded cells with an undivided nucleus (Figure 2B) with approximately a 2C DNA content (Figure 2C, bottom). After 8 hr at the restrictive temperature most c d c l 3 R A D mutant cells remained arrested while cell growth continued (Figure 2A, inset). In contrast, cdc l3 rad9 mutants failed to arrest after shift to the restrictive temperature. The double mutant cells continued to divide for up to about 8 hr at nearly the rate of CDC cells incubated at 36” (Figure 2D and inset; data not shown). The cell and nuclear morphology (Figure 2E) and DNA content (Figure 2F, bottom) of cdcl3 rad9 cells after 4 hr at 36” resembled that of the asynchronous, exponen- tially dividing cdc l3 rad9 culture at 2 3 ” . The cell viability of cdc l3 rad9 mutants at the fully restrictive temperature was also significantly lower than that of the c d c l 3 R A D strain (Table 2), and the maximum permissive temperature was also different in the two strains, though in an unexpected way (see below). Comparing the data presented in Table 2, we conclude that nearly all cdc l3 cells at the restrictive temperature require the R A D 9 gene for arrest and viability.

The cell cycle arrest of cdcl7 mutants is more complex than that of cdc l3 mutants yet reveals the phase specificity of the RAD9 checkpoint. We initially found that cdcl7 rad9 mutants after shift to 36”


cdcl3 cdcl3 rad9 . ..,".... .. ..% . . "%"-."""/"

69

DNA contentkell DNA contentkell FIGURE 2.-Cell cycle arrest of cdcl? and cdcl? rad9 strains. (A-C) cdcl?; (D-F) cdcl3 rad9. (A and D) Microcolony test of first cycle

arrest. Cells were grown in liquid medium at 23". plated on agar slabs and incubated at 36" for 4 hr. Insets: cells incubated at 36" for 8 hr. Photographs in A. D, and insets are same magnification. (B and E) Cell and nuclear morphology. Magnification is different from cells shown in A and D. (C and F) Flow cytometric analysis of DNA content in cells grown at 23" (top) or at 36" for 4 hr (bottom).

showed a partial defect in cell cycle arrest compared when shifted from 23" to 34" for 4 hr, in early S to c d c l 7 RAD strains (data not shown). We subse- phase when shifted to 38", and a mixture of cells in quently learned that the cdc l7 -1 mutant strain ar- early S to late S/G2 phase when shifted to 36" (CARSON rested with a DNA content of cells in late S/Gp DNA 1987; Figure 3C). Consequently, we tested c d c l 7 mu-


cdcl7 cdcl7 rad9

rl 1

I lF

1 40 88 120 160 208 1 40 80 120 168 200

DNA contentkell DNA content/cell FIGURE 3.-Cell cycle arrest of cdcl7 and cdcl7 rad9 strains. (A-C) cdcl l ; (D-F) cdcl7 rad9. (A and D) Microcolony test of first cycle

arrest at 34". (B and E) Microcolony test of first cycle arrest at 38". (C and F) Flow cytometric analysis of DNA content per cell in cells incubated at 23" (top) 34" for 4 hr (middle) or 38" for 4 hr (bottom).

tants for cell cycle arrest at 34" and 38". After shift and E) as large-budded cells with undivided nucleus to 38" for 4 hr both cdcl7 RAD and cdcl7 rad9 and an early S phase content of DNA (Table 2, Figure mutants arrested in the first cell cycle (Figure 3, B 3, C and F, bottom). In contrast, after shift to 34" for

71

i

t

I i D

DNA contentkell DNA contentlcell FIGURE 4.-Cell cycle arrest of cdcd and cdcd rad9 strains. (A and B) cdcd; (C and D) cdcd rad9. (A and C) Microcolony test to determine

first cycle arrest at 36"; (B and D) flow cytometric analysis of DNA content per cell in cells incubated at 23" (top) or 36" for 4 hr (bottom).

4 hr cdcl7 RAD mutants did arrest in the first cell cycle in late S/G2 phase (Figure 3, A and C, middle), whereas cdcl7 rad9 did not arrest in the first cell cycle (compare Figure 3, A and D) and did not arrest with a late S/G2 phase content of DNA (Figure 3F, middle). In addition, the cell viability was decreased more dramatically in the cdcl7 rad9 strain compared to cdcl7 RAD strain after incubation at 34" than after incubation at 38" (Table 2). We conclude that the cell cycle arrest and viability of cdcl7 mutants in late S/ GP phase requires the RAD9 gene but arrest of cdcl7 mutants in early S phase does not. (We speculate that the 2-3-fold lower viability of cdcl7 rad9 strains compared to cdcl7 RAD at 38" may reveal a requirement for some delay at the RAD9 checkpoint to repair DNA lesions formed at 38" after temperature shift down.)

