Impact of Pif1 translational mechanism on genetic interactions with YEN1ON and other DNA repair enzymes

  1. Tomás Lama Díaz
Dirixida por:
  1. Miguel González Blanco Director

Universidade de defensa: Universidade de Santiago de Compostela

Fecha de defensa: 02 de xaneiro de 2023

  1. Departamento de Bioquímica e Bioloxía Molecular

Tipo: Tese


Homologous recombination (HR) entails the formation of several branched recombination intermediates that must be timely disengaged to safeguard chromosome segregation and cell viability. Hence, cells are endowed with DNA helicases and structure-selective endonucleases (SSEs) that sever these physical connections between DNA joint molecules prior to cell division. Surprinsingly, the activation of SSEs is delayed until the final stages of cell cycle. This strategy, conserved from yeast to humans, suggests that this tight control could be crucial to prevent the unscheduled processing of DNA replication and repair intermediates. To understand the biological relevance of this strict regulatory system, we searched for genetic interactions between a constitutively active version of the YEN1 nuclease (YEN1-ON) and key helicases involved in DNA replication and repair. Here, we show that deletion of the conserved PIF1 helicase in a YEN1-ON strain results in a dramatic reduction of its viability under genotoxic stress. This suggests that the unscheduled nucleolytic processing of secondary DNA structures accumulated in the absence of PIF1 is detrimental for cells. PIF1 encodes both mitochondrial and nuclear isoforms of the enzyme, but widely employed separation-of-function alleles (nuclear or mitochondrial-specific) failed to recapitulate such genetic interaction. This prompted us to delve into the translational mechanism of Pif1, leading us to the refinement of the molecular mechanism of alternative translation initiation for PIF1 mRNA, the discovery of a new nuclear Pif1 isoform and the development of the first bona-fide nuclear-null Pif1 isoform.