INVESTIGATION Genome Rearrangements Caused by Depletion of Essential DNA Replication Prote ins in Saccharomyces cerevisiae Edith Cheng,* Jessica A. Vaisica, ** Jiongwen Ou,* Anastasia Baryshnikova,' Yong Lu, Frederick P. Roth,4.* and Grant W. Brown*.* Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada, 'Donnelly Centre for Cellular and Biomolecular Research, Toronto, Ontario M5S 3E1, Canada, *Banting and Best Department of Medical Research and Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5G 1L6, Canada, SDepartment of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, and *"Samuel Lunenfeld Research Institute, Mt. Sinai Hospital, Toronto, Ontario M5G 1X5, Canada ABSTRACT Genetic screens of the collection of ~4500 deletion mutants in Saccharomyces cerevisiae have identified the cohort of nonessential genes that promote maintenance of genome integrity. Here we probe the role of essential genes needed for genome stability. To this end, we screened 217 tetracycline-regulated promoter alleles of essential genes and identified 47 genes whose depletion results in spontaneous DNA damage. We further showed that 92 of these 217 essential genes have a role in suppressing chromosome rearrangements. We identified a core set of 15 genes involved in DNA replication that are critical in preventing both spontaneous DNA damage and genome rearrangements. Mapping, classification, and analysis of rearrangement breakpoints indicated that yeast fragile sites, Ty retrotransposons, tRNA genes, early origins features at breakpoints when essential replication genes that suppress chromosome rearrangements are down regulated. We propose mechanisms by which depletion of essential replication proteins can lead to double-stranded DNA breaks near these features, which are subsequently repaired by homologous recombination at repeated elements replication, and replication termination sites are common CCURATE transmission of the genome is essential for normal cell growth and survival. As such, cells have developed elaborate mechanisms to prevent errors in repli cation and to respond to spontaneous DNA damage that can lead to genomic instability (Kolodner et al. 2002; Branzei and Foiani 2007, 2009, 2010; Harper and Elledge 2007; Cimprich and Cortez 2008). The failure to repair the genome in an error-free manner can result in chromosome abnormal- ities that underlie many human diseases, including cancers (Kolodner et al. 2002; McKinnon and Caldecott 2007; Aguilera and Gomez-Gonzalez 2008). Therefore, defining the genes that contribute to genome maintenance will be useful in understanding disease development and in designing new strategies for therapeutics. However, to date, a comprehensive curation of genes that function to suppress genome instabil- ity is incomplete. Yeast is an ideal model for genomic studies due to the conservation of gene functions and biological pathways be- tween yeast and humans. Phenotypic screens conducted with the Saccharomyces cerevisiae nonessential gene deletion collec- tion (Giaever et al. 2002) have aided in the annotation and functional characterization of nonessential genes involved in the suppression of spontaneous DNA damage (Huang et al. 2003; Huang and Kolodner 2005; Shor et al. 2005; Alvaro et al. 2007) and in the suppression of spontaneous chromosome rearrangements (Smith et al. 2004; Yuen et al. 2007; Andersen et al 2008). However, since the deletion of essential genes causes lethality, similar genome-wide screening approaches to identify the complete set of genes that suppress spontaneous DNA damage and chromosome rearrangements require collec- tions of conditional alleles of essential genes. Systematic collections of conditional alleles have been generated in several ways, including the replacement of native promoters with a tetracycline-regulated promoter (Mnaimneh Copyright 2012 by the Genetics Society of America doi: 10.1534/genetics.112.141051 Manuscript received April 9, 2012; accepted for publication May 28, 2012 Supporting information is available online at http//www.genetics.org/content suppl/2012/06/05/genetics. 112.141051 DC1 'Corresponding author: Donnelly Centre for Cellular and Biomolecular Research, 160 College St, Toronto, ON MSS 3E1, Canada. E-mail: grant.brown@utoronto.ca Genetics, Vol. 192, 147-160 September 2012 147 et al. 2004; Yu et al. 2006), destabilization of target gene mRNAs through the insertion of a selectable marker in the 3'-UTR of essential genes (Schuldiner et al. 2005), systematic addition of a heat-inducible degron to the amino terminus of the protein product (Labib et al. 2000), systematic generation of novel temperature-sensitive alleles (Ben-Aroya et al 2008), and systematic integration of existing temperature- sensitive alleles (Li et al. 2011). Despite the availability of several essential gene collections, no one collection is com- plete, suggesting that complementary approaches using a number of screening strategies and multiple types of con- ditional alleles will be necessary to identify all of the es- sential genes that function to suppress genomic instability