We also found that two additional cdc mutants, cdc2 and cdc9, of the 12 cdc mutants tested required the RAD9 gene for cell cycle arrest; cdc2 rad9 and cdc9

rad9 cells failed to cell cycle arrest and lost cell viability rapidly after 4 hr at the restrictive temperature compared to cdc RAD strains [Table 2 and HARTWELL and WEINERT (1989)l. We measured DNA content at the restrictive temperature and found that cdc2 RAD and cdc9 RAD, but not the cdc rad9 mutants, arrest with an apparent 2C DNA content after 4 hr (data not shown). We conclude that arrest of cdc2 and cdc9 mutants in the late S/G2 phase requires the RAD9 gene. (From the data in Table 2 we can conclude that at least half of the cdc RAD cells for cdc2, cdc9 as well as for cdcl7 require the RAD9 gene for cell cycle arrest, though these quantitative estimates of cell cycle arrest may be underestimates; see MATERIALS AND

METHODS). We also observed synthetic lethality in cdc2 rad9,

cdc9 rad9, and cdcl7 rad9 mutants as measured by the maximum permissive temperature; the cdc rad9 mutants had a lower maximum permissive tempera-


I

I

FIGURE 5.-Cell cycle arrest in X-irradiated RAD and rad 17 cells. Time lapse photomicroscopy of X-irradiated wild type (A and B) and radl 7 (C and D) strains. (A and C) Cells plated and X-irradiated (2 krad) at t = 0 hr; (B and D) progeny of cells, t = 10 hr. Arrows indicate unbudded G I cells and their progeny, and asterisks indicate large-budded postanaphase cells and their progeny.

TABLE 3

Cell cycle response to hydroxyurea of RAD+, rad9 or rad17 cells

Percent arrest

Strain genotype' H u b morphologvC First cvcled Viabilitv'

Nuclear

RAD+ - 16 14 94 f 3.0 + 85 87 73 f 13.3

rad 9 - 12 8 95 f 1.6 + 83 83 76 2 10.1

rad I7 - 13 7 86 f 4.2 + 95 92 83 * 1.8

' Strains are RAD+ (7859-10-2); rad9 (7859-7-4); rad17 (208-7- 2).

HU, hydroxyurea at final concentration at 0.2 M. Percentage of cells with large bud and undivided nucleus. Percentage of microcolonies that contain either 2 or 4 buds. Mean value from triplicate samples; standard deviation shown.

ture for colony formation than did the cdc RAD mutants (Table 2). This result implies that cells with suboptimal levels of these CDC gene functions generate viable progeny only when the RAD9 checkpoint prevents cell division until errors are corrected.

The maximum permissive temperature for cdcl3 rad9 is exceptional because the cdcl3 rad9 double mutant formed colonies at a higher temperature (28") than did cdcl3 RAD mutant (25"). (The colony for-

TABLE 4

&segregation of radiation-sensitivity and defect in cell cycle arrest

Strain lethality arrest Arrest/lethality RAD Percent Percent

7870-1-1 42 46 1.09 + 1-2 81 4 0.05 - 1-3 66 63 0.95 + 1-4 66 1 0.02 - 2- 1 79 6 0.08 - 2-2 63 54 0.86 + 2-3 72 1 0.01 - 2-4 40 40 1 .oo + 3-1 73 2 0.03 - 3-2 64 49 0.77 + 3-3 59 10 0.17 - 3-4 42 40 0.95 +

RAD+ 48 42 0.92 + radl 7 71 6.9 0.10 -

Meiotic products from a diploid strain 7870 heterozygous for radl 7 were analyzed for cell cycle defect and radiation sensitivity. Cell lethality, cell cycle arrest, and radiation sensitivity were determined as described in MATERIALS AND METHODS. Results from 3 of the 12 tetrads analyzed are shown, and the averages of all 24 RAD+ and 24 rad I7 spores tested are given.

mation of cdcl3 rad9 cells is not due to suppressors of cdcl3-nor of rad9; data not shown.) We think the reason cdcl3 rad9 cells form a colony at intermediate temperature is an exaggeration of its unusual phenotype at high temperature (36") where cdcl3 rad9 cells sustain several cell divisions before cessation of division (see Figure 2); at the intermediate temperature cdcl3 rad9 cells sustain more divisions (about 20), enough to form a colony. We posit that lethal DNA damage that leads to cessation of cell divisions requires more cell divisions to accumulate at intermediate temperature us. high temperature. This hypothesis predicts that cdcl3 rad9 cells generated at the intermediate temperature should contain a high fraction of inviable cells. This prediction was confirmed; the viability of cdcl3 rad9 cells from colonies formed at the intermediate temperature (28") was only 27.5 f 5.5% (n = 6) (viability as tested by colony formation at 23") whereas the viability of cdcl3 rad9 cells grown at permissive temperature (23") was 86% (see Table 2)

