Abstract
DNA replication stress is a source of genomic instability. Here we identify changed mutation rate 1 (Cmr1) as a factor involved in the response to DNA replication stress in Saccharomyces cerevisiae and show that Cmr1—together with Mrc1/Claspin, Pph3, the chaperonin containing TCP1 (CCT) and 25 other proteins—define a novel intranuclear quality control compartment (INQ) that sequesters misfolded, ubiquitylated and sumoylated proteins in response to genotoxic stress. The diversity of proteins that localize to INQ indicates that other biological processes such as cell cycle progression, chromatin and mitotic spindle organization may also be regulated through INQ. Similar to Cmr1, its human orthologue WDR76 responds to proteasome inhibition and DNA damage by relocalizing to nuclear foci and physically associating with CCT, suggesting an evolutionarily conserved biological function. We propose that Cmr1/WDR76 plays a role in the recovery from genotoxic stress through regulation of the turnover of sumoylated and phosphorylated proteins.
Highlights
DNA replication stress is a source of genomic instability
In an effort to identify new factors involved in the maintenance of genome stability, a series of stable isotope labelling by amino acids in cell culture (SILAC)-based mass spectrometry (MS) experiments were performed under conditions wherein the replication protein Rfa[1] and the recombination protein Rad[52] were induced to relocalize to DNA repair foci by DNA damage before protein extraction and pull down using a yellow fluorescent protein (YFP) tag (Fig. 1a)
The physical association between Cmr[1] and the RPA complex, which has been reported in several independent large-scale studies[11,15,16], was confirmed by reverse pull down using changed mutation rate 1 (Cmr1)-YFP as the bait (Fig. 1c and Supplementary Data 1)
Summary
We identify changed mutation rate 1 (Cmr1) as a factor involved in the response to DNA replication stress in Saccharomyces cerevisiae and show that Cmr1—together with Mrc1/Claspin, Pph[3], the chaperonin containing TCP1 (CCT) and 25 other proteins—define a novel intranuclear quality control compartment (INQ) that sequesters misfolded, ubiquitylated and sumoylated proteins in response to genotoxic stress. Resumption of DNA replication after checkpoint activation relies both on the repair or bypass of the lesion and on the inactivation of checkpoint signalling The latter requires dephosphorylation of Rad[53] by the PP4 phosphatase Pph3-Psy2-Psy[4] Dia[2] directly binds Mrc[1] and promotes its ubiquitylation and proteasomal degradation in response to replication stress This recovery pathway appears to act in parallel with dephosphorylation of Rad[53], as DIA2 and PPH3 show negative genetic interaction in the presence of replication stress[8]. Our findings document a novel connection between the cellular response to DNA replication stress and turnover of replication stress factors
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