Establishing Genetic Interactions by a Synthetic Dosage Lethality Phenotype

Eugene S. Kroll,*?lKatherine M. Hyland,*” Philip Hieter” and Joachim J. Lit *Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 and tDepartment of Microbiology and Immunology, University of California at San Francisco, San Francisco, California 94143-0414 Manuscript received August 31, 1995 Accepted for publication February 1, 1996

ABSTRACT We have devised a genetic screen, termed synthetic dosage lethality, in which a cloned “reference” gene is inducibly overexpressed in a set of mutant strains carrying potential“target” mutations. To test the specificity of the method,two reference genes,CTFl3, encoding a centromere binding protein, and ORC6, encoding a subunit of the origin of replication binding complex, were overexpressed in a large collection of mutants defective in either chromosome segregation or replication. CTF13 overexpression caused synthetic dosage lethality in combination with ~$14-42(cbfl, ndclo), ~$17-61 (ch14), ctfl9-58 and ~$19-26.ORC6 overexpression caused synthetic dosage lethality in combination withcdc2-1, cdckl, cdcl4- 1, cdcl6-I and cdc46-1. These relationships reflectspecific interactions, as overexpression of CTF13caused lethality in kinetochore mutants and overexpressionof ORC6 caused lethality in replication mutants.In contrast, only one case of dosage suppression was observed. We suggest that synthetic dosage lethality identifies a broad spectrumof interacting mutations andis of general utility in detecting specific genetic interactions using a cloned wild-type gene as a starting point. Furthermore, synthetic dosage lethalityis easily adapted to the study of cloned genes in other organisms.

practical strategy for studying the function of a isolate synthetic lethal mutations, a strain containing the A protein of interest is to identify other proteins that reference mutation must harbor the wild-type copy of interact with it. This may lead to the isolation of new a gene on a counterselectable plasmid that covers the components participating in thesame pathway or iden- mutation of interest. After mutagenesis, synthetic lethal tification of previously characterized factors that can mutations are identified in strains that have become de- help elucidate the function of the protein understudy. pendent on theplasmid for viability. Dosagesuppression Identification of a genetic interaction between a muta- screens involve identification of wild-type genes that, at tion in the gene coding fora protein of interest and a increased copy number or when overexpressed, comple- mutation in an unlinked locus is a proven method for ment the phenotype caused by a reference gene muta- establishing functional links between proteins. A ge- tion. If the reference mutation is conditionally lethal, netic interaction can be manifested as a novel pheno- this canbe accomplished by transformation of a genomic type that is not attributable to either mutation alone. library on a high copy vector and selection ofviable Typically, genetic interaction screens use a mutation in transformants at the nonpermissive condition. Overex- the gene of interest as a starting point and rely on pression of some gene products are known to cause detri- finding phenotypes caused by the combination of two mental phenotypes including the loss of viability (LIU et mutations. Several powerful genetic interaction screens al. 1992). These genes would not be recovered in a dos- have been developed that are based on either loss or age suppression screen. However, since only a fraction gain ofviability as a