Caracterización de la entrada en un nuevo ciclo celular con separación incompleta de cromátidas hermanas en la levadura saccharomyces cerevisiae

Resumen

Genomic instability is one of the hallmarks of cancer with cancer cells carrying variations in ploidy, gross chromosomal rearrangements, loss-of-heterozigosity, sequence amplifications and deletions. Therefore, the maintenance of genome integrity is essential to prevent those disorders. Genes that prevent the appearance of phenotypes associated with cancer are known as tumour suppressor genes. Among them, two different types can be defined: gatekeepers, which negatively regulate cell division, and caretakers, which prevent genome instability. Genome integrity is constantly challenged by both exogenous and endogenous sources. Among the latter, the failure in segregating sister chromatids during mitosis is one of the most dangerous, and often causes the appearance of anaphase bridges. These are considered to lead to an irreversible mitotic catastrophe that has been related to carcinogenesis and intratumour heterogeneity. Failure to resolve sister chromatids arises due to deregulations in mechanisms that remove linkages established between sister chromatids. Main factors involved in sister chromatid resolution are the separase, which removes cohesin, and condensin and topoisomerase II, which remove topological entanglements as a consequence of the normal replication and transcription of the DNA. Mutants for those proteins lead to anaphase bridges comprised of most chromosomes. Despite this, cells often perform cytokinesis, leading to a phenotype in which most chromosomes are severed and, as a consequence, cells are unable to enter a new cell cycle. This severe phenotype has precluded those mutants from the study of the consequences that milder phenotypes of anaphase bridges formation during mitosis have in the cellular progeny. Therefore, the consequences for the dividing cells that present anaphase bridges have been poorly studied. In budding yeast it has been reported that the last region to segregate is the rDNA array, located on the right arm of the chromosome XII. In addition, segregation of this locus requires proper activation of Cdc14, a key phosphatase in mitotic exit which is conserved in humans and supposed to be a caretaker. Transient inactivation of Cdc14 in yeast, by means of thermosensitive alleles such as cdc14-1, leads to missegregation of the rDNA-bearing chromosome XII, with most of the genome properly segregated. Contrary to cohesin removal, condensin and topoisomerase II mutants, cells are able to resume the cell cycle upon reactivation of Cdc14. In addition, resumption of the cell cycle occurs in a synchronous manner despite the fact that half of the cells within a population fail to segregate the chromosome XII. In this thesis, I have used the Saccharomyces cerevisiae haploid cdc14-1 mutant to further characterize the anaphase bridge resulting of the temporary inactivation of Cdc14 and the cellular response to it. I have determined that, in spite of remaining together after mitotic exit, daughter cells complete cytokinesis. Furthermore, cells transit through G1 and enter S-phase, where they trigger a Rad52-dependent and Mre11-independent DNA damage response, concomitant with the activation of a Rad9-dependent G2/M cell cycle checkpoint. Synchrony of the cell cycle, non-resolution of specific genomic regions (i.e.: rDNA), the fact that there is a mixture with cells that segregated the chromosome XII and cells that missegregated it for the same population and experiment, the fact that daughter cells remain together after exiting from mitosis, and the existence of a parallel control strain (cdc15-2 mutant) makes the cdc14-1 mutant an outstanding model to study the cellular response to the presence of anaphase bridges during mitosis and its short- and long-term consequences for the cell progeny. In addition, the cdc14-1 mutant also provides an exceptional model to study how one-ended double-strand breaks are processed by the cellular machinery. Additionally, I have studied the consequences that the transient inactivation of Cdc14 has on diploid yeast cells. Remarkably, cellular viability is significantly compromised, and genome instability events affecting most chromosomes, mainly aneuploidy and loss-of-heterozigosity, was a common outcome. Therefore, Cdc14 seems to be an important caretaker in diploid yeast cells, as its transient inactivation, not mutation, leads to dramatic consequences in genome stability.