Nuevas funciones de los complejos MCM y Cohesina en el mantenimiento de la estabilidad genómica y la organización de la cromatina

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 29-10-2009The six minichromosome maintenance proteins (Mcm2‐7), which presumably constitute the core of the replicative DNA helicase, are present on chromatin in large excess relative to the numer of active replication forks. The function of this apparent surplus is not well understood, as most of them are displaced from the DNA during S‐phase, apparently without having played an active role in DNA replication. At least in yeast, Xenopus and Drosophila the concentration of MCM proteins can be reduced without affecting the DNA replication. This is known as the “MCM paradox”. In the first part of this work we tried to solve this classic controversy of eucharyotic cells. To evaluate the importance of MCM concentration in human cells, we have used RNA interference (RNAi) to modulate the expression of Mcm2‐7 genes and effectively reduce the concentration of Mcm2‐7 proteins on chromatin. We found conditions in which cells are capable of apparently normal replication with a very reduced concentration of Mcm2‐7 complexes. However, under these conditions of limited licensing, cells progressively accumulated DNA lesions and displayed chromosomal fragility. An analysis of origin density revealed that “excess” MCM proteins, although not necessarily active during an unperturbed S‐phase, might activate a reservoir of backup origins that are required to recover from DNA replication stress. Our data show that the chromatin‐bound “excess” Mcm2‐7 complexes are essential to maintain genomic integrity in human cells. Different lines of evidence show that Mcm2‐7 complex or its individual subunits could be involved in other processes apart from DNA replication. The systematic Knock‐Down of these proteins led us to identify an essential role for MCM proteins during the checkpoint activation. Our data indicate that this role would be limited to the replication checkpoint, where the Mcm2 and Mcm5 proteins appear to be essential to promote Chk1 activation upon diverse treatments that cause fork stalling. We show that after checkpoint induction these proteins interacted with the ATR allosteric activator TopBP1, which mails to relocate to affected replication forks in Mcm2 Knocked‐down cells. These data propose the participation of these two single proteins in the activation of the replication checkpoint by locating/estabilizing TopBP1 to stalled forks. Finally, we tried to identify new proteins associated with the MCM complex that could help to understand their different functions. This led us to identify Cohesin, a protein complex that mediates sister chromatid cohesion, as an MCM partner. To our surprise, MCM was not involved in cohesion establishment but Cohesin appeared to be essential for DNA replication. Cohesin is enriched at replication origins and its downregulation led to a decreased density of active origins, delaying S‐phase progression. Taking into account the ability of Cohesin to constrain the DNA topology and its presence in the nucleoskeleton, we demonstrate that Cohesin is an architectural element of the nucleus participating in the generation of chromatin loops. This function affects DNA replication, as some of the matrix‐bound regions are replication origins. This study contributes to our understanding of the roles of Cohesin in a variety of processes and adds new clues to the higher order chromatin organization in the nucleus