Copyright 0 1993 by the Genetics Society of America A Novel Mutation in DNA Topoisomerase I of Yeast Causes DNA Damage and RAD9-Dependent Cell Cycle Arrest Nikki A. Levin,*91Mary-Ann Bjornstit and Gerald R. Fink*" *Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142, and Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, and +Department $Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, Pennsylvania, 19107-5541 Manuscript received October 9, 1992 Accepted for publication December 10, 1992 ABSTRACT DNA topoisomerases, enzymes that alter the superhelicity of DNA, have been implicated in such critical cellular functions as transcription, DNA replication, and recombination. In the yeast Saccha- romyces cerevisiae, a null mutation in the gene encoding topoisomeraseI (TOPl) causes elevated levels of mitotic recombination in the ribosomal DNA (rDNA), but has little effect on growth. We have isolated a missense mutation in TOPl that causes mitotic hyper-recombination notonly in the rDNA, but also at other loci,in addition to causing a number of other unexpectedphenotypes. This topoisomerase I mutation (topl-103) causes slow growth, constitutive expression of DNA damage- inducible genes, and inviability inthe absenceof the double-strand break repair system. Overexpres- sion of topl-103 causes RAD9-dependent cell cycle arrest in GP.We show that the Topl-103 enzyme nicks DNA in vitro, suggesting that it damages DNA directly. We propose that Topl-103 mimics the action of wild-type topoisomerase I in the presence of the anti-tumordrug, camptothecin. YPE I DNA topoisomerases remove superhelical (CHRISTMAN,DIETRICH and FINK 1988). Deletion of T turns from DNA by making a transient single- TOPl in combination with a conditional mutation in stranded break in the DNA backbone and then acting the TOP2 gene, which encodes the yeast type I1 to- as a swivel point about which the strands may rotate poisomerase, results in recombinational excision of [Figure 1; reviewed in CHAMPOUX (1990)l. A major rDNA circles (KIM and WANG 1989).The association role of topoisomerases is believed to be reduction of between topoisomerase mutations and increased re- superhelicity that is generated by such processes as combination levels is also observed for a third yeast transcription and DNA replication (LIU and WANG topoisomerase gene, TOP3, which has been shown to 1987). In fact, a number of studies have implicated elevate mitotic recombination between delta ele- topoisomerases in these important cellular functions ments, which are the terminal repeats of the retro- [reviewed in YANACIDAand STERNCLANZ (1990)l. In transposon Ty (WALLISet al. 1989). the yeast Saccharomyces cereuisiae, topoisomerase mu- In order to explore the role of topoisomerase I in tants have been shown to build up supercoils on highly suppressing recombination, we have isolated new mu- transcribed plasmids (BRILLand STERNCLANZ 1988; tant alleles of the yeast TOPl gene.Among these GIAEVERand WANC1988), andtopoisomerase activity mutants, we identified one allele, designated topl-103, has been shown to be required for DNA replication that has a number of unexpected phenotypes. topl- and transcription (GOTOand WANG 1985). 103 causes an extremely high level of mitotic rDNA The yeast TOPl gene is a member of the highly recombination, giving an rDNA recombination rate conserved group of eukaryotic type I topoisomerases approximately 250-fold higher than wild type and 10- (GOTOand WANG1985; THRASHet al. 1985). Despite fold higher than that found in a topl deletion strain. the evidence for involvement of topoisomerase I in topl-103 also elevates recombination atother loci, essential functions such as transcription and DNA unlike previously studied mutations in TOPl or TOP2. replication, TOPl is notrequired for growth, and The topl-103 mutant grows slowly and expresses deletion of this gene results in only a modest growth DNA damage-induciblegenes constitutively. The defect. Deletion of TOPI does, however, cause a large topl-103 mutantrequires an intact double-strand increase in mitotic recombination in the tandemly break repair system for viability. In addition, overex- repeated ribosomal DNA arrays, a chromosomal do- pression of topl-103 causes cell cycle arrest in GS that main that is normally suppressed for recombination is dependent on the RAD9 gene product, a sensor of DNA damage. In the absence of RAD9, cells overex- ' Present address: Division of Molecular Cytometry, Department of Lab- pressing topl-103 traverse the cell cycle without re- oratory Medicine, MCEi-230, University of California, San Francisco, Cali- fornia94143. pairing DNA damage, resulting in rapid cell death. To whom correspondence should be addressed. We show here that Topl-103 enzyme nicks DNA Genetics 133: 799-814 (April, 1993) 800 N. A. Levin, M.-A. Bjornsti and G. R. Fink A FIGURE 1.