Investigation of the function and control of Dia2, a regulator of genomic stability in budding yeast.

Abstract

Maintenance of genomic integrity can be particularly challenged during DNA replication, which is critical for cellular viability and proliferation. Cancer cells exhibit loss of genomic integrity, thus it is critical to understand the pathways involved in genome maintenance. We have identified the F-box protein Dia2 as a novel and previously unappreciated mediator of genome stability. F-box proteins are substrate specificity subunits of SCF ubiquitin ligases for ubiquitin mediated proteolysis, although most remain uncharacterized in their function or targets. Deletion of the DIA2 gene in Saccharomyces cerevisiae leads to genomic integrity defects, and the Dia2 protein associates with chromatin and origins of replication, indicating it performs a chromatin-associated role in DNA replication. Interestingly, the Yra1 protein was identified to physically interact with Dia2 and promotes Dia2 binding to replication origins yet is not a proteolytic substrate of SCF-Dia2. The Dia2 protein itself is subject to proteolysis, but is stabilized by the activation of the replication checkpoint and this suggests it plays a role during periods of replication stress and DNA damage during S phase. Surprisingly, Dia2 turnover is not controlled by an autocatalytic mechanism involving its F-box domain, but instead relies on a region upstream of its F-box that controls both its stability and nuclear localization. Replication checkpoint activation leads to inhibition of late-firing origins, stabilization of replication forks, as well as stabilization of the Dia2 protein. Our observations indicate that SCF-Dia2 activity performs ubiquitin ligase activity at one or both of these sites that are regulated by the checkpoint. These studies establish a novel link between DNA replication and genomic integrity to the SCF ubiquitin ligase via Dia2.