Alberto Sanchez-Diaz

GINS maintains association of Cdc45 with MCM in replisome progression complexes at eukaryotic DNA replication forks

The components of the replisome that preserve genomic stability by controlling the progression of eukaryotic DNA replication forks are poorly understood. Here, we show that the GINS (go ichi ni san) complex allows the MCM (minichromosome maintenance) helicase to interact with key regulatory proteins in large replisome progression complexes (RPCs) that are assembled during initiation and disassembled at the end of S phase. RPC components include the essential initiation and elongation factor, Cdc45, the checkpoint mediator Mrc1, the Tof1–Csm3 complex that allows replication forks to pause at protein–DNA barriers, the histone chaperone FACT (facilitates chromatin transcription) and Ctf4, which helps to establish sister chromatid cohesion. RPCs also interact with Mcm10 and topoisomerase I. During initiation, GINS is essential for a specific subset of RPC proteins to interact with MCM. GINS is also important for the normal progression of DNA replication forks, and we show that it is required after initiation to maintain the association between MCM and Cdc45 within RPCs.

Functional proteomic identification of DNA replication proteins by induced proteolysis in vivo

Evolutionarily diverse eukaryotic cells share many conserved proteins of unknown function. Some are essential for cell viability, emphasising their importance for fundamental processes of cell biology but complicating their analysis. We have developed an approach to the large-scale characterization of such proteins, based on conditional and rapid degradation of the target protein in vivo, so that the immediate consequences of bulk protein depletion can be examined. Budding yeast strains have been constructed in which essential proteins of unknown function have been fused to a ‘heat-inducible-degron’ cassette that targets the protein for proteolysis at 37 °C (ref. 4). By screening the collection for defects in cell-cycle progression, here we identify three DNA replication factors that interact with each other and that have uncharacterized homologues in human cells. We have used the degron strains to show that these proteins are required for the establishment and normal progression of DNA replication forks. The degron collection could also be used to identify other, essential, proteins with roles in many other processes of eukaryotic cell biology.

Mcm10 associates with the loaded DNA helicase at replication origins and defines a novel step in its activation

Mcm10 is essential for chromosome replication in eukaryotic cells and was previously thought to link the Mcm2‐7 DNA helicase at replication forks to DNA polymerase alpha. Here, we show that yeast Mcm10 interacts preferentially with the fraction of the Mcm2‐7 helicase that is loaded in an inactive form at origins of DNA replication, suggesting a role for Mcm10 during the initiation of chromosome replication, but Mcm10 is not a stable component of the replisome subsequently. Studies with budding yeast and human cells indicated that Mcm10 chaperones the catalytic subunit of polymerase alpha and preserves its stability. We used a novel degron allele to inactivate Mcm10 efficiently and this blocked the initiation of chromosome replication without causing degradation of DNA polymerase alpha. Strikingly, the other essential helicase subunits Cdc45 and GINS were still recruited to Mcm2‐7 when cells entered S‐phase without Mcm10, but origin unwinding was blocked. These findings indicate that Mcm10 is required for a novel step during activation of the Cdc45–MCM–GINS helicase at DNA replication origins.

APCste9/srw1 promotes degradation of mitotic cyclins in G1 and is inhibited by cdc2 phosphorylation

Fission yeast ste9/srw1 is a WD‐repeat protein highly homologous to budding yeast Hct1/Cdh1 and Drosophila Fizzy‐related that are involved in activating APC/C (anaphase‐promoting complex/cyclosome). We show that APCste9/srw1 specifically promotes the degradation of mitotic cyclins cdc13 and cig1 but not the S‐phase cyclin cig2. APCste9/srw1 is not necessary for the proteolysis of cdc13 and cig1 that occurs at the metaphase–anaphase transition but it is absolutely required for their degradation in G1. Therefore, we propose that the main role of APCste9/srw1 is to promote degradation of mitotic cyclins when cells need to delay or arrest the cell cycle in G1. We also show that ste9/srw1 is negatively regulated by cdc2‐dependent protein phosphorylation. In G1, when cdc2–cyclin kinase activity is low, unphosphorylated ste9/srw1 interacts with APC/C. In the rest of the cell cycle, phosphorylation of ste9/srw1 by cdc2–cyclin complexes both triggers proteolysis of ste9/srw1 and causes its dissociation from the APC/C. This mechanism provides a molecular switch to prevent inactivation of cdc2 in G2 and early mitosis and to allow its inactivation in G1.

