A study of chromosome segregation where persistent DNA junctions are present between sister chromatids carried out in

Abstract

The fidelity of chromosome inheritance is of utmost importance to all living organisms. During every cell division precisely one copy of the parental genome must be segregated into each of the two daughter cells in order to allow the stable existence of progeny. This is achieved initially by the complete and faithful duplication of the chromosomes during DNA replication. Following replication each chromosome is comprised of two identical sister chromatids. Next, sister chromatids are fully resolved from each other, ensuring that no physical connection exists between them. Finally, the sisters are segregated to opposite ends of the dividing cell, guaranteeing that when cell division is completed, both daughter cells have a full complement of genetic information. After replication sister chromatids are held together by three defined kind of linkages: 1) proteinaceous linkages, mediated by the cohesin complex: a ring shaped structure that embraces both sister chromatids. 2) Topological linkages, mostly intertwines between double helixes. 3) DNA-DNA linkages formed by regions of the chromosome that have not been fully replicated, or by intermediates of the homologous recombination (HR) DNA repair process, i.e. recombination intermediates. Complete replication of the chromosomes, cleavage of cohesin and removal of catenations are essential to segregate chromosomes correctly. If the cell leaves these linkages unresolved, they lead to the formation of DNA filaments connecting the nuclei of both daughter cells, known as chromosome bridges. If the chromosome bridges are not resolved they can be severed during cytokinesis leading to double strand breaks of the DNA molecule and genome instability. Formation of chromosome bridges is a key characteristic of tumorous cells and it is considered as one the first events in the transformation from a somatic to a cancerous cell, since it favours gross chromosomal rearrangements, amplification of oncogenes and elimination of tumour suppressor genes. However, it is unknown if recombination intermediates can lead to the formation of chromosome bridges. Recombination intermediates appear as a consequence of the DNA repair process that uses homologous recombination pathway. In this pathway the undamaged sister chromatid provides a template which facilitates the restoration of the original sequence of a broken DNA molecule.

In this thesis the budding yeast Saccharomyces cerevisiae has been used as a model organism to study the sources of chromosome bridges. A haploid cdc15-2 mutant has been used as a reference strain. This mutant can be blocked in telophase with the genome correctly segregated between the two daughter cells. Using this strain it has been study whether persistent recombination intermediates, more specifically those that depend on structure specific endonucleases (SSEs) for their resolution, can lead to chromosome bridges. To this purpose, the steady-state levels of these joint molecules (JMs) were modified by deleting different combinations of the yeast SSEs genes together with exogenously forcing the cells to bypass replication stress by utilising HR. It was found that both Mus81-Mms4 and Yen1, but not Slx4-related SSEs, are essential and compensate each other in preventing and resolving a specific type of chromosome bridge, which mostly comprises noncanonical (discontinuous) forms of the Holliday Junction (HJ) molecule.

In addition, it was found that the SSE Yen1 is targeted to the nucleus by the mitotic master phosphatase Cdc14, acting as a `last resort¿ endonuclease to deal with any remaining HJs that might compromise chromosome segregation. This result highlights the essential role of early-activated Cdc14; through the FEAR network, Cdc14 effects the removal of all kinds of non-proteinaceous linkage that preclude faithful sister chromatid segregation in anaphase.