phenotype (ROSE et al. 1990). of genes in yeast are toxic upon amplification or overex- Among those most widely used are second site suppres- pressionin a wild-type strain background (LIU et al. sion (HARTMAN and ROTH 1973), synthetic lethality 1992), this is not generally a problem. (GUARENTE1993), and dosage suppression screens Various molecular genetic strategies lead to the isola- (RINE 1991). tion of a cloned wild-type gene of known sequence and Synthetic lethality screens, which identify two nonalle- unknown function. In this study, we have set up a ver- lic and nonessential mutations that arelethal for the cell sion of a synthetic lethality screen in which a cloned only when both are present, have proved to be very use- “reference” gene is overexpressed in a set of mutant ful in finding genes encoding interacting proteins. To strains carrying potential “target” mutations. This provides a means for identifylng genetic interactions using Corresponding author: Philip Hieter, Department of Molecular Biol- a cloned wild-type gene (rather than a mutant) as the ogy and Genetics,Johns Hopkins Medical School, The Johns Hopkins University, 725 N. Wolfe St., Baltimore, MD 21205. starting point in a screen (Figure 1).We reasoned that E-mail: [email protected] although increasing the amountof activityof one factor I These authors contributed equally to this work. (encoded by the reference gene) may not produce a

Genetics 143: 9.5-102 (May, 1996) 96 E. S. Kroll et al. a. VectoronlyVector + "Reference"gene upon overexpression of the RED3 gene (FRIEDMAN et al. 1994). Overproduction of the MCM3 gene product Transform "Target" mutant collection accentuates the defects caused by the mcm2 mutation under noninducing conditions (glucose) (YAN et al. 1991). Itis also interesting to note thatMCM2 overexpression suppresses the phenotype causedby the mcm3 mutation, and the mcm2 mcm3 double mutant is GALl REF ne nonviable (YAN et al. 1991). The goal of this work was to assess the feasibility of employing the synthetic dosage lethality phenotype for the identification of meaningful genetic interactions. This study was designed to determine the functional I- Target 1- mutationTarget specificitymutation of themethod by conditionallyoverex- pressing two known reference genes, CTFl3 and ORC6, Induce overexpression of in the context ofa large collection ofpreviously charac- "Reference" gene (galactose) terized target mutations, in this case a set of ctfand cdc mutants. We also determined the relative spectrum of interactions identified by synthetic dosage lethality (at permissive temperature) as opposed to dosage suppression (at nonpermissive temperature) in a collection of previously characterized temperature-sensitive mutants. Our results indicate that screening for synthetic dosage lethality provides a convenient and relevant approach for the identification of genes encoding functionally interacting proteins.

MATERIALSAND METHODS

Yeast strains and media: Table 1 lists the genotypes of all Vectoronly EFaene Genetic interaction strains used in this study. The chromosome transmission fi- +I + None delity (ctf) mutants tested were all isogenicderivatives of YPH278,as described by SPENCERet al. (1990), cdc strains +/ - Syntheticdosage lethality were described by LI and HERSKOWITZ(1993). Standard yeast -1 + Dosagesuppression mediaused was as described (ROSE et al. 1990). Synthetic complete (SC) media lacking leucine and containing as a -1 - None carbon source either 2% glucose (SC glucose - Leu) or 2% FIGURE1.