Abstract: 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 an SCF (Skp1-Cdc53/Cullin-F-box) E3 ubiquitin ligase complex. Systematic analysis of genetic interactions between dia2Delta and ~4,400 viable gene deletion mutants revealed synthetic lethal/synthetic sick interactions with a broad spectrum of DNA replication, recombination, checkpoint, and chromatin remodeling pathways. dia2Delta 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, dia2Delta strains display a high rate of gross chromosomal rearrangements (GCRs) that involve the rDNA locus and an increase in extra-chromosomal 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 SCFDia2 ubiquitin ligase serves to modify or degrade protein substrates that would otherwise impede the replication fork in problematic regions of the genome.