-Model of eukaryotic topoisomerase I mechanism. (A) Topoisomerase I binds to super- coiledDNA with high affinity in a noncovalent interaction. The enzyme makes a single-stranded nick in DNA, simultaneously becoming covalently bound to the 3‘ end of the broken DNA strand. This topoisomerase-DNA intermediate is called the “cleavable complex.” The enzyme provides a Cleavable complex swivel about which the broken DNA strand may rotate to relieve superhelical tension. The enzyme then religates the broken DNA strands, releasing B itself from the DNA. After multiple rounds of nicking and resealing, the supercoiled DNA is con- verted to the completely relaxed form. (B) The anti-tumor drug camptothecin (CPT) inhibits the CPT religation step of the reaction, resulting in accu- + mulation of the cleavable complex. (After LW Cleavable complex 1989.) in vitro, suggesting that it can damage DNA directly. personal communication). pGAL::lacZ (pLR1 A23) is a 2-pm We propose that Topl-103mimics the actionof wild- plasmid containing URA3 and GALl-promoted lac2 (WEST, YOCUM and PTASHNE1984). pWE3 is a CEN-ARS plasmid type topoisomeraseI in the presence the anti-tumor of carrying URA3 and GALI-promoted TOPI(ENG et at. 1988). drug, camptothecin. This drugcauses topoisomerase pNKY3Ois an integrative plasmid for creating a I to become trapped during the relaxation reaction as rad5O::URA3 disruption (E. ALANIand N. KLECKNER,per- part of a covalent enzyme-DNA complex [Figure 1; sonal communication).pRR330 is an integrative plasmid for reviewed in Liu (1989,1990)l. The enzyme-bound creating a rad9::URA3 disruption (SCHIESTLet al. 1989). CB17 consists of the 3.8-kb HindIIl fragment of TOPl nicks generated camptothecin result in hyper-re- by filled in with Klenow and cloned into the SmaI site of YEp24 combination, induction of DNA damage responses, (M. F. CHRISTMAN,personal communication). CB32 and and cell cycle arrest, in close parallel to the effects of CB39 are integrative vectors carrying rDNA sequences topi-103. marked by URA3 and HIS3, respectively (CHRISTMAN,DIE- TRICH and FINK1988). CB45 carriesADE2 on a 6-kbBamH1 fragment cloned into the BamHI site of pBR322 and the MATERIALS AND METHODS 6.4-kb HindIII fragment of rDNA cloned into the HindIII site. CB51 and CB52 contain the 3.8-kb HindIIl fragment Media, strains and genetic techniques: Strains are de- of TOPI and of topl-727, respectively,cloned into the scribed Table l. YPD, SD, SC and 5-FOA media are as in HindIII site of YCp405. CB107 is the same as pWE3, but described in GUTHRIEand FINK(1 99 1). “Sectoring plates” with topl-727 replacing TOPl. are SD supplemented with 75 p~ adenine hemisulfate, 75 pNL2 contains the 3.2-kb HindIII-NruI fragment of p~ histidine, 400 p~ tryptophan and 1.7 mM leucine. The levelsof adenine and histidine are lower than those in TOPIcloned into the 7.0-kb HindIII-NruI fragment of standard media in order toachieve darker red coloration of YCp50. pNL2-102, pNL2-103 and pNL2-106 are pNL2 ade2 mutants. Yeast matings, tetrad dissection, lithium ace- containing the mutant alleles topl-102, topl-103 and topl- tate transformation and replica-plating are as in GUTHRIE 106, respectively, in place of TOPI. pNL8 is an integrative and FINK(1 99 1). vector consisting of the 4.3-kb HindIII-ApaI fragment of Plasmids: YCp50is a CEN-ARS vector marked with URA3 pNL2-103 cloned into the 3.5-kb HindIII-ApaI fragment (ROSE et al. 1987). YCp405 is a CEN-ARS vector marked of YIp5.pNL14 is a CEN-ARS plasmid consistingof the 2.6- with LYS2 (MA et al. 1987). YEp24 is a 2-pm vector marked kb MluI-NsiI fragment of topl-I03 from pNL2-103 cloned with URA3 (BOTSTEINet al. 1979). YIp5 is an integrative into the 12.8-kb MluI-NsiI fragment of CB51. pNL30 is a vector carrying URA3 (STRUHLet al. 1979). YIp357r is an CEN-ARS plasmid containing the 2.3-kb Bsu36I fragment integrative vector carrying the URA3, amp’ and lac2 genes of pNL14 cloned into the9.1-kbBsu36I fragment of pWE3 (MYERSet al. 1986). pRS303 and pRS305 are integrative to generate CALI-promoted topl-103. pNL46 is an integra- vectors carrying HIS3 and LEU2, respectively (SIKORSKY tive plasmid marked with LEU2 for fusing the GALl pro- and HIETER1989). moter to alleles of TOPI, consisting of the 2.0-kb EagI- pCT80ATOP is an integrative plasmid used for creating EcoRV fragment of pNL3O cloned into the 5.5-kb EagI- a topl-7::LEU2 disruption (THRASHet al. 1985). pSR 16 and SmaI fragment of pRS305. pNL48 is an integrative plasmid pSR18 are integrative plasmids marked with URA3 for gen- for marking the rDNA with LYS2, consisting of a 2.8-kb erating lac2 fusions to DIN1 and DIN3, respectively (RUBY EcoRI fragment ofrDNA cloned into the EcoRI siteof and SZOSTAK1985). pR12Sclig2 is an ARS plasmid carrying pUC9 carrying LYS2. pNL5 1 is a CEN-ARS plasmid carrying URA3 and CDC9 (BARKER and JOHNSTON 1983). URA3 and topl-103,727 made by cloning the 865-bp Sphl YEpl3(RAD52) is a 2-pmplasmid carrying LEU2 and fragment of CB52 into the 9.3-kb SphI fragment of pNL2- RAD52 (SCHILDet al.