Originally published as Genetics Published Articles Ahead of Print on June 4, 2006.

Genetics, Vol. 174, 1709-1727, December 2006, Copyright © 2006
doi:10.1534/genetics.106.057836

The F-Box Protein Dia2 Overcomes Replication Impedance to Promote Genome Stability in Saccharomyces cerevisiae

Deborah Blake^*,, Brian Luke^,1, Pamela Kanellis^*,, Paul Jorgensen^,2, Theo Goh^,3, Sonya Penfold^,4, Bobby-Joe Breitkreutz, Daniel Durocher^*,, Matthias Peter and Mike Tyers^*,,5

^* Department of Medical Genetics and Microbiology, University of Toronto, Toronto, Ontario M5S 1A8, Canada, Samuel Lunenfeld Research Institute, Toronto, Ontario M5G 1X5, Canada and Institute of Biochemistry, 8093 Zürich, Switzerland

⁵ Corresponding author: Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Room 1080, 600 University Ave., Toronto, ON M5G 1X5, Canada.
E-mail: tyers{at}mshri.on.ca

The maintenance of DNA replication fork stability under conditions of DNA damage and at natural replication pause sites is essential for genome stability. Here, we describe a novel role for the F-box protein Dia2 in promoting genome stability in the budding yeast Saccharomyces cerevisiae. Like most other F-box proteins, Dia2 forms a Skp1-Cdc53/Cullin-F-box (SCF) E3 ubiquitin–ligase complex. Systematic analysis of genetic interactions between dia2 and 4400 viable gene deletion mutants revealed synthetic lethal/synthetic sick interactions with a broad spectrum of DNA replication, recombination, checkpoint, and chromatin-remodeling pathways. dia2 strains exhibit constitutive activation of the checkpoint kinase Rad53 and elevated counts of endogenous DNA repair foci and are unable to overcome MMS-induced replicative stress. Notably, dia2 strains display a high rate of gross chromosomal rearrangements (GCRs) that involve the rDNA locus and an increase in extrachromosomal rDNA circle (ERC) formation, consistent with an observed enrichment of Dia2 in the nucleolus. These results suggest that Dia2 is essential for stable passage of replication forks through regions of damaged DNA and natural fragile regions, particularly the replication fork barrier (RFB) of rDNA repeat loci. We propose that the SCF^Dia2 ubiquitin ligase serves to modify or degrade protein substrates that would otherwise impede the replication fork in problematic regions of the genome.

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