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

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 a Skp1-Cdc53/Cullin-F-box (SCF) E3 ubiquitin-ligase complex. Systematic analysis of genetic interactions between dia2D 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. dia2D 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, dia2D 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. Collapse of replication forks results in the formation of DNA double-strand breaks (DSBs) that can then lead to illegitimate recombination and genome rearrangements, both of which are thought to be underlying causes of many human cancers (Lengauer et al. 1998). Physical impediments to replication fork progression include tightly bound non-nucleosomal protein-DNA complexes, DNA secondary structures, and regions of DNA damage, whereas inadequate dNTP pools cause forks to slow and eventually stall in a non-locus-specific manner (Branzei and Foiani 2005). Accumulated DNA damage or stalled replication forks elicit a checkpoint response that results in a delay of the cell cycle, induction of damage responsive genes, and the repair or bypass of the DNA lesion (Melo and Toczyski 2002;Branzei and Foiani 2005). The DNA damage checkpoint is activated upon detection of DNA lesions in G 1 and G 2 phase, and in S-phase the latter sometimes is referred to as the intra-S checkpoint. A second response in S-phase, referred to as the replication checkpoint, is the response to delayed DNA synthesis as caused by lowered dNTP pools upon inhibition of ribonucleotide reductase by hydroxyurea (HU). It is likely that the replication and intra-S checkpoint pathways are integrated such that the key signal is stalled or slowed replication forks, due to either dNTP shortage or collision with DNA damage. The only essential function of the S-phase checkpoint is to stabilize the fork when cells undergo replicative stress (Terceroet al. 2003) and thereby prevent the accumulation of recombinog...

Powering through ribosome assembly

“…F-box proteins provide target specificity to the Skp1-Cullin-F-box (SCF)-complex-mediated ubiquitination of proteins that are destined for degradation by the proteasome. The nucleolar F-box protein Dia2 interacts physically or genetically with 15 ribosome assembly factors (Ho et al 2002;Blake et al 2006;Pan et al 2006), and its partners Cdc53 and Hrt1 bind three additional ones (Ho et al 2002;Davierwala et al 2005). In addition, Dia2 interacts with the cullin Rtt101 and Mms1.…”

Activation of the S-Phase Checkpoint Inhibits Degradation of the F-Box Protein Dia2

“…We and others have shown that dia2⌬ cells exhibit synthetic growth phenotypes on normal media in combination with checkpoint mutants (4,17,28,43). We tested a series of these double mutants for their sensitivity to HU, which induces replication stress.…”

“…The dia2⌬ mutant is hypersensitive to DNA damage, exhibits chromosome loss and rearrangement, and accumulates DNA damage foci (4,17,28). Dia2 is a bona fide F-box protein in that it assembles with Skp1, Cdc53, and Rbx1 into a functional SCF ubiquitin ligase complex (15,(17)(18)(19).…”

“…Dia2 is a bona fide F-box protein in that it assembles with Skp1, Cdc53, and Rbx1 into a functional SCF ubiquitin ligase complex (15,(17)(18)(19). The dia2⌬ strain progresses more slowly through S phase than wild-type cells when challenged with the DNA-damaging agent methyl methanesulfonate (MMS), suggesting that Dia2 promotes passage of replication complexes through areas of damaged DNA (4).…”

Synthetic Lethal Interactions Identify Phenotypic “Interologs” of the Spindle Assembly Checkpoint Components

“…org/supplemental/). To date, there are 79 MAD1 synthetic lethal interactions identified in S. cerevisiae (Wang and Burke 1995;Hardwick et al 1999;Krogan et al 2004;Lee and Spencer 2004;Tong et al 2004;Measday et al 2005;Montpetit et al 2005;Blake et al 2006;Daniel et al 2006;Pan et al 2006). We used reciprocal best blastp (Altschul et al 1997) analysis to identify putative C. elegans orthologs for 37 of these 79 genes (supplemental Table 1).…”