-The synthetic dosage lethality (SDL) assay. (a) galactose and 2% raffinose (SC Gal + Raf - Leu) were used. A collection of mutants carrying potential target mutations Standard yeast transformation procedures were utilized as described (ROSE et al. 1990). Yeastwere cultured at 25" and are transformed with two plasmids under noninducing conditions. One plasmid contains the cloned wild-type copy of a shifted to other temperatures as indicated. Plasmid constructs: Overexpression was achievedusing "reference gene" under the control of an inducible GALl promoter. The second plasmid is a vector-only control. (b) constructs with inducible GALl promoters. pJL749 contains the GALl promoter driving the expression of ORC6 in the Overexpression of the reference gene is accomplished by LEU2, 2-p based vector,pRS425 (SIKORSKIand HIETER1989), streaking on galactose plates. (c) Growth of four independent as previously described (LI and HERSKOWITZ1993). pJL772 is transformants was scored at the temperatures indicated (see identical to pJL749 except that it lacks the ORC6 sequence. Table 1).Lethality (or slow growth) well dosage suppres- as as pKF88 was constructed in the expression vector p415GEU2. sion were noted. growth; -, no growth. +, p415GEU2has a 460-bp GALl promoter fragment cloned into the KpnI site, two tandem copies of the El tag sequence, noticeable phenotype in a wild-type strain, when com- inserted into theBamHI site of pRS415 (SIKORSKIand HIETER bined with decreased or alteredactivity of another gene 1989) and the URA? promoter that serves as a transcription encoding an interacting protein a(in hypomorphic mu- terminator, and was obtained from pRS314GU (R. SIKORSKI, tant strain),a lethal phenotype might result that could personal communication). The GALl promoter directs transcription from its own ATG toward the polylinker. The CTF13 be conveniently detected in a screen. In fact, several OW was cloned into this expression vector to create pKF88, cases of synthetic dosage lethality or toxicity have pre- (as described for pKF80 by DOHENYet al. 1993) and was shown viously been reported. For example, overexpression of to complement a ctfl? deletion mutation. the ORC6 gene lowers the nonpermissive temperature Synthetic dosage lethality screen (Figure 1): Strains were transformed with all four constructs: pJL772 (vector alone), spectra of replication mutants cdcdl and cdc46-1 (LI pJL749 (vector plus ORCQ, p415GEU2 (vector alone), and and HERSKOWITZ1993). Certain linker-inserted condi- pKF88 (vector plus CTFl?). Transformants were plated at 25" tional mutations in the HOP1 gene cause partial spore on SC glucose medium lacking leucine to select for cells car- inviability, andspore inviability is furtherincreased rying the plasmid. Individual transformantswere isolated and Synthetic Dosage Lethality 97

TABLE 1 List of yeast strains used in this study

~~~ Strain Genotype Source YRD501 MATaleu2-3,112 ura3-52 trpl-289 cdc28-1 LI and HERSKOWITZ(1993) YRD510 MATa leu2-3,112 ura3-52 trpl-289 cdc4 m543 MATa leu2-3,112 ura3-52 trpl-289 his3A200 cdc7-4 YRD560 MA Ta leu2-3,112 ura3-52 cdc28-4 YRD619 MATa leu2-3,112 ura3-52 trpl-289 his3A200 ade2 Pep4::TRPl cdc15-2 YRD664 MATO leu2-3,112 ura3-52 trpl-289 cdc34-2 YJL304 MATa leu2-3,112 ura3-52 tql-289 YJL 179 MATa leu2-3,112 ura3-52trpl-289 cdc46-1 YJL338 MATa leu2-3,112 ura3-52 trpl-289 cdc2-1 YJL340 MATa leu2-3,112 ura3-52 trpl-289 ade2 cdc6-1 YJL353 MATa leu2-3,112 ura3-52 tql-289 cdcl7-1 YPH278 MATa his3-A200 leu2-AI lys2-801 ura3-52 ade2-101 SPENCERet al. (1990) CFIII(CEN3.L)URA3 SUPl 1 YCTFl 0 MATa his3-A200 leu2-A1 lys2-801 ura3-52 ade2-101 CFIII(CEN3.L) URA3SUP11 ctj7-10 YCTF9 MATa his3-A200 leu2-A1 