Inn1 couples contraction of the actomyosin ring to membrane ingression during cytokinesis in budding yeast.

By rapidly depleting each of the essential budding yeast proteins of unknown function, we identified a novel factor that we call Inn1, which associates with the contractile actomyosin ring at the end of mitosis and is needed for cytokinesis. We show that Inn1 has a C2 domain at the amino terminus of the protein that is required for ingression of the plasma membrane, whereas the remainder of the protein recruits Inn1 to the actomyosin ring. The lethal effects of deleting the INN1 gene can be suppressed by artificial fusion of the C2 domain to other components of the actomyosin ring, restoring membrane ingression on contraction of the actomyosin ring. Our data indicate that recruitment of the C2 domain of Inn1 to the contractile actomyosin ring is crucial for ingression of the plasma membrane during cytokinesis in budding yeast.

A critical analysis of total sialic acid and sialoglycoconjugate contents of bovine milk-based infant formulas

BACKGROUND Several infant formulas were bovine milk-based products. Mature bovine milk has a very low sialoglycoconjugate content compared with human milk from the first phases of lactation. METHODS The present study was undertaken to determine total sialic acid and oligosaccharide, glycoprotein, and ganglioside sialic acid contents of bovine milk-based formulas. RESULTS Starter formulas, designed for the first days/weeks after birth, have very similar sialic acid contents (233-266 mg/L fresh milk). We found more oligosaccharide-bound sialic acids (167-174 mg/L fresh milk) than those bound to proteins (53-84 mg/L fresh milk) in these formulas. The ganglioside sialic acid contents of starter formulas (952-1135 micrograms/L fresh milk) vary slightly from formula to formula. However, all the above-mentioned contents are lower than in human colostrum or transitional milk. CONCLUSIONS Infants fed starter formulas have total sialic acid and oligosaccharide, glycoprotein, and ganglioside sialic acid intakes of 36, 28, 50, and 20%, respectively, of those fed human colostrum or transitional milk. By contrast, follow-on formulas, used from 4 to 5 months of age, provide total sialic acid and oligosaccharide, glycoprotein, and ganglioside sialic acid contents similar to those furnished by mature human milk. Since the reference standard for optimal nutrition in the early months of infancy is human milk, a supplementation with sialic acid-containing glycoconjugates of infant formulas recommended for the first days after delivery could be advisable when breast-feeding is not possible.

Inn1 and Cyk3 regulate chitin synthase during cytokinesis in budding yeasts

Summary The chitin synthase that makes the primary septum during cell division in budding yeasts is an important therapeutic target with an unknown activation mechanism. We previously found that the C2-domain of the Saccharomyces cerevisiae Inn1 protein plays an essential but uncharacterised role at the cleavage site during cytokinesis. By combining a novel degron allele of INN1 with a point mutation in the C2-domain, we screened for mutations in other genes that suppress the resulting defect in cell division. In this way, we identified 22 dominant mutations of CHS2 (chitin synthase II) that map to two neighbouring sites in the catalytic domain. Chs2 in isolated cell membranes is normally nearly inactive (unless protease treatment is used to bypass inhibition); however, the dominant suppressor allele Chs2-V377I has enhanced activity in vitro. We show that Inn1 associates with Chs2 in yeast cell extracts. It also interacts in a yeast two-hybrid assay with the N-terminal 65% of Chs2, which contains the catalytic domain. In addition to compensating for mutations in the Inn1 C2-domain, the dominant CHS2 alleles suppress cytokinesis defects produced by the lack of the Cyk3 protein. Our data support a model in which the C2-domain of Inn1 acts in conjunction with Cyk3 to regulate the catalytic domain of Chs2 during cytokinesis. These findings suggest novel approaches for developing future drugs against important fungal pathogens.