Arrest of cdc mutants in early S phase does not require the RAD9 gene: The efficient arrest of cdcl7 RAD and cdcl7 rad9 mutants at 38" in early S phase described above prompted a further test of whether arrest in early S phase does not in general require the RAD9 gene. We found that cell cycle arrest in cdc8 mutants at the restrictive temperature and in hydroxyurea-treated cells, conditions that block DNA replication in early S phase, did not require the RAD9 gene (Tables 2 and 3 and Figure 4; and see Figure 1). We also examined the microtubule morphology in cdc8- or hyrdoxyurea-arrested cells and observed that

cdc Mutants and the RAD9 Checkpoint 73

TABLE 5

Requirement for RADZ7 in cell cycle arrest of cdc mutants

Arrest Viabilityf

morpholo- Nuclear

gyd Strain Restrictive

Maximum

genotypea temperatureb Cell cycle phase' 23" R T First cyclee 23" RT permissive

temperatureg

cdc 13 cdcl3 rad17

cdc2 cdc2 rad17

cdc9 cdc9 rad 17 cdcl 7 cdcl7 rad17

cdcl7 cdcl7 rad17

cdc8 cdc8 radl 7

cdc6 cdc6 radl 7 cdc28 cdc28 radl 7 cdc7 cdcl rad 17

cdcl6 cdcl6 rad 17

cdc20 cdc20 rad17

cdcl5 cdcl5 rad 17

CDC+ radl 7

36 36

36 36

36 36

34 34

38 38

36 36

36 36

36 36

36 36

36 36

36 36

36 36

36

GP Asyn

Gz Asyn

GP Gn-postanaphase

GP As y n

S S

S S

Late S Asyn

G , Gl

G,-S GI-S

Gz GP

G2 GP Postanaphase Postanaphase

As yn

9 11

26 22

15 20

11 22

11 22

13 13

18 18

31 19

14 19

15 24

25 35

13 16

15

87 18

83 70

93 47

82 63

63 65

92 91

93 68

83 84

78 68

67 69

94 86

86 86

10

90 20

95 63

90 79

66 44

64 68

91 85

89 83

87 86

30 36

81 81

87 90

75 85

14

90 f 2.1 88 f 3.2

94 f 1.7 64 f 4.8

95 f 1.7 56 * 3.9

82 f 7.8 83 f 5.3

82 f 7.8 83 f 5.3

98 f 1.5 87 f 3.2

98 k 2.6 86 f 2.8

85 f 6.3 91 f 1.9

84 f 11.9 90 f 2.2

98 f 0.8 91 f 3.3

95 f 1.9 95 f 0.8

82 f 6.2 85 f 2.9

93 k 2.5

73 f 10.8 25 53 f 11.2 30

67 f 4.6 30 0.9 f 0.5 25

10 f 0.5 32 I .4 f 0.9 28

81 f 4.2 32 13 f 0.9 30

47 f 8.9 32 36 f 6.0 30

39 f 5.6 30 33 f 17.3 30

16 f 2.8 30 4.0 f 2.6 28

61 f 1.3 32 62 f 6.1 32

32 f 0.8 28 37 f 6.2 28

68 f 2.9 30 31 f 2.9 30

38 f 5.0 30 55 f 2.5 30

42 f 1.3 30 50 f 6.2 30

89 f 1.2 36

a Strains are shown in Table 1. Restrictive temperature (RT) for cdc mutation. Conclusions from these and previously published observations (and see Figure 1). Percentage of cells with a large bud and undivided nucleus, except cdc28 (unbudded). Percentage of microcolonies that contain either 2 or 4 buds except for cdc28 that contain 1 or 2 buds (microcolony assay).

f Mean value from 3-6 cultures; standard deviation shown. g Maximum permissive temperature of colony formation.

both RAD and rad9 cells contained predominantly short spindles, as expected for cells arrested in medial nuclear division (BYERS and GOETSCH 1974). We conclude that arrest of the cell cycle in early S phase when DNA replication is inhibited does not require the RAD9 gene.

Arrest of cdc6 mutants: cdc6 was the only cdc mutant tested that showed an equivocal requirement for RAD9 for cell cycle arrest at the restrictive temperature; fewer cdc6 rad9 cells were arrested as large buds with an undivided nucleus than in cdc6 RAD cells although measurements of cell viability and first cycle arrest were similar in the two strains. In addition, cdc6 rad9 cells did have a slightly lower maximum permissive temperature than cdc6 RAD strains. We also measured the DNA content in the cdc6 RAD strain by flow cytometry (data not shown), and found that cdc6 RAD

cells shifted to 36" for 4 hr contained less DNA than cdc mutants that arrest in late S/G:, (e.g., in cdcl3- arrested cells in Figure 2C, bottom) but more DNA than that of mutants arrested in early S phase (e.g., as in cdc8 in Figure 4B) after 4 hr at the restrictive temperature. From our results we conclude that most cdc6 RAD cells at the restrictive temperature do not require RAD9 for arrest.

