The Bul2/Rsp5 ubiquitin ligase complex promotes cohesion-mediated fork re-start

Abstract

A complete and accurate replication of DNA is essential to maintain genome integrity. In response to replication stress cells have evolved a specialized branch of the DNA damage checkpoint response, called DNA replication checkpoint, that senses replication fork stalling and orchestrates a response aimed at delaying cell cycle progression, stabilizing fragile replication structures and promoting DNA repair. A genome-wide genetic screen carried out in S. cerevisiae to discover factors mediating fork stability identified BUL2, coding for the adaptor of the Bul2/Rsp5 ubiquitin ligase complex. We found that bul2 and rsp5 mutants are sensitive to replication stress induced by hydroxyurea (HU) and that bul2 sensitivity is suppressed by the ablation of the Ubp2 ubiquitin protease, which removes Rsp5-dependent ubiquitin chains. 2D gel and whole genome sequencing analyses revealed that cells impaired in Bul2/Rsp5 function were defective in fork re-start and progression upon HU treatment. A Mass Spectrometric analysis of Bul2 physical interactors identified Mec1, a key apical checkpoint kinase that acts as a replication stress sensor through recruitment to stalled replication forks via its partner Ddc2. Our data pointed out that this interaction is fundamental to drive Bul2/Rsp5 activity to stalled replication forks. Among Bul2 physical interactors we also scored Smc1 and Smc3 cohesin subunits. We identified a genome wide defect in cohesin mutants in re-starting replication forks upon HU treatment, which was epistatic to the defects conferred by BUL1 BUL2 double deletion. Moreover, cohesin mutants’ sensistivity to HU was suppressed by deletion of UBP2, pointing at a role of Bul2/Rsp5-medited ubiquitylation events in promoting cohesin function to survive to replication stress. We propose that Bul2/Rsp5, through interaction with Mec1/Ddc2, is directed to stalled replication where it mediates cohesin ubiquitylation in turn required to promote stalled fork stabilization and restart.