Genetic characterization of RPAMN as a system to study replication-fork associated double-strand breaks

Máster de Genética Molecular y Biotecnología.The repair of broken replication forks is poorly understood. Here, we have characterized a chimera formed by the ssDNA binding protein complex RPA fused to the micrococcal nuclease (RPAMN) as a putative system to study the repair of doubled-strand breaks (DSBs) at the replication forks in vivo in Saccharomyces cerevisiae. We found that RPAMN requires Rad52 for viability, suggesting that RPAMN generates DSBs at the advancing forks, which are repaired by homologous recombination. Accordingly, RPAMN viability partially depends on Exo1 and Sgs1, which are required for DSB resection. Despite the accumulation of DSBs, the DNA damage checkpoint is dispensable in RPAMN cells. We also show that RPAMN is deficient in the formation of DNA repair centers, which suggests that RPA might be involved in targeting the recombinogenic lesions to the DNA repair centers.Peer Reviewe