Arrest of some cdc mutants in G2/M and of all cdc mutants in other phases of the cell cycle does not require the RAD9 gene: We tested if the RAD9 gene is required for cell cycle arrest of each of 7 cdc mutants that arrest either in GI (cdc28), at the GI-S boundary (cdc7), in postanaphase ( cdc l5 ) , or in G2/M (cdc lb , cdc20, cdc23) at their restrictive temperatures (see Figure 1). The results from cell cycle arrest and cell viability tests for each of these cdc mutants were


A. R4DtX-ray B. radlZX-my c. WDtUv D. radl?,UV E. m , u v

h"nT 0 1 2 3 0 1 2 3 0 1 2 3

L 0 1 2 3

h-rrrrr 0 1 2 3

Time (hours) FIGURE 6.-Kinetics of cell cycle delay in X- or UV-irradiated GP cells in RAD, rad9 and radl 7 strains. GP cells were prepared by incubation

with MBC, irradiated with UV or X rays, released from the MBC block and analyzed for cell cycle progression using nuclear and cell morphology. The percentage of cells arrested was calculated in which the daughter bud was greater than half the diameter (and usually equal to diameter of) the mother bud and with an undivided nucleus. Symbols: open circles, unirradiated; closed circles, irradiated. Dose of X rays was 4 krad and of UV was 40 J/m'. (A and C) RAD; (Band D) radl7; (E) rad9. Strains: in A, RAD (7870-1-1); B, rad17 (7870-1-4); C, RAD (7859-10-2); D, rad17 (208-7-2), E, rad9 (7859-7-4). We also observed a UV-induced delay in the two additional RAD strains tested [7870-l and 7815-6-4; Weinert and Hartwell (1988)], and a failure to delay in the other rad17 (7870-1-4) and rad9 [7815-6-4AB; WEINERT and HARTWELL (1988)l strains tested. The cell viability in MBC-arrested and unirradiated cells was >80% in all strains tested.

similar in RAD and rad9 strains (Table 2). We conclude that the arrest of these cdc mutants does not require the RAD9 gene.

RADl 7 mutants provide a further test of the phase and signal specificity of the RAD9 checkpoint: We wanted to corroborate our findings on the role of the RAD9 checkpoint by analyzing a second mutation that, like rad9, inactivates the checkpoint. We show here first that a mutation in radl 7 results in a defect in cell cycle arrest after irradiation, and then we report the results of tests of cell cycle arrest in cdc mutants containing the radl 7 mutation.

We initially identified the RADl 7 gene as essential for the RAD9 checkpoint because rad17 cells, like rad9 cells, failed to arrest following X-irradiation. The cell cycle response to DNA damage from X rays was performed using the methodology and rational developed in earlier studies using the rad9 mutation (WEI- NERT and HARTWELL 1988). The response of GI and postanaphase haploid cells to X rays provides a convenient diagnostic for monitoring cell cycle arrest after DNA damage, In wild-type haploid strains, cells in GI and postanaphase when X-irradiated arrest as large budded cells (2 budded microcolony) and as 2 large-budded cells (4 budded microcolony), respectively. These cells are arrested essentially irreversible (see Figure 5) as the result of a signal generated from DNA double-strand breaks that persists unrepaired because in budding yeast their repair requires an intact homologous chromosome (BURNBORG and WIL- LIAMSON 1978; GAME 1983). In mutants which have lost regulation (e .g . , rad9), cells in GI and postanaphase when X-irradiated will not arrest but continue to divide for several generations.

Wild-type and rad17 cells were exposed to a dose of X rays that generates a few doublestrand breaks

per cell, and the response of individual cells was determined by photomicroscopy (Figure 5). We found that 83% (n = 42) of wildtype and 3.4% ( n = 59) of rad I7 unbudded (GI) cells when X-irradiated arrested with a large bud (Figure 5). The unbudded rad17 cells when X-irradiated typically continued to divide for 1-3 generations, a response typical of rad9 unbudded cells exposed to X rays (WEINERT and HARTWELL 1988).

We also examined budded cells exposed to X rays and observed that in rad17 mutants they also failed to arrest. Budded cells consist of both doublestrand break repair-proficient GP cells and repair-deficient postanaphase cells, and their cell cycle responses to DNA double-strand breaks differ; GB cells delay, repair the break and resume cell division, whereas postanaphase cells arrest cell division in the G:! phase in the next cell cycle. We found that 54% (n = 108) of the wild type and 4% (n = 94) of the rad17 budded cells exposed to X rays arrested as two adjacent large- budded cells (see Figure 5). Because the fraction of postanaphase cells among the budded cells in RAD and rad17 cultures were similar (43% and 49%, respectively, calculated from the nuclear morphologies of fixed cells from an asynchronous population of cells), we conclude that wild-type but not rad17 postanaphase cells exposed to X rays undergo cell cycle arrest. Below we describe additional experimental strategies to analyse the cell cycle delay in GP cells.

The cell cycle defect of rad17 cosegregates with radiation sensitivity: T o test if the defect in cell cycle arrest and radiation sensitivity are due to a single mutation we analyzed the meiotic products from a diploid strain heterozygous for radl7. The two phenotypes cosegregated in all 12 tetrads analyzed (Table 4).


