Genome rearrangements caused by depletion of essential DNA replication proteins in Saccharomyces cerevisiae

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 of replication, and replication termination sites are common features at breakpoints when essential replication genes that suppress chromosome rearrangements are downregulated. 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.

Figure 1

Depletion of yeast essential genes results in elevated levels of spontaneous Ddc2 foci formation. (A) A total of 217 Tet alleles that express Ddc2-YFP and display a G2/M or S phase cell cycle arrest phenotype were grown in the presence of doxycycline (10 μg/ml) for 4 hr to inhibit the transcription of each essential gene. Representative DIC and YFP images are shown for the wild-type, DPB11 and NSE1 strains. Ddc2-YFP foci are indicated with white arrows. (B) The percentage of cells with Ddc2-YFP foci is plotted for 47 Tet alleles that showed an increase in Ddc2 foci of at least three standard deviations above the average observed in wild type. Bars are shaded according to the GO process annotation of each gene of interest.

Figure 2

Depletion of yeast essential genes results in elevated levels of illegitimate mating. (A) MATα Tet alleles were grown on YPD or YPD containing doxycycline (10 μg/ml) for 24 hr and a standard mating test was performed using MATα and MATa tester strains. Representative images of strains with elevated levels of illegitimate diploid formation following growth in doxycycline are shown. (B) The resulting number of illegitimate diploid colonies that grew without doxycycline treatment was subtracted from the number that grew with doxycycline treatment and was used to subcategorize the strains into four groups. For each group, the distribution of percentage of budded cells with Ddc2-YFP foci was plotted. Bold lines represent the median values, boxes represent the upper and lower quartiles, whiskers represent 1.5 times the interquartile range, and outliers are indicated by circles. (C) Comparison of Tet alleles with elevated levels of Ddc2 foci and >10 illegitimate mating diploid colonies.

Figure 3

Classification of rearrangement events that lead to illegitimate mating. (A) Schematic diagram of the three expected classes of genetic events resulting in illegitimate mating. Using diagnostic selection media, mutations in the MAT locus, whole chromosome III loss, and loss of the right arm of chromosome III can be distinguished as class 1, 2, and 3 genetic events, respectively (Lemoine et al. 2005, 2008). (B) We classified ∼200 illegitimate diploids for each strain. The frequencies of the three classes of rearrangements are plotted for the 15 strains with the most elevated levels of illegitimate mating. (C) Ratios of the three classes of rearrangements are plotted for the indicated strains.

Figure4

Comparative genome hybridization microarray analysis of class 3 illegitimate diploids. Genomic DNA was isolated from class 3 (chromosome III arm loss) illegitimate diploids and hybridized to a Saccharomyces cerevisiae whole genome tiling microarray to identify copy number variations. In each histogram, the y-axis represents log2 ratios of probe signal intensities, comparing the indicated strain to a legitimate MATa/α diploid, and the x-axis represents chromosome coordinates. Black arrows indicate breakpoint locations on each chromosome, black circles represent the locations of centromeres, and the chromosome number is indicated to the right of each histogram. A representative histogram for each of the major types of rearrangements observed is shown. (A) Class 3-1 diploid, in which no copy number variation of chromosome III was evident. (B) Class 3-2 diploids have a loss of sequence (red) from the right arm of chromosome III and duplication of sequences (blue) from chromosome XV. (C) Class 3-3 diploids have an amplification of the left arm sequence and a deletion of the right arm sequence of chromosome III. (D) Class 3-4 diploids have a loss of sequence from the right arm of chromosome III without copy number variation on nonhomologous chromosomes. (E) Class 3-5 diploids exhibit a loss of sequence from the right arm of chromosome III and loss of sequence from the right arm of chromosome V.

Figure 5

Mechanisms of repair in replication deficient mutants. (A) Schematic of boundaries of rearrangement that occur on chromosome III of replication mutants are shown. (B) Following a double stranded break and resection to Ty retrotransposons (arrows) or δ long terminal repeats (triangles), chromosome fragments can be repaired in illegitimate diploids through several mechanisms. Class 3-1: Chromosome III is repaired by BIR using the homologous chromosome of the tester strain. Class 3-2: Ectopic break-induced replication (BIR) mediated by strand invasion at a Ty retrotransposon on a nonhomologous chromosome XV of the tester strain (gray) yields nonreciprocal translocations. Class 3-3: Ectopic BIR involving strand invasion at a different locus of chromosome III results in shortened fragments of chromosome III with two left arms. Class 3-4: Chromosome III fragments are directly repaired by telomere acquisition. Class 3-5: Chromosome fusions can be created through single stranded annealing (SSA) of chromosome III and chromosome V fragments with boundaries at Ty retrotransposons or through a BIR and half-crossover event.

Figure 6

Southern blot confirmation of chromosome rearrangements predicted in microarray analysis. (A) Intact chromosomal DNA was isolated from class 3 (chromosome arm loss) illegitimate diploids. Genomic DNA was separated on a contour-clamped homogenous electric field (CHEF) gel and chromosome III was detected through hybridization with a radio-labeled probe specific to the left arm of chromosome III. Smaller chromosome III fragments were also detected in samples with chromosome rearrangements. Representative Southern blots for a wild-type diploid, classes 3-3, 3-4, and 3-1 diploids of the indicated strains are shown, respectively. (B) Representative Southern blots of a wild-type and a class 3-2 illegitimate diploid. Chromosomes on the left and right were detected with probes for chromosomes III and XV, respectively. In addition to chromosome III and XV, a nonreciprocal translocation (nrt) was visualized. (C) Representative Southern blot of a wild-type and a class 3-5 illegitimate diploid. Chromosomes on the left and right were detected with probes for chromosome III and V, respectively. In addition to chromosomes III and V, a chromosome fusion (fus) event was detected.

Figure 7

Mechanisms by which genome instability occurs in replication-deficient mutants. (A) tRNA genes and replication forks are clustered in proximity to Ty retrotransposons. The transcription machinery creates obstacles for replication fork progression and could lead to DSB formation and resection to the repeated elements. (B) Secondary structure formation involving repeated Ty retrotransposons (arrows) on the lagging strand causes replication fork stalling, subsequent replisome dissociation, and the formation of double stranded breaks (DSBs). (C) Failure to resolve termination structures at converging replication forks that flank Ty retrotransposons is a potential source of DSBs.