The Mitotic Exit Network and Cdc14 phosphatase initiate cytokinesis by counteracting CDK phosphorylations and blocking polarised growth.

Polarisation of the actin cytoskeleton must cease during cytokinesis, to support efficient assembly and contraction of the actomyosin ring at the site of cell division, but the underlying mechanisms are still understood poorly in most species. In budding yeast, the Mitotic Exit Network (MEN) releases Cdc14 phosphatase from the nucleolus during anaphase, leading to the inactivation of mitotic forms of cyclin‐dependent kinase (CDK) and the onset of septation, before G1‐CDK can be reactivated and drive re‐polarisation of the actin cytoskeleton to a new bud. Here, we show that premature inactivation of mitotic CDK, before release of Cdc14, allows G1‐CDK to divert the actin cytoskeleton away from the actomyosin ring to a new site of polarised growth, thereby delaying progression through cytokinesis. Our data indicate that cells normally avoid this problem via the MEN‐dependent release of Cdc14, which counteracts all classes of CDK‐mediated phosphorylations during cytokinesis and blocks polarised growth. The dephosphorylation of CDK targets is therefore central to the mechanism by which the MEN and Cdc14 initiate cytokinesis and block polarised growth during late mitosis.

Hof1 and Rvs167 have redundant roles in actomyosin ring function during cytokinesis in budding yeast.

The Hof1 protein (Homologue of Fifteen) regulates formation of the primary septum during cytokinesis in the budding yeast Saccharomyces cerevisiae, whereas the orthologous Cdc15 protein in fission yeast regulates the actomyosin ring by using its F-BAR domain to recruit actin nucleators to the cleavage site. Here we show that budding yeast Hof1 also contributes to actin ring assembly in parallel with the Rvs167 protein. Simultaneous deletion of the HOF1 and RVS167 genes is lethal, and cells fail to assemble the actomyosin ring as they progress through mitosis. Although Hof1 and Rvs167 are not orthologues, they both share an analogous structure, with an F-BAR or BAR domain at the amino terminus, capable of inducing membrane curvature, and SH3 domains at the carboxyl terminus that bind to specific proline-rich targets. The SH3 domain of Rvs167 becomes essential for assembly of the actomyosin ring in cells lacking Hof1, suggesting that it helps to recruit a regulator of the actin cytoskeleton. This new function of Rvs167 appears to be independent of its known role as a regulator of the Arp2/3 actin nucleator, as actin ring assembly is not abolished by the simultaneous inactivation of Hof1 and Arp2/3. Instead we find that recruitment to the bud-neck of the Iqg1 actin regulator is defective in cells lacking Hof1 and Rvs167, though future studies will be needed to determine if this reflects a direct interaction between these factors. The redundant role of Hof1 in actin ring assembly suggests that the mechanism of actin ring assembly has been conserved to a greater extent across evolution than anticipated previously.

HBP2: a new mammalian protein that complements the fission yeast MBF transcription complex.

Abstract. The mammalian HBP2 gene has been isolated by functional complementation of cells unable to undergo DNA replication in fission yeast. HBP2 is a protein with a high mobility group (HMG) domain that belongs to the Sox (Sry-related HMG box) family of transcription factors. As in other members of the family, the HMG box in HBP2 is a DNA-binding domain that is essential for its function in Schizosaccharomyces pombe. Expression of HBP2 in fission yeast activates Cdc10-dependent transcription at G1/S and allows growth of cdc10-129, res1Δ and rep2Δ mutant cells at the restrictive temperature. The mammalian HBP2 activates transcription at G1/S in the fission yeast Sch. pombe.