Regulación y función de la endonucleasa específica de estructura Mus81-Mms4/EME1

Resumen

The maintenance of genome stability during eukaryotic chromosome replication and the fidelity of DNA synthesis are essential for cell survival and to prevent pathological conditions that in metazoans could lead to cancer and other diseases. Together with many other proteins, different specific endonucleases contribute to genomic stability thanks to their ability to cleave DNA secondary structures that arise during replication-associated repair processes or during replication restart after fork blocks. In this PhD thesis, we focused our work on the study of one of these endonucleases: the conserved heterodimeric complex Mus81-Mms4/EME1. This endonuclease is composed of a catalytic subunit, Mus81/MUS81, and a non-catalytic subunit, EME1/Mms4 (EME1/Eme1 in mammals, Schizosaccharomyces pombe and plants, Mms4 in Saccharomyces cerevisiae and Drosophila), both of which are required for the activity of the complex. In this PhD thesis, using the budding yeast S. cerevisiae as a model organism, we have studied the function and regulation of Mus81-Mms4 in proliferating cells. We have found that this endonuclease is strictly regulated during the mitotic cell cycle by Cdc28 (CDK)- and Cdc5 (PLK)- dependent phosphorylation of the non-catalytic subunit Mms4. The phosphorylation of this subunit occurs only after bulk DNA synthesis and before chromosome segregation, and is absolutely necessary for the endonuclease function of the Mus81-Mms4 complex. Mus81-Mms4 regulation determines its activity through the cell cycle, so that it is low during G1 and S phases and high at the G2/M transition and during mitosis. Moreover, despite being required for dealing with DNA lesions that hinder replication, Mus81-Mms4 activation is not induced by the presence of DNA damage at replication forks. Full Mus81-Mms4 activity is only acquired when cells finish S-phase and the endonuclease executes its function after the bulk of genome replication is completed. The mode of regulation of Mus81-Mms4 restricts its activity to a short period of the cell cycle, thus preventing its function during chromosome replication and the potential negative consequences for genome stability derived from its nucleolytic action. Yet, the controlled Mus81-Mms4 activity provides a fail-safe mechanism that guarantees the resolution of DNA intermediates that may remain after replication and require processing before chromosome segregation.