Abstract
DNA replication stress is a source of genome instability and a replication checkpoint has evolved to enable fork stabilisation and completion of replication during stress. Mediator of the replication checkpoint 1 (Mrc1) is the primary mediator of this response in Saccharomyces cerevisiae. Mrc1 is partially sequestered in the intranuclear quality control compartment (INQ) upon methyl methanesulfonate (MMS)-induced replication stress. Here we show that Mrc1 re-localizes from the replication fork to INQ during replication stress. Sequestration of Mrc1 in INQ is facilitated by the Btn2 chaperone and the Cdc48 segregase is required to release Mrc1 from INQ during recovery from replication stress. Consistently, we show that Cdc48 colocalizes with Mrc1 in INQ and we find that Mrc1 is recognized by the Cdc48 cofactors Ufd1 and Otu1, which contribute to clearance of Mrc1 from INQ. Our findings suggest that INQ localization of Mrc1 and Cdc48 function to facilitate replication stress recovery by transiently sequestering the replication checkpoint mediator Mrc1 and explains our observation that Btn2 and Cdc48 are required for efficient replication restart following MMS-induced replication stress.
Footnotes
Abbreviations: MMS, methyl methanesulfonate; HU, hydroxyurea; PI, propidium iodide; IAA, Indole-3-acetic acid; YFP, yellow fluorescent protein; CFP, cyan fluorescent protein; sfGFP, super folder green fluorescent protein; yEmRFP, yeast enhanced monomeric red fluorescent protein; BiFC, bimolecular fluorescence complementation; INQ, intranuclear quality control compartment; PTM, post- translational modification; SUMO, small ubiquitin-like modifier; STUbL, SUMO- targeted ubiquitin ligase; DUB, de-ubiquitylation enzyme; iodo-deoxyuridine, IdU; chloro-deoxyuridine, CldU.
