ABSTRACT: Genomic instability is a common feature found in cancer cells. Accordingly, many tumor suppressor genes identified in familiar cancer syndromes are involved in the maintenance of the stability of the genome during every cell division, and are commonly referred to as caretakers. Inactivating mutations and epigenetic silencing of caretakers are thought to be the most important mechanism that explains cancer-related genome instability. However, little is known of whether transient inactivation of caretaker proteins could trigger genome instability and, if so, what types of instability would occur. In this work, we show that a brief and reversible inactivation, during just one cell cycle, of the key phosphatase Cdc14 in the model organism Saccharomyces cerevisiae is enough to result in diploid cells with multiple gross chromosomal rearrangements and changes in ploidy. Interestingly, we observed that such transient inactivation yields a characteristic fingerprint whereby trisomies are often found in small-sized chromosomes and gross chromosome rearrangements, often associated with concomitant loss of heterozygosity (LOH), are mainly detected on the rDNA-bearing chromosome XII. Taking into account the key role of Cdc14 in preventing anaphase bridges, resetting replication origins and controlling spindle dynamics in a well-defined window within anaphase, we speculate that its transient inactivation causes cells to go through a single mitotic catastrophe with irreversible consequences for the genome stability of the progeny. The details of these experiments are in press in Genetics (Oliver Quevedo, Cristina Ramos-Pérez, Thomas D. Petes, and Félix Machín) but will be described briefly below. The diploid S. cerevisiae strain FM1468 is homozygous for a temperature-sensitive allele of CDC14 which encodes a phosphatase that is important in regulating the cell cycle. The strain was constructed by mating cdc14-1 derivatives of two sequence-diverged haploids, one isogenic with W303-1A (JSC12) and one isogenic with YJM789. The resulting diploid is heterozygous for about 55,000 SNPs (St. Charles, J. and Petes, T. (2013) PLoS Genetics 9: e1003434; PMID 23593029). In addition, the strain is homozygous for the ade2-1 ochre mutation and heterozygous for an insertion of SUP4-o, a gene encoded a tRNA ochre suppressor. The SUP4-o gene is located near the right telomere of chromosome IV and is inserted on the YJM789-derived homolog. The FM1468 strain forms pink colonies because a single copy of SUP4-o partially suppresses the red colony phenotype associated with the ade2 mutation. In the wild-type genetic background, a mitotic crossover between SUP4-o and the centromere of IV produces a red/white sectored colony with the red sector containing no copies of SUP4-o and the white sector containing two copies of SUP4-o (St. Charles and Petes, 2013). To map the position of the crossover and to examine recombination events resulting in loss of heterozygosity (LOH) throughout the genome, we used SNP-specific microarrays (St. Charles et al., 2012; Genetics 190: 1267-1284; PMID 22267500). These arrays contained about 50,000 oligonucleotides, allowing us to monitor LOH at about 13,000 positions in the genome. Each of these single-nucleotide polymorphism (SNP) was represented by four 25-base nucleotides, two for the Watson and Crick strands of the YJM789-derived SNP and two for the Watson and Crick strands of the W303-1A-derived SNP. The sequences and locations of each of these oligonucleotides has been published (St. Charles et al., 2012). As described below, by measuring the level of hybridization to these oligonucleotides, we identified the location of LOH events throughout the genome in FM1468. It should also be noted that the microarrays detect changes in chromosome number in addition to detecting LOH events. A total of 13 sectored colonies were examined. Each colony was given a number and letter indicate the sectors. For example, 1A and 1B are different sectors of one FM1468 colony. The key to the color of these colonies is: 1A (white), 1B (pink); 2A (pink), 2B (red); 3A (white), 3B (pink); 4A (white), 4B (pink); 5A (white), 5B (red); 6A (white), 6B (pink); 7A (white), 7B (pink); 8A (pink), 8B (red); 9A (white), 9B (pink); 10A (white), 10B (pink), 10C (red); 11A (white), 11B (pink), 11C (red); 12A (white), 12B (red); 13A (white), 13B (red). Note that colonies 10 and 11 were tri-colored colonies, and microarrays were done on all three sectors. In summary, microarry analysis was done on 28 sectors derived from 13 colonies.