Here we describe a series of screens to identify essential genes that function to suppress genome instability, using the collection of tetracycline-regulated promoter replacement alleles (Tet alleles) of essential genes (Mnaimneh et al 2004). We screened 217 Tet alleles of essential genes whose depletion caused accumulation in S or G2 phases of the cell cycle (Yu et al. 2006) and identified 47 with elevated levels of spontaneous DNA damage. A second screen performed with the same Tet alleles identified 92 essential genes that suppress the formation of chromosome rearrangements, whole chromosome deletions, and gene conversions. We quantified the levels of each type of mutation in 15 strains that exhibited both elevated levels of spontaneous DNA damage and chro- mosome rearrangements following the depletion of an essen- tial gene. Mapping of rearrangement breakpoints in seven representative mutants from this set revealed several unique previously described for Rad52-YFP (Lisby et al. 2004; Lisby and Rothstein 2004; Chang et al. 2005). Ddc2 foci were quantified in at least 100 cells for each strain. Ddc2 foci in wild-type cells were analyzed four times and used to calcu- late a standard deviation. Tet allele strains that had Ddc2 foci levels that were at least three standard deviations greater than wild type were scored as positive. illegitimate mating assays Tet allele strains and the R1158 wild-type strain were grown in parallel for 24 hr on YPD solid media either containing or lacking 10 Hg/ml of doxycycline. A standard mating assay was performed with tester strains MCY13 (MATa , legiti- mate mating) and MCY14 (MATa, illegitimate mating) on the same media conditions that the strains were grown. Diploids were isolated by replica plating on minimal media. In the quantitative form of this mating assay, Tet allele strains and R1158 wild-type strain were grown in parallel for 24 hr in YPD liquid media containing or lacking 10 ug/ml doxycycline. Strains were mixed with fivefold excess of MCY13, MCY14, or 1225a (MATa his4 thr4) tester strains and plated on YPD solid media. After 5 hr, cells were col- lected, resuspended in water, and plated on diploid selection media. Independent illegitimate diploids were isolated after the mating of the Tet allele strains with the 1225a tester strain. For each mating experiment, 100 diploids were iso- lated and tested for their ability to grow in the presence or absence of histidine or threonine. This assay was repeated two times. Viability of each strain following growth in doxycycline was confirmed by plating on YPD. Only MCM7 (10 %), NUF2 (30%), and UBC9 (50% ) had <100% viability following growth in doxycycline. rearrangement structures. Sequence features, including Ty ret- rotransposons and DNA replication origins and termination zones, correlated with the rearrangements identified. We pro- pose a central role for DNA replication proteins in suppressing the formation of chromosome breaks that promote chromo- Array comparative genome hybridization Genomic DNA was extracted (Qiagen) from independent illegitimate diploids and wild-type diploids isolated from the mating assay. CGH on a microarray was performed as previously described (Dion and Brown 2009) using S. cerevisiae whole genome tiling microarrays (Affymetrix). Signal in tensities of the experimental and wild-type control sam- ples were normalized and compared using tiling analysis software (Affymetrix). Genomic patterns were mapped and analyzed using the integrated genome browser software (Affymetrix). some rearrangements. Materials and Methods Yeast strains and media Tet allele strains were constructed as described previously (Mnaimneh et al. 2004). The genotype of the wild-type Tet allele strain, R1158, is MATa URA3::CMV-tTA his341 leu240 met1540. Using standard genetic methods, 217 MATa Tet allele strains were engineered to contain YFP-Ddc2 marked with a nourseothricin (Nat) resistance gene. Gen otypes for strains used in this study are listed in Table S6. The essential genes that were studied are listed in Table S1 and Table S2. Standard yeast media and growth conditions were used unless otherwise specified (Sherman 1991). CHEF gel electrophoresis and Southern blot analysis Contour-clamped homogenous electric field (CHEF) gels were used to examine intact chromosomes of illegitimate diploids isolated from the mating assay. CHEF gel analysis was performed as described previously (Desany et al 1998). A 1.2 % agarose gel was run at 8 V/cm using pulse times of 120 sec for 30 hr at 14° in 0.5x TBE buffer. PCR- Fluorescence microscopy Tet allele strains were grown in YPD liquid media at 30°, Samples were divided into two cultures and grown in par allel in the presence and absence of 10 Hg/ml doxycycline for 4 additional hours at 23°. Intracellular localization of Ddc2-YFP was determined by fluorescence microscopy as purified fragments were radio labeled by random priming (Stratagene) and used as hybridization probes for Southern blot analysis. PCR primers designed for probe construction are listed in Table S7. E. Cheng et al 148