Mechanisms mediating replication checkpoint inactivation

Abstract

The replication checkpoint plays a key role in the maintenance of genome integrity by sensing problems in replication fork progression and orchestrating a cellular response that delays cell cycle progression and protects replication fork stability. While the mechanisms through which the checkpoint senses and signals replication stress have been extensively characterized, little is known about the pathways that inactivate checkpoint signalling to re-establish cellular physiology. We identified BUL2 in a budding yeast genome-wide genetic screen for factors involved in the checkpoint response to replication stress. Bul2 is the adaptor component of the Bul2/Rsp5 ubiquitin ligase complex, involved in the inactivation of different targets through polyubiquitination and subsequent degradation or subcellular sorting. Along with BUL2 we isolated the Pph3/Psy2 and Ptc2 phosphatase coding genes, suggesting a direct involvement of Bul2/Rsp5 in the checkpoint response. We found that bul2 and rsp5 mutants show checkpoint inactivation defects and are sensitive to replication stress induced by hydroxyurea treatment, in a fashion synergistic with pph3 and ptc2 mutants. This evidence suggests that the Bul2/Rsp5 complex contributes to cell viability mediating timely checkpoint shut-off in a mechanism alternative to Rad53 dephosphorylation. Mass spectrometric analysis of Bul2 physical interactors identified Mec1, the apical replication checkpoint kinase. Mec1 acts as a stalled fork sensor through its recruitment to RPA-coated ssDNA stretches via its partner Ddc2. We found that the interaction between the Bul2/Rsp5 and Mec1/Ddc2 complexes is enhanced in response to replication stress. We propose that Mec1/Ddc2 directs Bul2/Rsp5 activity to stalled replication forks thus driving the local polyubiquitination and inactivation of key checkpoint signal transducers. The impact of persistent checkpoint signalling on replication fork dynamics and genome integrity will be discussed.