Delay at the RAD9 checkpoint in RAD, rad9 and radl 7 cells after X- or UV-irradiation: The photom- icroscopic studies described above indicate that GI and postanaphase cells after irradiation require the R A D l 7 gene for arrest at the RAD9 checkpoint in the G2 phase. The RAD17 gene, like the RAD9 gene, should also be required for arrest in GB of cells irradiated in G2. T o test this prediction, cells were synchronized in the G:! phase by treatment with the microtubule inhibitor MBC, X- or UV-irradiated, the tubulin inhibitor was removed and the kinetics of exit from the G2 phase determined (see MATERIALS AND METHODS). We found that, as expected, X- or UV- irradiated wild-type G2 cells delayed in G2 compared to unirradiated wild-type G2 cells [Figure 6, A and C; see WEINERT and HARTWELL (1988, 1990)], and X- or UV-irradiated rad17 cells exhibited little delay in G2; rather they completed chromosome segregation nearly as rapidly as unirradiated rad I7 GP cells (Figure 6, B and D). A UV-induced delay of cell division in wild-type cells has been shown previously (ESPOSITO 1968). We also found that UV-irradiated rad9 G2 cells did not delay (Figure 6E), extending our previous finding that rad9 mutant G2 cells did not delay after x-irradiation (WEINERT and HARTWELL 1988, 1990). We conclude that the checkpoint for arrest after irradiation requires both the RAD9 and R A D l 7 genes.

The role of the RAD1 7 gene in arrest of cdc mutants: Finally, we tested if the cell cycle arrest of cdc mutants at their restrictive temperatures required R A D l 7. The cell cycle phenotypes of cdc RAD and cdc rad l 7 mutants (Table 5) were similar to those of cdc RAD and cdc rad9 mutants (Table 2); (1) the arrest and viability of cdc2, cdc9 and cdc l3 at the restrictive temperature of 36" required an intact RAD17 gene; (2) the arrest and viability of cdcl7 required RAD17 at 34" but not at 38"; (3) the arrest and viability of cdc6 mutants was slightly greater in cells with an intact RAD17 gene; and (4) the maximum permissive temperature was lower in cdc2, cdc6, cdc9, and cdcl7 mutants that were r a d l 7 , and higher in cdcl3 rad17 than in cdcl3 RAD. Results from the tests for cell cycle arrest and viability of the other cdc mutants examined were similar in RAD and rad17 mutants. Finally incubation with hydroxyurea induced arrest efficiently in rad l 7 mutants (Table 3).

The phenotypes of cdcl3 rad9 and cdcl3 rad17 mutants differed slightly; cdcl3 rad17 mutants shifted to 36" retained higher cell viability, were capable of sustaining more cell divisions after shift to 36", and formed colonies at a higher maximum permissive temperature than did cdcl3 rad9 cells (compare Tables 2 and 5; data not shown). Perhaps the rad17 allele retains partial function that may account for the different phenotypes.

DISCUSSION

The role of the RAD9 checkpoint in cell division appears specific: Previously we showed that rad9 mutants failed to arrest after X-irradiation or inactivation of DNA ligase, but unperturbed rad9 cells were viable and had cell cycle kinetics similar to that of wild-type strains. We concluded that the RAD9 checkpoint, when activated by DNA lesions, acts as a negative regulator to arrest cells before mitosis until damage is repaired. Here we tested more comprehensively the function(s) of the RAD9 checkpoint primarily by determining if cell cycle arrest of any of 12 cdc mutants requires the RAD9 and RAD17 genes. Our results are fully consistent with the hypothesis that the RAD9 checkpoint is activated only by DNA damage and arrests cells only in late S/G2 phase (the distinction between the late S and GS phase is addressed below). This conclusion is based on phenotypes of the cdc mutants that require the RAD9 checkpoint for arrest and those that do not. First, the four cdc RAD mutants (cdc2, 9, 13 and 1 7 ) that required intact RAD9 and RAD17 genes for arrest each arrest in the late S/G2 phase at the restrictive temperature. This indicates that the function of the RAD9 checkpoint is phase- specific. Importantly, in each of the four cdc RAD mutants chromosome damage occurs when cell division is limited by the function of any of the mutant CDC gene products (HARTWELL and SMITH 1985>, and three of these four CDC genes encode known DNA replication enzymes (DNA ligase and DNA polymerase I and 111, see Figure l). These observations are consistent with the hypothesis that DNA lesions formed at the restrictive temperature in each of these cdc mutants activate the RAD9 checkpoint to arrest cell division. We suggest that the DNA lesions may be nicks, gaps or double-strand breaks formed either as a result of errors made during DNA replication by the defective replication enzymes or as gaps present at stalled replication forks in cells that have not completed replication.

Additional observations suggest the RAD9 checkpoint is required only in arrest of cell division in the late S/G2 phase and is activated only by DNA damage. This conclusion is supported by negative evidence that the RAD9 and RAD17 genes are not required for the cell cycle arrest of cdc mutants that arrest in either GI, GI-S, early S phase or postanaphase, or for cells arrested in S phase by hydroxyurea. The RAD9 checkpoint is also not required for arrest of cdc mutants in the Gz/M phase in which chromosome damage does not occur when the CDC gene product is limiting for cell division, or for arrest in G2 by microtubule poisons (WEINERT and HARTWELL 1988, 1990) (Table 3, Fig- ure 6).