lys2-801 ura3-52 ade2-101 CFIII(CEN3.L) URA3 SUPll ctfs-9 YCTFSO MATa his3-A200 leu2-Al lys2-801 ura3-52 ade2-101 CFIII(CEN3.L) URA3 SUPll ctfl3-30 YCTF42 MATa his3-A200 leu2-Al lys2-801 ura3-52 ade2-101 CFIII(CEN3.L) URA3 SUPll ctfl4-42 YCTF61 MATa his3-A200 leu2-Al lys.2-801 ura3-52 ade2-101 CFIII(CEN3.L) URA3SUP11 ctfl7-61 YCTF26 MATa his3-A200 leu2-Al lys2-801 ura3-52 ade2-101 CFIII(CEN3.L) URA3 SUPl 1 ctfl926 YCTF58 MATa his3-A200 leu2-Al lys2-801 ura3-52 ade2-101 CFIII(CEN3.L) URA3 SUP11 ctfl958 YCTFlO3 MATa his3-A200 leu2-Al lys2-801 ura3-52 ade2-101 CFIII(CEN3.L) URA3SUPl 1 ctfl0-103 YCTFl8 MATa his3-A200 leu2-Al lys2-801 ura3-52 ade2-101 CFIII(CEN3.L) URA3 SUPl 1ctfl2-18 YCTFl22 MATa his3-A200 leu2-A1 lys2-801 ura3-52 ade2-101 CFIII(CEN3.L) URA3 SUPl 1ctfl5-122 YCTFl24 MATa his3-A200 leu2-Al lys2-801 ura3-52 ade2-101 CFIII(CEN3.L) URA3SUPll ctfl6-124

WH206 MATa his7leu2ade2 ade3 canl sap3 gall ural HARTWELL (1970) WH223 MATa his7 leu2 ade2 ade3 canl sap3 gall URA cdcl4-1 WH224 MATa his7 leu2 ade2 ade3 canl sap3 gall ural cdcl5-1 WH218 MATa his7 leu2 ade2 ade3 canl sap3 gall URAl cdcl61 WH219 MATa his7 leu2 ade2 ade3 canl sap3 gall ural cdc20-1 YPH221 MATa his7 leu2 ade2 ade3 canl sap3 gall ural cdc23-1 WM8 MATa his3-A200 leu2-Al lys2-801 ura3-52 ade2-101 W. MICHAUDand P. HIETER, cdc27-1::TRPl unpublished data purified. Four independent colonies were streaked onto SC RESULTS Gal + Raf - Leu plates and placed at 25, 30 and 37” for 3- 5 days. Other temperatures were utilized when necessary to Rationale: The basic concept of the synthetic dosage clarify a result (see Table 2). The effects of overexpression of ORC6 or CTFl3, which were induced upon growing in the lethality screen (Figure 1) involves use of an inducibly presence of galactose, were assayed at this range of tempera- overexpressed form of a cloned “reference” gene to tures so that both synthetic dosage lethality and dosage sup identify strainsbearing “target” mutations in genesen- pression could be detected. Each strain with ORC6 or CTFl3 coding potentially interacting proteins. Two reference overexpressed was analyzed specifically for lethality as compared to the same strain with vector alone. In addition, poten- genes were used:ORC6, encoding a subunit of the repli- tial dosage suppression effects were assessedfor the tempera- cation originrecognition complex (LI and HERSKOWITZ ture sensitive strains. 1993), and CTFl3, encoding a kinetochore binding pro- 98 E. S. Kroll et al.

streaking on galactose-containing media, growth was Vector CTFI3 Vector ORC6 I I directly compared for each mutant containing either overexpression plasmid or the respective vector alone. Each pair (overexpression construct/vector alone) was therefore intrinsically controlled. We used three temperatures for testing growth of transformants, 25, 30 and 37", though occasionally other temperatures were tested. If a transformant overexpressing the reference gene did not form visual colonies in the streak by 5 days on galactose, or acquired a dosagedependenttem- perature sensitivity at any temperature compared with ~$14-42 ctj-14-42 its vector alone control, we called it a synthetic dosage lethal event (+/- in Figure 1 and Table 2). Visual scoring was performed without the aid of a microscope, Vector I CTF13 Vector I ORC6 consequently no attempt was made to distinguish between early division arrest and formation of microcol- onies. The visual screen is therefore rapid and, although qualitative in nature, unambiguous. In some cases, macroscopic colonies formed containing anoverexpression construct that were noticeably smaller, on average less then half the diameter, than colonies with control vector alone. We documented these transformants as displayinga "slow growth" phenotype [ (+) in Table 21. In addition, for the ts-mutants within the set, dosage suppression was also noted, as evidenced by ~d~46-I cdc46-I rescue of viability of a ts-mutant by the reference gene FIGURE2.