Abstract
Maintenance of genomic integrity is important for cellular viability and proliferation. During DNA replication, cells respond to replication stress by activating checkpoint pathways that stabilize replication forks and prevent cell cycle progression. The Saccharomyces cerevisiae F-box protein Dia2 is a ubiquitin ligase component required for genomic stability and may help replication complexes negotiate damaged DNA or natural fragile sites. We recently implicated Dia2 in the replication stress response. We demonstrated that Dia2 is targeted for ubiquitin-mediated proteolysis and that activation of the S-phase checkpoint inhibits Dia2 protein turnover. S-phase checkpoint mutants fail to stabilize the Dia2 protein and checkpoint mutants that lack Dia2 exhibit increased sensitivity to replication stress. We also showed that Dia2 protein turnover is not the result of an autocatalytic mechanism. Instead, an N-terminal 20 amino acid motif that is also required for nuclear localization is necessary for Dia2 proteolysis. Dia2 mutants lacking this motif but modified with an exogenous strong nuclear localization signal are both nuclear and stable and disrupt cell cycle dynamics. In summary, our studies suggest that inhibition of Dia2 proteolysis is a novel target of the S-phase checkpoint. We think that this work will help to identify the mechanisms that function downstream of checkpoint activation and that intersect with cell cycle control pathways.
Highlights
Accurate DNA replication is critical to faithful chromosome segregation and cell viability
There has been remarkable progress in identifying key proteins required for activation of checkpoint signaling pathways [1,2], but little is known about the molecular targets of these pathways and how they inhibit progression through S phase. The goal of this commentary is to describe recent work from my laboratory [12] that suggests the proteolytic regulation of the budding yeast ubiquitin ligase component Dia2, a protein previously determined to be required for genomic stability [13,14,15], is a target of the replication checkpoint pathway
The dia2 deletion strain shows synthetic interactions with a number of mutants in DNA replication and checkpoint proteins [13,14,15,16]. These phenotypes indicate that Dia2 is important to maintain a stable genome and that it is likely to have a role in DNA replication or the replication stress response
Summary
Accurate DNA replication is critical to faithful chromosome segregation and cell viability. The progression of DNA synthesis may be hindered by the presence of damaged DNA or genotoxic stresses, which can lead to replication fork stalling or fork collapse Such events have the potential to induce genomic instability, one of the hallmarks of cancer cells. There has been remarkable progress in identifying key proteins required for activation of checkpoint signaling pathways [1,2], but little is known about the molecular targets of these pathways and how they inhibit progression through S phase The goal of this commentary is to describe recent work from my laboratory [12] that suggests the proteolytic regulation of the budding yeast ubiquitin ligase component Dia, a protein previously determined to be required for genomic stability [13,14,15], is a target of the replication checkpoint pathway
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