The role(s) of the RAD9 checkpoint in cell division may be tested and refined further through studies of


other mutants that delay the cell cycle. For example, recently GERRING, SPENCER and HIETER (1991) found that chll (ctf l) mutants delay nuclear division in the G2/M phase and that the delay does not require the RAD9 gene. chll mutants also have an increased rate of chromosome loss but have normal levels of mitotic recombination compared to wild-type cells, which indicates that DNA lesions are not formed in cells limited by CHLl function. The delay in chll mutants may be due to another checkpoint that is required for cell cycle arrest when microtubule function or assembly is defective because chll mutants exhibit synthetic lethality in combination with mad2, a checkpoint mutant (e.g., the chll mad2 double mutant has lower cell viability than either single mutant) (LI and MURRAY

Is the RAD9 checkpoint required for arrest in late S phase as well as for arrest in the GZ phase? The RAD9 checkpoint is clearly required for arrest in the GP phase after DNA replication is completed; RAD cells, but not rad9 or rad1 7 cells, arrested with the microtubule inhibitor MBC delay nuclear division when they are X- or UV-irradiated (Figure 6) (Wei- nert and Hartwell 1988, 1990). In the cdc mutants that require the RAD9 checkpoint for arrest we are unable to determine if DNA replication has been completed. The methods employed here and in previous studies (CULLOTTI and HARTWELL 197 1 ; CON- RAD and NEWLON 1983; BURKE and CHURCH 199 1) are not sufficient to determine whether 10% of the genome, for example, is unreplicated in cells that arrest at the RAD9 checkpoint. We are therefore left with two possible interpretations on the extent of DNA replication in these cdc mutants and consequently on when the RAD9 checkpoint acts; cdc mutant cells at the restrictive temperature either contain unreplicated DNA and arrest in what we call late S phase, or they contain completely replicated DNA but the sister chromosomes contain errors in the form of gaps or nicks and cells arrest in what we call the G2

phase. Additional experimental approaches must be developed to determine whether the presence of any unreplicated DNA arrests cells at an earlier S phase checkpoint or at the RAD9 checkpoint.

We consider two models that describe the relationships between checkpoint controls and DNA replication, and consider the implications of these models for the results presented here. By either model we imagine that the RAD9 checkpoint can be activated by nicks or gaps present either at replication forks or in fully replicated but defective sister chromosomes.

Model 1. The RAD9 checkpoint is required for arrest both in late S phase and in the G2 phase: This model suggests the RAD9 checkpoint is required when most but not all chromosome sequences have been replicated, and the single-strand gaps or nicks at the stalled

199 1).

replication forks may be sufficient to activate the RAD9 checkpoint. This interpretation predicts that, in the experiments reported here, DNA polymerase I mutants (encoded by cdcl7) at 34" do not complete DNA replication, and arrest occurs because the function of DNA polymerase I is required to complete DNA replication. By the same reasoning CDC2, CDC9 and CDCl3 gene products are required to complete DNA replication in the corresponding mutants at the restrictive temperature. This model also implies that an earlier S phase checkpoint is active when, for example, 50% of the genome is replicated (e.g., cdcl7 at 38 ") but becomes inactive when 90% of the genome is replicated. We and others have identified mutations in budding and fission yeasts that inactivate the earlier S phase checkpoint (our unpublished results; ROWLEY, HUDSON and YOUNG^^^^; Rowley, Subramani and YOUNG 1992; AL-KHODAIRY and CARR 1992; ENOCH and NURSE 1990). Perhaps sufficient "liscensing factor" (LASKEY, FAIRMAN and BLOW 1989) is released only when 90% of the genome has been replicated and inactivates the earlier S phase checkpoint. After 90% of the genome has been replicated, only the RAD9 checkpoint prevents mitosis until replication and repair of any damage are completed.

Model 2. The RAD9 checkpoint is required for arrest only in the G2 phase: This model suggests the RAD9 checkpoint is required for arrest when sister chromosomes are fully replicated but contain nicks or small gaps due to replication errors. By this model, the RAD9 checkpoint is required only when all regions in the genome have been duplicated; replication forks have completed the elongation phase of DNA replication but the defective replication enzymes have left DNA lesions that require repair. This model predicts that, in the experiments reported here, the DNA polymerase I mutants at 34" complete the elongation phase of DNA replication but leave DNA lesions. Cell cycle arrest is due to the DNA lesions whose repair may require DNA polymerase I. (If DNA polymerase I is required in this repair, the DNA polymerase I protein may exist in both a replication and a repair complex.) By the same reasoning, cdc2, cdc9 and c d c l 3 mutants also complete DNA replication at the restrictive temperature but leave DNA lesions whose repair also requires the function of the corresponding CDC gene product.

