Camilla Skettrup Colding:
Regulation of replication recovery and genome integrity: Cdc48-mediated turnover of Mrc1 and Cmr1 in INQ

Date: 27-04-2016    Supervisor: Michael Lisby

Preserving genome integrity is essential for cell survival. To this end, mechanisms that supervise DNA replication and respond to replication perturbations have evolved. One such mechanism is the replication checkpoint, which responds to DNA replication stress and acts to ensure replication pausing, replication fork stability and if necessary, DNA repair. In Saccharomyces cerevisiae, the replication checkpoint is activated by recruitment of the sensor kinase Mec1 to the stalled fork and subsequent Mec1- mediated phosphorylation and activation of the checkpoint effector kinase Rad53. Checkpoint activation is mediated by Mrc1, which ensures Mec1 presence at the stalled replication fork thus facilitating Rad53 phosphorylation. When replication can be resumed safely, the replication checkpoint is deactivated and replication forks restart. One mechanism for checkpoint deactivation is the ubiquitin-targeted proteasomal degradation of Mrc1.

In this study, we describe a novel nuclear structure, the intranuclear quality control compartment (INQ), which regulates protein turnover and is important for recovery after replication stress. We find that upon methyl methanesulfonate (MMS)-induced replication stress, INQ contains the Cdc48 chaperone, proteasomes and 23 other proteins that specifically relocalize to INQ upon MMS treatment and proteasomal inhibition. Among these are several proteins involved in replication and cell cycle control, including Mrc1. Mrc1 relocalization to INQ requires SCF-Dia2-mediated ubiquitylation and the INQ-resident chaperone Btn2, which directly interacts with Mrc1. INQ-localized Mrc1 also interacts with the Cdc48 cofactors Ufd1 and Ubx5 and these interactions facilitate Cdc48-mediated degradation of Mrc1 in INQ. In addition, INQlocalized Mrc1 interacts with the Cdc48 cofactor and de-ubiquitylation enzyme Otu1 and the small ubiquitin-like modifier (SUMO) de-conjugating enzyme Ulp2 and we propose that these interactions lead to the de-ubiquitylation, de-SUMOylation and release of Mrc1 from INQ. Together, Cdc48-mediated degradation of Mrc1 in INQ and de-modification-induced release of Mrc1 from INQ facilitate replication recovery after MMS-induced replication stress.

Our data reveal that control of Mrc1 turnover through the interplay between posttranslational modifications and INQ localization adds another layer of regulation to the replication checkpoint. We also add replication recovery to the list of processes regulated by Cdc48 and propose that Cdc48 acts more generally to regulate turnover of INQ-localized proteins. Further studies are needed to establish whether and how other nuclear processes are regulated through Cdc48-mediated turnover in and of INQ.