Jacob Kwasi Autzen:
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Protein quality control and DNA replication checkpoint mechanisms are key to promoting a functional proteome and genome integrity and thereby ensuring cellular survival. In yeast several different compartments have been characterized that function in protein quality control. One such compartment is the intranuclear quality control compartment (INQ), which was recently shown to be a site where multiple proteins accumulate in response to proteasome inhibition and/or genotoxic stress. The proteins Mrc1 and Pph3, which function in the DNA replication checkpoint, are two such proteins, where Mrc1 functions both in the activation of the checkpoint effector kinase Rad53 as well as in the resolution of the checkpoint through Mrc1 degradation, and Pph3 functions in recovery from the checkpoint through dephosphorylation of Rad53.
In this study, we investigate the involvement of the INQ in checkpoint recovery, as well as characterize the involvement of several factors such as the segregase Cdc48, the chaperone Btn2, and the E3 ligase Dia2, which play key roles in the regulation of Mrc1 INQ re-location and fate. We performed a proteomics analysis of the Mrc1 interactome in a wild-type strain as well as in several strains mutant for proteins that affect Mrc1 INQ re-localization and dissociation. We identified several previously know Mrc1 interactors, but also several new interactors such as the helicase Rrm3 or the subunits of casein kinase II. Furthermore, we observed major changes in the interactome of mutants that affect Mrc1 INQ re-localization or mediate Mrc1 inclusion dissolution. Of note we saw a major increase in Mrc1 interaction with Rad53 in response to Cdc48 depletion and a decrease in the interaction of Mrc1 with fork components in a btn2Δ mutant strain, suggesting that INQ directly impact checkpoint signaling.
We found that upon DNA replication stress or proteasomal inhibition, Mrc1 re-locates to the INQ in a manner that depends on Btn2 and Dia2. As such, our findings suggest that the INQ facilitates checkpoint recovery by transiently sequestering the replication checkpoint mediator Mrc1. We also found that Btn2 as well as Dia2 presence affected the re-localization of Cmr1 and Pph3 to the INQ in addition to Mrc1, suggesting a more generalized role for Dia2 in INQ re-localization than previously believed. Additionally, Btn2 overexpression alone was sufficient to induce INQ focus formation for Mrc1, and this overexpression was not deleterious to cell viability.
In human, Mrc1 appears to be generally conserved as the homolog Claspin, is also necessary for unperturbed replication as well as functions in a checkpoint pathway with the Mec1 and Rad53 homologs, ATR and CHK1, respectively. Fluorescence tagging of the human homologs of the INQ re-locating proteins Cmr1 and Mrc1, WDR76 and Claspin, respectively, indicated a potential conservation of INQ function in the nucleoli in response to proteasomal inhibition but not replication stress. The formation of WDR76 and Claspin inclusions in the nucleoli in response to proteasomal inhibition depended on the human homolog of Cdc48, p97, and possibly also clearance of WDR76 and Claspin from the inclusions. However, further studies are needed to fully elucidate this potential conservation and the role of p97 in nucleoli.