This model implies that cell cycle checkpoints distinguish between completion of DNA replication (checked by the S phase checkpoint) and completion of DNA repair before mitosis (checked by the RAD9 checkpoint). How the cell determines that DNA replication is complete under this model is a matter of further speculation; perhaps the absence of replication forks or differences in nucleotide metabolism signals


“the end of DNA replication” and leads to inactivation of the earlier S phase checkpoint.

In either model, the R A D 9 checkpoint may actually act in early S phase as well as in G2. However if R A D 9 acts in early S phase then it is functionally redundant with a second earlier S phase checkpoint. Alternatively the R A D 9 checkpoint may not act until the late S/G2 phase because, for example, the function of the R A D 9 checkpoint is itself dependent upon passage beyond the earlier S phase checkpoint.

The order of events and the RAD9 checkpoint: Figure 1 shows the role of the R A D 9 checkpoint relative to stages of cell division that require the function of CDC gene products (reviewed in PRINGLE and HARTWELL 1981). The function of the cdc mutants that arrest in late S/G2 phase at the R A D 9 checkpoint are likely required for steps in S phase as well as in late S/G2 phase. First, genetic studies of cdcl7 mutants reveal that CDC17 (POLI) is required for steps in both early S phase and in late S/G2 phase (CARSON 1987; BURKE and CHURCH 1991; this report). CDC2 (POLIII) and CDCB (DNA ligase), and perhaps CDC13 as well, also encode essential DNA replication enzymes which must function during S phase. The mutant alleles or experimental conditions used here apparently allowed mutant cells to complete the step in early S phase but not the step in late S/G2 phase.

The model in Figure 1 does distinguish between two classes of cdc mutants that arrest in late S/G2/M phase at the restrictive temperature; one class requires the R A D 9 checkpoint for cell cycle arrest (cdc2, cdc9, c d c l 3 and cdcl7 arrest in late S/G2 phase) and the other does not (cdcl6, cdc20 and cdc23 arrest in G2/ M phase). The functions of the CDC gene products from the first class appear to be in DNA replication as discussed above, but the functions of the second class, C D C l 6 , C D C 2 0 and CDC23, are unknown. These three CDC gene products are unlikely to be involved in DNA replication because, unlike the mutants in DNA replication enzymes, DNA lesions are not formed in cells limited for their function (HART- WELL and SMITH 1985; SIKORSKI et al. 1990). Fur- thermore, results from a recent study suggest that the CDC20 gene product may be involved in microtubule assembly or stability (SETHI et al. 1992). These observations provide further evidence that supports the conclusion here that the R A D 9 checkpoint is activated only by DNA lesions.

The basis for cell cycle arrest in cdcl6, cdc20 and cdc23 in G2/M remains unknown. These mutants are not arrested due to the R A D 9 checkpoint though they may be arrested at a step in nuclear division that is before or interdependent with the R A D 9 checkpoint (located at or before the R A D 9 checkpoint). Support for this hypothesis comes from the observation that

these cdc mutants retain viability when arrested at the restrictive temperature and then X-irradiated (F. FABRE, personal communication). This is the result predicted if the cdc mutants were arrested at or before a step that is still subject to control by the R A D 9 checkpoint. This indirect evidence suggests the R A D 9 checkpoint may arrest nuclear division up until the time of chromosome segregation at anaphase, as the model in Figure 1 implies.

The function of CDC6: From the results in Tables 2 and 5, we concluded that the arrest of cdcb R A D cells does not require the R A D 9 checkpoint. A role for the CDC6 gene product in initiation of DNA replication has been proposed from results of recip- rocal shift experiments with hydroxyurea (HARTWELL 1976), and from more recent studies on the rate of loss of minichromosomes containing additional ARS elements in cdcb mutants (HOGAN and KOSHLAND 1992). Several studies have found that some DNA is replicated in cdc6 mutants at the restrictive temperature [CULOTTI and HARTWELL (1 97 1) and our unpublished observations; but see HOGAN and KOSHLAND (1 992) and BUENO and RUSSELL (1 992)] in apparent contradiction to conclusion that CDCG is involved in initiation of DNA replication. Initiation of DNA replication may be leaky in cdcb mutants, and different experimental protocols may lead to different conclusions on the extent of DNA replication. To reconcile the apparent contradiction from these observations it has been proposed that cdcb mutants may replicate some DNA at the restrictive temperature but do so incorrectly (HARTWELL 1976); under this hypothesis after shift from the restrictive to the permissive temperature DNA replication would need to be reintiated correctly. Nevertheless, the results reported here that arrest of cdc6 mutants does not require the R A D 9 checkpoint are consistent with a role for CDC6 in initiation of DNA replication.

Recently BUENO and RUSSELL (1 992) reported that overexpression of CDC6 leads to arrest in the G2/M phase that is independent of the R A D 9 checkpoint. They made the intriguing suggestion that the CDCB protein may act both in initiation of DNA replication and as a component linking DNA replication to cell cycle controls. A relationship between DNA replication, CDCG and cell cycle checkpoint controls may be further evaluated by study of the S phase checkpoint mutants.