-Examples of the synthetic dosage lethalityphe- at anotherwise nonpermissive temperature. Results are notype. Colony growth phenotypes of ctfl4-42 and cdc46-1 listed in Table 2with specificexamples shown in Figure mutant strains overexpressing either CfF13or ORC6are compared with "vector alone" controls at 25". In each case, four 2. The genetic interactions identified are summarized independent transformants are streaked out to reveal the iso- in Figure 3. lated colony growth phenotype. The relevant genotypes are Overexpression of CTF13 : Overexpression of CTFl3 indicated below each plate. Upon overexpression of CTFI3, caused synthetic dosage lethality (indicated as +/- in the ctfl4-42 mutant strain does not form visible colonies at Table 2) in fourmutant strains asfollows: YCTF42 25", whereas the same mutant does form colonies with the vector alone control or upon overexpression of ORC6. In (ctfl4-42) at 25",YCTF61 (ctfl7-61) at 37",YCFT26 contrast, the cdc46-I mutant strain does form colonies upon (ctfl9-26) at 37", and YCTF58 (ctfl 958) at all tempera- overexpression of CTFI3 and with the vector alone control tures tested. These four mutantstrains define mutations but does not form visible colonies upon overexpression of in three genes that are known to be involved [CTFI4 ORC6 at 25". (NDCIO)] (GOHand KILMARTIN 1993;J'ANG et al. 1993), or strongly implicated [ CTFl7 (CHL4)and CVI (Do- tein (DOHENYet al. 1993). The target mutations were HEW et al. 1993; KOUPRINAet al. 1993) in kinetochore represented by a setof mutants implicated in two differ- function. ent pathways-DNA replication (cdc2,4, 6, 7,17, 34, No examples of dosage suppression of conditional 46) and chromosome segregation/mitosis [cdcl4, 15, lethality (indicated as -/+ in Table 2) were observed 16, 20, 23, 27; ctf7, 8, 12, 13, 14 (ndclo), 17(ch14), 193. upon overexpression of CTF13. However, because it is In addition, several mutants with phenotypes affecting known that CTF14 (NDCIO)encodes the110-kD subunit eitherone or both pathwayswere included as well of the kinetochore complex CBF3, we tested the effects (cdc28-1, cdc28-4, ctj15and ctfl6). Mutant strains (Table of overexpression of NDClO (CTF14) in the four mu- 1) were transformed under noninducingconditions tants listed above. NDCIO (CTF14) overexpression did with four different plasmids: overexpression constructs not cause dosage lethality in any of these strains. Inter- containing either ORC6 [pJL749 (Lr and HERSKOWITZ estingly, however, NDCIO (CTF14) overexpression was 1993)l or CTFI3 [pKF88 (DOHENYet al. 1993)] under able to dosage suppress the temperature-sensitive phe- control of CALI promoter, and the two corresponding notype of YCTF30 (ctfl3-30) at 37". vectors on which the overexpression constructs were Overexpression of ORC6: Overexpression of ORC6 based, pJL772 and p415GEU1,respectively. Trans- caused synthetic dosage lethality in five mutant strains, formants were colony-purified under noninducing con- as follows:YJL179 (cdc46-I) at 30",YJL338 (cdc2-I) at ditions (glucose-containing media). Upon induction by 30",YJL340 (cdc6-I) at 32", WH223 (cdcl4-I) at 30", Sy nthetic Dosage Lethality Dosage Synthetic 99

TABLE 2 Synthetic dosage lethality/dosage suppression

