Degradation of Mrc1 promotes recombination-mediated restart of stalled replication forks.

The DNA replication or S-phase checkpoint monitors the integrity of DNA synthesis. Replication stress or DNA damage triggers fork stalling and checkpoint signaling to activate repair pathways. Recovery from checkpoint activation is critical for cell survival following DNA damage.

Recovery from the DNA Replication Checkpoint.

Checkpoint recovery is integral to a successful checkpoint response. Checkpoint pathways monitor progress during cell division so that in the event of an error, the checkpoint is activated to block the cell cycle and activate repair pathways. Intrinsic to this process is that once repair has been achieved, the checkpoint signaling pathway is inactivated and cell cycle progression resumes.

Artificial targeting of misfolded cytosolic proteins to endoplasmic reticulum as a mechanism for clearance.

We report that misfolded cytosolic proteins can be cleared from mammalian cells by directing them to endoplasmic reticulum (ER). NAT1 R64W and Parkin R42P are naturally occurring misfolded variants of cytosolic enzymes that acetylate arylamines and ubiquitinate proteins, respectively. We demonstrate that proteasome inhibition causes ER accumulation of NAT1 R64W and its ubiquitinated species, and that these products are cleared from cells following inhibition release.

Analyzing cell cycle-dependent degradation and ubiquitination in budding yeast.

Cell cycle progression is tightly regulated to prevent uncontrolled growth and division. Specific cell cycle factors are responsible for driving the cell from one cell cycle stage to the next. Many of these proteins are targeted for degradation by the ubiquitin proteasome system when their function is no longer required or may disrupt cell cycle progression.

Cell cycle regulation by protein degradation.

Cell division is controlled by a highly regulated program to accurately duplicate and segregate chromosomes. An important feature of the cell cycle regulatory program is that key cell cycle proteins are present and active during specific cell cycle stages but are later removed or inhibited to maintain appropriate timing. The ubiquitin-proteasome system has emerged as an important mechanism to target cell cycle proteins for degradation at critical junctures during cell division.

The Saccharomyces cerevisiae F-box protein Dia2 is a mediator of S-phase checkpoint recovery from DNA damage.

Cell-cycle progression is monitored by checkpoint pathways that pause the cell cycle when stress arises to threaten the integrity of the genome. Although activation of checkpoint pathways has been extensively studied, our understanding of how cells resume the cell cycle when the stress is resolved is relatively limited. In this study, we identify the Saccharomyces cerevisiae F-box protein Dia2 as a novel player in the S-phase checkpoint recovery pathway.

The Hect domain E3 ligase Tom1 and the F-box protein Dia2 control Cdc6 degradation in G1 phase.

The accurate replication of genetic information is critical to maintaining chromosomal integrity. Cdc6 functions in the assembly of pre-replicative complexes and is specifically required to load the Mcm2-7 replicative helicase complex at replication origins. Cdc6 is targeted for protein degradation by multiple mechanisms in Saccharomyces cerevisiae, although only a single pathway and E3 ubiquitin ligase for Cdc6 has been identified, the SCF(Cdc4) (Skp1/Cdc53/F-box protein) complex.

The stomatin-like protein SLP-1 and Cdk2 interact with the F-Box protein Fbw7-γ.

Control of cellular proliferation is critical to cell viability. The F-box protein Fbw7 (hAgo/hCdc4/FBXW7) functions as a specificity factor for the Skp1-Cul1-F-box protein (SCF) ubiquitin ligase complex and targets several proteins required for cellular proliferation for ubiquitin-mediated destruction. Fbw7 exists as three splice variants but the mechanistic role of each is not entirely clear.

Hect E3 ubiquitin ligase Tom1 controls Dia2 degradation during the cell cycle.

The ubiquitin proteasome system plays a pivotal role in controlling the cell cycle. The budding yeast F-box protein Dia2 is required for genomic stability and is targeted for ubiquitin-dependent degradation in a cell cycle-dependent manner, but the identity of the ubiquitination pathway is unknown. We demonstrate that the Hect domain E3 ubiquitin ligase Tom1 is required for Dia2 protein degradation.

TORC1 kinase and the S-phase cyclin Clb5 collaborate to promote mitotic spindle assembly and DNA replication in S. cerevisiae.

