In silico protein interaction screening uncovers DONSON's role in replication initiation.

CDC45-MCM2-7-GINS (CMG) helicase assembly is the central event in eukaryotic replication initiation. In yeast, a multi-subunit "pre-loading complex" (pre-LC) accompanies GINS to chromatin-bound MCM2-7, leading to CMG formation. Here, we report that DONSON, a metazoan protein mutated in microcephalic primordial dwarfism, is required for CMG assembly in vertebrates.

DNA polymerase ε-dependent modulation of the pausing property of the CMG helicase at the barrier.

The proper pausing of replication forks at barriers on chromosomes is important for genome integrity. However, the detailed mechanism underlying this process has not been well elucidated. Here, we successfully reconstituted fork-pausing reactions from purified yeast proteins on templates that had binding sites for the LacI, LexA, and/or Fob1 proteins; the forks paused specifically at the protein-bound sites.

Crystal structure of the homology domain of the eukaryotic DNA replication proteins Sld3/Treslin.

The initiation of eukaryotic chromosomal DNA replication requires the formation of an active replicative helicase at the replication origins of chromosomal DNA. Yeast Sld3 and its metazoan counterpart Treslin are the hub proteins mediating protein associations critical for the helicase formation. Here, we show the crystal structure of the central domain of Sld3 that is conserved in Sld3/Treslin family of proteins.

Sld7, an Sld3-associated protein required for efficient chromosomal DNA replication in budding yeast.

Genetic screening of yeast for sld (synthetic lethality with dpb11) mutations has identified replication proteins, including Sld2, -3, and -5, and clarified the molecular mechanisms underlying eukaryotic chromosomal DNA replication. Here, we report a new replication protein, Sld7, identified by rescreening of sld mutations. Throughout the cell cycle, Sld7 forms a complex with Sld3, which associates with replication origins in a complex with Cdc45, binds to Dpb11 when phosphorylated by cyclin-dependent kinase, and dissociates from origins once DNA replication starts.

CDK-dependent complex formation between replication proteins Dpb11, Sld2, Pol (epsilon}, and GINS in budding yeast.

Eukaryotic chromosomal DNA replication requires cyclin-dependent kinase (CDK) activity. CDK phosphorylates two yeast replication proteins, Sld3 and Sld2, both of which bind to Dpb11 when phosphorylated. These phosphorylation-dependent interactions are essential and are the minimal requirements for CDK-dependent activation of DNA replication.

CDK-dependent phosphorylation of Sld2 and Sld3 initiates DNA replication in budding yeast.

In eukaryotic cells, cyclin-dependent kinases (CDKs) have an important involvement at various points in the cell cycle. At the onset of S phase, active CDK is essential for chromosomal DNA replication, although its precise role is unknown. In budding yeast (Saccharomyces cerevisiae), the replication protein Sld2 (ref.

GINS is a DNA polymerase epsilon accessory factor during chromosomal DNA replication in budding yeast.

GINS is a protein complex found in eukaryotic cells that is composed of Sld5p, Psf1p, Psf2p, and Psf3p. GINS polypeptides are highly conserved in eukaryotes, and the GINS complex is required for chromosomal DNA replication in yeasts and Xenopus egg. This study reports purification and biochemical characterization of GINS from Saccharomyces cerevisiae.

High-dimensional and large-scale phenotyping of yeast mutants.

One of the most powerful techniques for attributing functions to genes in uni- and multicellular organisms is comprehensive analysis of mutant traits. In this study, systematic and quantitative analyses of mutant traits are achieved in the budding yeast Saccharomyces cerevisiae by investigating morphological phenotypes. Analysis of fluorescent microscopic images of triple-stained cells makes it possible to treat morphological variations as quantitative traits.

GINS, a novel multiprotein complex required for chromosomal DNA replication in budding yeast.

Eukaryotic chromosomal DNA replication requires a two-step assembly of replication proteins on origins; formation of the prereplicative complex (pre-RC) in late M and G1 phases of the cell cycle, and assembly of other replication proteins in S phase to load DNA polymerases to initiate DNA synthesis. In budding yeast, assembly of Dpb11 and the Sld3-Cdc45 complex on the pre-RC at origins is required for loading DNA polymerases. Here we describe a novel replication complex, GINS (Go, Ichi, Nii, and San; five, one, two, and three in Japanese), in budding yeast, consisting of Sld5, Psf1 (partner of Sld five 1), Psf2, and Psf3 proteins, all of which are highly conserved in eukaryotic cells.

S-Cdk-dependent phosphorylation of Sld2 essential for chromosomal DNA replication in budding yeast.

Cyclin-dependent protein kinases (Cdks) in eukaryotic cells work as a key enzyme at various points in the cell cycle. At the onset of S phase, active S-phase Cdks (S-Cdks) are essential for chromosomal DNA replication. Although several replication proteins are phosphorylated in a Cdk-dependent manner, the biological effects of phosphorylation of these proteins on the activation of DNA replication have not been elucidated.