CF13 overexpression ORC6 overexpression (vector/vector + CTFl3) (vector/vector + ORC6) 25" 30" 37" 25" 30" 37" genotype 30" Relevant 25"Strain 37" 30" 25"

YRD 501 cdc28-l +/+ +/+ "- +/+ +/+ -/- YRD 510 cdc4 +/+ +/+ +/+-/- +/+ "- YRD 543 cdc7-4 +/+ +/+ -/- +/+ +/+ -/- YRD 560 cdc28-4 +/+ +/+ -/- +/+ +/+ -/- YRD 619 cdc15-2 +/+ +/+ -/- +/+ +/+ -/- YRD 664 cdc34-2 +/+ +/+ -/- +/+ +/+ -/- YJL 179 cdc46-1 +/+ +/+ -/- +/+ +/- -/- YJL304 me+ +/+ +/+ +/++/+ +/+ +/+ YJL 338 cdc2-1 +/+ +/+ -/- +/+ +/- -/- YJL 340 cdcdl" +/+ +/+ -/- +/+ +/(+I -/- YJL 353 CdCl7-1 +/+ +/+ -/- +/+ +/+ -/- YPH 278 CT2" +/+ +/++/+ +/++/+ +/+ + /+ +/+ +/+ +/+YCTFlO +/+ ctp-lo +/+ +/+ +/+ +/+ YCTF42 ctfl4-42 +/- -/- -/- +/+-/- -/- YCTF61 ctfl7-61 +/+ +/(+I +/++/- +/+ +/+ YCTF26 ctflP26 +/+ +/(+I +/++/- +/+ +/(+) YCTF58 ctflP58' +/-+/- +/- +/+ +/+ +/+ YCTFlO3 ctfl @I 03 +/+ -/- -/- +/+ -/+ -/- YCTFl8 ~$12-18 +/+ +/+ -/- +/+ +/+ -/- YCTFl22 ctf15-122 +/+ +/+ "- +/+ +/+ -/- YCTFl24 ctfl6-124 +/+ +/+ +/+-/- +/+ "- YPH206 CDC+ +/++/+ +/+ +/++/+ +/+ YPH223 cdcl4-1 +/+ +/+ -/- +/+ +/- -/- YPH224 cdcl5-1 +/+ +/+ +/+-/- +/+ -/- WH225 cdcldl +/+ +/+ -/- +/+ +/- -/- YPH2 19 cdc20-1 +/+-/- -/- +/+-/- -/- YPH221 cdc23-1 +/+ -/- -/- +/(+I -/- -/- YRS281 cdc27-1 +/+ +/+ -/- +/+ +/(+I -/- +, visible colony formation; -, absence of visible colonies; and (+), small colony size in the presence of the CTF13 or ORC6 overexpression constructs when compared to vector alone. Note that the synthetic dosage lethality phenotype,-, is emphasized here for establishing meaningful genetic interactions; however,we also include the slow growth designation,(+) , for complete- ness. As described in the text,slow growth is a qualitative and variable phenotype that refers to casesin which colonies with the overexpression construct were on average half the size of colonies with vector alone. "At 32" strain YJL340 (cdcbl) transformed with plasmid overexpressing ORC6 gene did not grow, whereas the same strain, transformed with vector alone grew normally (+/-). Strain YCTF58 (ctflP58) transformed with plasmid overexpressing ORC6 gene grew slowly at 17" as compared to the same strain transformed with vector alone (+/( +)). and WH225 (cdcl6-1) at 30". At 30°, cdc6-1 exhibited interesting to note that ctjl@103isan allele of the CDC6 only a slow growth phenotype. We therefore retested gene (we have renamed this allele cdc6-103) and that the dosage phenotype at 32" and confirmed our pre- suppression of this allele byORC6 overexpression is viously reported result (LI and HERSKOWITZ1993). Sev- opposite to the synthetic dosage lethality effect ob- eral strains exhibited a weaker, but clearly detectable served for the cdc6-I allele (see above). phenotype [indicated as +/ (+) in Table 21, evidenced by slow growth described above. Overexpression of as DISCUSSION ORC6 caused a synthetic slow growth phenotypein three strains asfollows: YCTF26 (ctflP26)at 37", Here we describe a broadly applicable visual second- WH221 (cdc23-I) at 25", and YRS281 (cdc27-I) at 30". ary screen thatemploys a phenotype we term "synthetic In addition, we observed one example of dosage sup- dosage lethality" resulting fromthe overexpression of a pression. The temperaturesensitivity ofYCTF103 (ctfl0- particular reference geneon thebackground of various 103) at 30" was suppressed by ORC6 overexpression. At target mutations. We attempted toassess the usefulness 37", overexpression of ORC6 was unable to suppress of this phenomenon for the identification of specific the temperature-sensitive phenotype of YCTF103. It is genetic interactionsby devising a test screen that would 100 E. S. Kroll et al.

