The F-Box Protein Dia2 Regulates DNA Replication

Koepp, Deanna M.; Kile, Andrew C.; Swaminathan, Swarna; Rodriguez-Rivera, Veronica

doi:10.1091/mbc.e05-09-0884

Cited by 42 publications

(70 citation statements)

References 35 publications

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“…A high concentration of the replication inhibitor HU modestly reduced the plating efficiency of a dia2D strain. The sensitivity of the dia2D mutant to MMS, HU, and camptothecin has recently been reported in other studies (Ohya et al 2005;Koepp et al 2006;Pan et al 2006). To determine if these defects reflected a general sensitivity to DNA damage, we also tested sensitivity of the dia2D strain to UV light, which induces thymidine dimers and other photo products, and to X rays, which produce DSBs.…”

Section: -The F-box Protein Dia2 Forms An Scf Complex and Is Requiredmentioning

confidence: 77%

“…Two recent reports have suggested replication-associated functions for Dia2 (Koepp et al 2006;Pan et al 2006). On the basis of apparent premature S-phase entry of synchronous cultures of a dia2D strain and detection of replication origin sequences in crosslinked Dia2 immunoprecipitates, Koepp et al (2006) proposed that Dia2 prevents precocious firing of replication origins.…”

Section: Discussionmentioning

confidence: 99%

“…On the basis of apparent premature S-phase entry of synchronous cultures of a dia2D strain and detection of replication origin sequences in crosslinked Dia2 immunoprecipitates, Koepp et al (2006) proposed that Dia2 prevents precocious firing of replication origins. Dia2 thus might function in a manner analogous to the CDK inhibitor Sic1, which is known to prevent Clb5/6-Cdc28 activation and subsequent origin firing (Lengronne and Schwob 2002).…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2006

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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...

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Section: -The F-box Protein Dia2 Forms An Scf Complex and Is Requiredmentioning

confidence: 77%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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

et al. 2006

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“…All of these gene products are known to help maintenance of fork integrity and to facilitate its navigation through tightly bound nonhistone protein barriers (7,14,15). Dia2 binds to replication origins, is present at replication forks and interacts with Mrc1, that is a component of the fork protection complex (18,19,43). Therefore, it is reasonable to expect that in the absence of these proteins replication forks are likely to be more fragile and error-prone to induce addition or deletions of base pairs.…”

Section: Discussionmentioning

confidence: 99%

The Intra-S Phase Checkpoint Protein Tof1 Collaborates with the Helicase Rrm3 and the F-box Protein Dia2 to Maintain Genome Stability in Saccharomyces cerevisiae

Bairwa

Mohanty

Stamenova

et al. 2011

Journal of Biological Chemistry

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The intra-S phase checkpoint protein complex Tof1/Csm3 of Saccharomyces cerevisiae antagonizes Rrm3 helicase to modulate replication fork arrest not only at the replication termini of rDNA but also at strong nonhistone protein binding sites throughout the genome. We investigated whether these checkpoint proteins acted either antagonistically or synergistically with Rrm3 in mediating other important functions such as maintenance of genome stability. High retromobility of a normally quiescent retrovirus-like transposable element Ty1 of S. cerevisiae is a form of genome instability, because the transposition events induce mutations. We measured the transposition of Ty1 in various genetic backgrounds and discovered that Tof1 suppressed excessive retromobility in collaboration with either Rrm3 or the F-box protein Dia2. Although both Rrm3 and Dia2 are believed to facilitate fork movement, fork stalling at DNA-protein complexes did not appear to be a major contributor to enhancement of retromobility. Absence of the aforementioned proteins either individually or in pair-wise combinations caused karyotype changes as revealed by the altered migrations of the individual chromosomes in pulsed field gels. The mobility changes were RNase H-resistant and therefore, unlikely to have been caused by extensive R loop formation. These mutations also resulted in alterations of telomere lengths. However, the latter changes could not fully account for the magnitude of the observed karyotypic alterations. We conclude that unlike other checkpoint proteins that are known to be required for elevated retromobility, Tof1 suppressed high frequency retrotransposition and maintained karyotype stability in collaboration with the aforementioned proteins.

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“…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).…”

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confidence: 99%

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

Kile

Koepp

2010

Molecular and Cellular Biology

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A stable genome is critical to cell viability and proliferation. During DNA replication, the S-phase checkpoint pathway responds to replication stress. In budding yeast, the chromatin-bound F-box protein Dia2 is required to maintain genomic stability and may help replication complexes overcome sites of damaged DNA and natural fragile regions. SCF (Skp1/Cul1/F-box protein) complexes are modular ubiquitin ligases. We show here that Dia2 is itself targeted for ubiquitin-mediated proteolysis and that activation of the S-phase checkpoint pathway inhibits Dia2 protein degradation. S-phase checkpoint mutants fail to stabilize Dia2 in response to replication stress. Deletion of DIA2 from these checkpoint mutants exacerbates their sensitivity to hydroxyurea, suggesting that stabilization of Dia2 contributes to the replication stress response. Unlike the case for other F-box proteins, deletion of the F-box domain in Dia2 does not stabilize the protein. Rather, an N-terminal domain that is also required for nuclear localization is necessary for degradation. When a strong nuclear localization signal (NLS) is added to dia2 mutants lacking this domain, the Dia2 protein is both stable and nuclear. Together, our results suggest that Dia2 protein turnover does not involve an autocatalytic mechanism and that Dia2 proteolysis is inhibited by activation of the replication stress response.

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The F-Box Protein Dia2 Regulates DNA Replication

Cited by 42 publications

References 35 publications

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

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

The Intra-S Phase Checkpoint Protein Tof1 Collaborates with the Helicase Rrm3 and the F-box Protein Dia2 to Maintain Genome Stability in Saccharomyces cerevisiae

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

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