Unusual properties of cdcl3 mutants: Three of the four cdc mutants that required RAD9 and RAD17 for arrest (cdc9, cdc2 and c d c l 7 ) undergo but one extra round of cell division in rad9 strains after shift to the restrictive temperature compared to cdc R A D strains. In contrast, cdc l3 rad9 and cdcl3 rad17 sustain many cell divisions at the fully restrictive temperature for cdc l3 ( e .g . , see Figure 2D). Why cdc l3 rad9


cells can continue cell division in the present of DNA lesions may be related to the chromosomal distribution of the DNA lesions. This distribution of DNA lesions has been determined in cells limited for CDC gene products by examining the increase in mitotic recombination at specific intervals along the arms of chromosomes (CARSON 1987; KRANTZLER 1986). The results indicate that in cells limited for CDC13 function the DNA lesions appear localized to telomere- proximal regions whereas in cells limited for CDC9 function, or in irradiated cells, DNA lesions appear more random. Identifying the DNA lesion in cdcl3- defective cells may provide clues to the role of CDC13 in DNA replication as well as information on the quantity and quality of DNA lesion detected by the RAD9 checkpoint.

The target(s) of inhibition by the RAD9 Check- point: Because the RAD9 gene is nonessential, we suggested that the RAD9 checkpoint most likely arrests cell division by inhibiting another gene product(s) whose activity or function is essential. The iden- tity of the target gene(s) in budding yeast is unknown. The cdc2+ gene product (whose homolog is CDC28 in budding yeast) has been implicated as a target of checkpoint control in fission yeast and in frog embryos (ENOCH and NURSE 1990; DASSO and NEWPORT 1990; ROWLEY, HUDSON and YOUNG 1992; ROWLEY, SUB- RAMANI and YOUNG 1992; AL-KHODAIRY and CARR 1992). Cdc2+ encodes a protein kinase, is essential for mitosis in all eucaryotic cells, and can be regulated by phosphorylation (NURSE 1990). Genetic and biochemical evidence from studies in fission yeast and frog embryos suggest that the cdc2 protein kinase is phosphorylated and thereby inactivated after DNA damage or when DNA replication is inhibited. This mechanism of checkpoint control is attractive in budding yeast as well because the genetic regulation and pat- terns of phosphorylation of ~34"'~+in fission yeast and

MORENO and REED 1989; SURANA et al. 199 1 ; GHIARA et al. 1991; AMON et al. 1992; SORGER and MURRAY 1992; NURSE 1990). The role of phosphorylation of ~ 3 4 ' ~ " ~ ' was tested, however, and mutants of CDC28 that cannot be phosphorylated (due to site-specific mutations in CDC28 that eliminate the sites of phosphorylation) were still capable of cell cycle arrest in early S phase or at the RAD9 checkpoint in the late S/G2 phase (AMON et al. 1992; SORGER and MURRAY 1992). Therefore, either the RAD9 checkpoint does not act by altering phosphorylation of ~ 3 4 ' ~ ' ~ ~ or it acts by an additional, redundant mechanism. The relationship between checkpoint controls in budding yeast and fission yeast remain unclear.

Synthetic lethality and checkpoint controls: A synthetic phenotype suggests that gene products either interact or possess redundant functions (e.g., ROTH-

P34cDc28 in budding yeast are very similar (RUSSELL,

BLATT et al. 1989; STRAUSS and GUTHRIE 1991; RY- KOWSKI et al. 198 1; RICHARDSON et al. 1990). The study of checkpoint mutants suggests a third interpretation of synthetic phenotypes. Checkpoints appear to regulate the kinetics of a cellular process and therefore they may compensate for a defect in synthesis, assembly, or function of the process. For example the RAD9 checkpoint minimizes the consequences of errors in DNA replication (e.g., in DNA ligase mutants) by imposing a delay on mitosis until errors are corrected. Double mutants defective for both DNA ligase and for the RAD9 checkpoint die presumably due to cell division with the uncorrected errors; this is called synthetic lethality because at the same temperature both single mutants are viable. Whether any particular instance of a synthetic phenotype is due to the existence of checkpoint controls, to the interaction of gene products, or to functional redundancy of gene products is not easily determined. The existence of a checkpoint may be suspected where the synthetic phenotype is gene-nonspecific; for example we observed synthetic lethality between all combinations of four DNA replication mutants with either of two checkpoint mutants. Of course mutations in gene products that form a multimeric complex may also show synthetic phenotypes which are gene-nonspecific. UIti- mately, evidence for a checkpoint control relies on evidence that a mutation relieves the dependence of events (HARTWELL and WEINERT 1989).

We thank TERESA NAUJACK for capable technical assistance. This research was supported by U.S. Public Health Service grant GM 17709 from the National Institute of General Medical Sciences (L.H.) and GM45276-01 (T.A.W), and by a fellowship from the Jane Coffin Childs Foundation (T.A.W.).

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cell cycle arrest of cdc mutants and specificity of the rad9 - genetics

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