dosage synthetic dosage the 50-kD subunit of the origin recognition complex of supression lethality/sensitivity the origin recognition complex, a six protein complex that binds the core consensus sequence of yeast origins cdc6-1 (cff IO) cdc6 - 1 (cff 7 0) (BELLand STILLMAN1992) and participates in the initia- 7 replication Cdc46- genes tion of DNA replication (FOX et al. 1995; LUNGet al. 1995). Overexpression of the ORC6 gene uncovered cd~2-7 five cases of the synthetic dosage lethality phenotype. Three out of five mutations identified correspond to cdcl4-1 genes with a role in DNA replication in Saccharomyces cermisiae, WC46, WC6, and cDC2. WC46is oneof the \ family of five MCM genes (TYE 1994), which were first cdcl6-1 segregation identified by the phenotype of impaired maintenance genes Cfff 9-26,-58 of plasmid minichromosomes in yeast. These genes are CTF13 thought to help preparecells earlyin G1 for the proper Cff14-42(ndc70, Cbf2) initiation of replication at the Gl/S boundary (Su et al. .cff77-61 (ch14) 1995; WANGand Lr 1995). CDC6is also required for FIGURE 3.-Schematic representation of genetic interac- plasmid maintenance and the onset of chromosomal tions between overexpressed ORCG and CTFl3 as reference replication (BUENOand RUSSELL1992; HOGANand genes and the subset of target mutants. Only those mutants KOSHLAND1992). Our observation of synthetic dosage that were positive in synthetic dosage lethalityor dosage suppression screens are shown. The positive target mutants are interactions between ORC6 and both CDC6 and CBC46 divided into two groups in respect of the pathway that they further implicates these genes in the process of replica- define or are implicated. tion initiation. It is particularly satisfying and reassuring that the genetic connections revealed by this synthetic allow us to check the specificity of a genetic interaction. dosage lethality screen have recently been corroborated We reasoned that this screen could potentially reveal a by observations of synthetic lethality and dosage sup- genetic interaction between a cloned reference gene pression among the genes encoding Orc2p, Orc5p, the under study and a subset of target mutations repre- Mcm proteins and the Cdc6 protein (Loo et al. 1995). sented in established or newly generated mutant collec- It is also noteworthy that while the overexpression of tions. The CTF13 gene is an essential gene required for ORC6 reported in this paper was toxic in combination chromosome segregation in yeast. It encodes the 58-kD with the cdc6-1 allele, the same operation suppressed subunit of CBF3, the multiprotein complex that binds the temperaturesensitivegrowth defect in the combina- to centromere DNA (LECHNER and CARBON1991; DO- tion with the cdc6-IO? allele. This allele specificity of HEW et al. 1993). Overexpression of the CTFI3 gene the overexpression phenotype suggests that systematic led to lethality in an ndcl0 (ctjl4-42) mutant, which screening fordosage lethality, as well asdosage suppres- was previously shown to exhibit perturbed kinetochore sion, is likely to increase the chance of identifying a integrity (DOHENYet al. 1993; GOH and KILMARTIN particular pair of interacting genes. The CDC2 gene 1993). ADC10 (CBF2) has been shown to encode the product is the catalytic subunit of DNA polymerase S, 110-kD subunit of the CBF3 complex (GOHand KILMAR- one of the essential polymerases at the eukaryotic repli- TIN 1993; JIANG et al. 1993).Furthermore, overex- cation fork. The observation of synthetic dosage lethal- pression of hrDCl0 led to suppression of temperature ity between CDC2 and ORC6 encourages further investi- sensitivity of a ~$3-30mutant. Thus, the synthetic le- gation into the interactionsof ORC withthe replicating thality phenotype of ctjl4-42 allele upon overexpression polymerases. of CTFI3 appears to define a meaningful genetic inter- Mutants carrying cdcl4-1, or cdcI6-I, and, to a lesser action. Overexpression ofCTF13 also led to lethality extent, cdc23-I, or cdc27-1 mutations were also sensitive in mutants bearing mutations in two other loci, CTFI 7 to overexpression of ORC6. The cdc14-1 mutant arrests (CHZd4) and CTF19. These mutations havepreviously in late anaphase/telophase. Interestingly, as shown for been shown to relieve a CEN-mediated transcription the cdc6-1 mutant, the instability of a centromeric plas- block in uivo (DOHENYet nl. 1993), suggesting a role in mid in the cdcl4-l mutant could also be partially sup- kinetochore function. In addition,ch14-I, another allele pressed by the addition of several origins of replication of the CTFl7gene, has been shown to allowstable main- on that plasmid (HOGAN andKOSHLAND 1992). This tenance of a dicentric plasmid (KOUPRINAet al. 1993). phenomenon supports the validity of the observation Since the genetic evidence reveals involvement of these that cdcl4-l and ORC6 genetically interact by the syn- genes in kinetochore function in yeast, we conclude thetic dosage lethality criterion. The CDC16, CDC23and that the synthetic dosage lethal interactions with the WC27gene products are components of a spindleasso- CTF13 gene, as assayed in this study, are meaningful. ciated multiprotein complex essential for transition The ORC6gene (LI and HERSKOWITZ1993) encodes from metaphase to anaphase (LAMB et al. 1994; TUGEN- D osage Lethality Synthetic Dosage 101