The Target of Rapamycin complex 1 (TORC1) is a central regulator of eukaryotic cell growth that is inhibited by the drug rapamycin. In the budding yeast Saccharomyces cerevisiae, translational defects associated with TORC1 inactivation inhibit cell cycle progression at an early stage in G1, but little is known about the possible roles for TORC1 later in the cell cycle. We investigated the rapamycin-hypersensitivity phenotype of cells lacking the S phase cyclin Clb5 (clb5Δ) as a basis for uncovering novel connections between TORC1 and the cell cycle regulatory machinery.

The replication stress response and the ubiquitin system: a new link in maintaining genomic integrity.

Maintenance of genomic integrity is important for cellular viability and proliferation. During DNA replication, cells respond to replication stress by activating checkpoint pathways that stabilize replication forks and prevent cell cycle progression. The Saccharomyces cerevisiae F-box protein Dia2 is a ubiquitin ligase component required for genomic stability and may help replication complexes negotiate damaged DNA or natural fragile sites.

Activation of the S-phase checkpoint inhibits degradation of the F-box protein Dia2.

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.

The C. elegans L1CAM homologue LAD-2 functions as a coreceptor in MAB-20/Sema2 mediated axon guidance.

The L1 cell adhesion molecule (L1CAM) participates in neuronal development. Mutations in the human L1 gene can cause the neurological disorder CRASH (corpus callosum hypoplasia, retardation, adducted thumbs, spastic paraplegia, and hydrocephalus). This study presents genetic data that shows that L1-like adhesion gene 2 (LAD-2), a Caenorhabditis elegans L1CAM, functions in axon pathfinding.

Yra1 is required for S phase entry and affects Dia2 binding to replication origins.

The Saccharomyces cerevisiae F-box protein Dia2 is important for DNA replication and genomic stability. Using an affinity approach, we identified Yra1, a transcription-coupled mRNA export protein, as a Dia2 interaction partner. We find that yra1 mutants are sensitive to DIA2 expression levels.

Fbw7 isoform interaction contributes to cyclin E proteolysis.

The ubiquitin proteasome system plays important roles in regulating cell growth and proliferation. Many proteins that function in ubiquitin-mediated destruction have been linked to tumorigenesis. The putative tumor-suppressor protein Fbw7 (hAgo/hCdc4) is a specificity factor for the Skp1-Cul1-F-box protein ubiquitin ligase complex and targets a number of proto-oncogene products for ubiquitin-mediated destruction, including the cell cycle regulator cyclin E.

Ubiquitin receptor proteins hHR23a and hPLIC2 interact.

Ubiquitin receptor proteins play an important role in delivering ubiquitylated protein substrates to the proteasome for degradation. HHR23a and hPLIC2 are two such ubiquitin receptors that contain ubiquitin-like (UBL) domains, which interact with the proteasome, and ubiquitin-associated (UBA) domains, which interact with ubiquitin. Depending on their abundance UBL/UBA family members can either promote or inhibit the degradation of other proteins, which suggests their participation in the delivery of substrates to the proteasome is highly regulated.

Arylamine N-acetyltransferase aggregation and constitutive ubiquitylation.

Arylamine N-acetyltransferases (NAT1 and NAT2) acetylate and detoxify arylamine carcinogens. Humans harboring certain genetic variations within the NAT genes exhibit increased likelihood of developing various cancer types, especially urinary bladder cancer. Such DNA polymorphisms result in protein products with reduced cellular activity, which is proposed to be due to their constitutive ubiquitylation and enhanced proteasomal degradation.

The F-box protein Dia2 regulates DNA replication.

Ubiquitin-mediated proteolysis plays a key role in many pathways inside the cell and is particularly important in regulating cell cycle transitions. SCF (Skp1/Cul1/F-box protein) complexes are modular ubiquitin ligases whose specificity is determined by a substrate-binding F-box protein. Dia2 is a Saccharomyces cerevisiae F-box protein previously described to play a role in invasive growth and pheromone response pathways.

Structure of the Cul1-Rbx1-Skp1-F boxSkp2 SCF ubiquitin ligase complex.

SCF complexes are the largest family of E3 ubiquitin-protein ligases and mediate the ubiquitination of diverse regulatory and signalling proteins. Here we present the crystal structure of the Cul1-Rbx1-Skp1-F boxSkp2 SCF complex, which shows that Cul1 is an elongated protein that consists of a long stalk and a globular domain. The globular domain binds the RING finger protein Rbx1 through an intermolecular beta-sheet, forming a two-subunit catalytic core that recruits the ubiquitin-conjugating enzyme.