DREICH et al. 1995). The observation that components screen is that it does not require isolation of a mutation involved in late mitosis genetically interact with a pro- in a reference gene as a means to learn about its func- tein implicated in the initiation of DNA replication is tion or as astarting point in a genetic interaction in accordancewith our growing awareness that “preini- screen. Thus, thebasic synthetic dosage lethality screen tiation” events occurring late in mitosis help prepare should be readily adaptable to a variety ofexperimental the cell for replication in the following cell cycle (re- organisms or cultured cells. Furthermore, this screen viewed in Su et al. 1995; WANGand LI 1995). Although can be helpful when a mutation in a gene of interest the genetic interactionswe observe may indicate simply does not produce any noticeable phenotype or when a that a smooth and unperturbed transition into G1 is heterologous gene is being studied. Other applications required for the proper executionof these preinitiation include employing the synthetic dosage lethality pheno- events, they also raise the possibility of a more direct type in a screen of random mutants, e.g., as a primary link between the machinery controlling mitosis and the mutagenesis screen. For this purpose, the screen could machinery governing replication initiation. The CDC14 conceivably employ both a synthetic dosage lethality gene, for example, encodesputative a phosphotyrosine screen (by plating under noninducing conditions and phosphatase. In addition to its potential mitotic sub- then shifting to inducingconditions) and overex- strates, we should now consider ORC and otherreplica- pression suppression screen (by plating under inducing tion components to be among its possible targets. conditions andthen shifting to noninducingcondi- Since most (21 out of 24) of the target mutants had tion), in parallel. a conditional phenotype (temperature sensitivity), we In general, interpretation of genetic interactionsulti- could also score for anoverexpression suppression phe- mately requires direct biochemical experiments. How- notype, which is an established method for identifica- ever, the simplicity and high throughput of genetic in- tion of potential interactors. Overall, only one mutant teraction screening employing the synthetic dosage strain was suppressed by ORC6 overexpression (cdc6- lethality phenotype may direct further analysis of such 103) and nomutants were suppressed by CTF13 overex- interactions by other, more specific means. The syn- pression [although it was subsequently shown that thetic dosage lethality screen is yet another useful ADClO (CTFZ4)overexpression could suppress the tem- methodfor broadening the pool of potentialinter- perature sensitivity caused by the ctfl3-30 mutation]. In actors and thus further facilitating the characterization contrast, we identified four synthetic dosage lethality of a protein’s function. positives for the CTF13 gene and five positives for the We thank SUSANMICHAELIS andJAsPER RINE for helpful comments ORC6 gene. Thus, thesynthetic dosage lethality screen on the manuscript and members of our labs for helpful discussions. may in generalcast a wider net for identifymg potential P.H. was supported in part by National Institutes of Health grant CA- genetic interactions. 16519, and J.J.L. was supported in part by the Lucille P.Markey Several scenarios are conceivable to explain how Charitable Trust and the Rita Allen Foundation. overexpression of a protein couldlead to a detrimental effect if an interacting protein is mutated. First, if a LITERATURE CITED target mutation is a partial-loss-of-function mutation, BELL,S. P., and B. STII.I.MAN,1992 ATP-dependent recognition of the overexpressed protein may titrate the rest of the eukaryotic origins of DNA replication by a multiprotein complex. activity away or moreeffectively compete for a common Nature 357: 128-134. binding site. Such a phenomenon might be due to er- BUENO,A., and P. 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