Interaction of Cdc2 and CdclS with a fission yeast ORC2-like protein

Leatherwood, Janet; López-Girona, Antonia; Russell, Paul

doi:10.1038/379360a0

Cited by 131 publications

(114 citation statements)

References 28 publications

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“…Orp2 also coprecipitated specifically with GST-Cdc18. We previously reported an interaction between Cdc18 and Orp2 when both proteins were overexpressed (Leatherwood et al, 1996). In the experiment shown in Figure 2, endogenous Orp2 associates with GST-Cdc18, suggesting that the association between Orp2 and Cdc18 might be requisite for Cdc18 function in vivo.…”

Section: Gst-cdc18mentioning

confidence: 67%

“…Plasmid pAL24 was constructed by inserting the 2.8-kb PstI-SacI fragment from pJL205 plasmid, containing the nmt1 promoter upstream of the GST open reading frame, into pJK210, an integrative vector (Keeney and Boeke, 1994). The cdc18 open reading frame was amplified by polymerase chain reaction from pRep4-Cdc18-HA using primers AL19 (5Ј-CGGGATCCAT GGGCCATGTGTC-3Ј) and oJL19 (5Ј-CTAG-CAGTAC TGGCAAGGGA GAC-3Ј) (Leatherwood et al, 1996). The polymerase chain reacction product was digested with BamHI and the 1.8-kb fragment was ligated into BamHI-digested pAL24 to create pAL27.…”

Section: Plasmids Strains and General Methodsmentioning

confidence: 99%

“…Purification of GST fusion proteins expressed in fission yeast was performed as described previously (Leatherwood et al, 1996). Cells were disrupted with glass beads in buffer L (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 5 mM EDTA, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 5 g/ml pepstatin, 5 g/ml leupeptin, and 5 g/ml aprotinin), and lysates were centrifuged at 16,000 ϫ g for 5 min at 4°C.…”

Section: Protein Methodsmentioning

confidence: 99%

“…The Cdc2 substrates involved in control of initiation of DNA replication are unknown, although a clue was recently provided through the discovery of Orp2 as a protein that interacts with Cdc2 in vivo (Leatherwood et al, 1996). Orp2 is required for DNA replication in S. pombe and is related to Orc2p, a component of origin recognition complex in budding yeast (Gavin et al, 1995).…”

Section: Introductionmentioning

confidence: 99%

“…Orp2 is required for DNA replication in S. pombe and is related to Orc2p, a component of origin recognition complex in budding yeast (Gavin et al, 1995). Orp2 also interacts with Cdc18 protein (Leatherwood et al, 1996). The physical association between these proteins suggests that Orp2 and/or Cdc18 may be important substrates of Cdc2.…”

Section: Introductionmentioning

confidence: 99%

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Negative Regulation of Cdc18 DNA Replication Protein by Cdc2

López-Girona

Mondésert

Leatherwood

et al. 1998

MBoC

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Fission yeast Cdc18, a homologue of Cdc6 in budding yeast and metazoans, is periodically expressed during the S phase and required for activation of replication origins. Cdc18 overexpression induces DNA rereplication without mitosis, as does elimination of Cdc2-Cdc13 kinase during G2 phase. These findings suggest that illegitimate activation of origins may be prevented through inhibition of Cdc18 by Cdc2. Consistent with this hypothesis, we report that Cdc18 interacts with Cdc2 in association with Cdc13 and Cig2 B-type cyclins in vivo. Cdc18 is phosphorylated by the associated Cdc2 in vitro. Mutation of a single phosphorylation site, T104A, activates Cdc18 in the rereplication assay. The cdc18-K9 mutation is suppressed by a cig2 mutation, providing genetic evidence that Cdc2-Cig2 kinase inhibits Cdc18. Moreover, constitutive expression of Cig2 prevents rereplication in cells lacking Cdc13. These findings identify Cdc18 as a key target of Cdc2-Cdc13 and Cdc2-Cig2 kinases in the mechanism that limits chromosomal DNA replication to once per cell cycle.

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Section: Gst-cdc18mentioning

confidence: 67%

Section: Plasmids Strains and General Methodsmentioning

confidence: 99%

Section: Protein Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Negative Regulation of Cdc18 DNA Replication Protein by Cdc2

López-Girona

Mondésert

Leatherwood

et al. 1998

MBoC

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The fission yeast Cdc1 protein, a homologue of the small subunit of DNA polymerase delta, binds to Pol3 and Cdc27.

et al. 1996

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cdc1+ is required for cell cycle progression in Schizosaccharomyces pombe. Cells carrying temperature‐sensitive cdc1 mutants undergo cell cycle arrest when shifted to the restrictive temperature, becoming highly elongated. Here we describe the cloning and sequencing of cdc1+, which is shown to encode a 462 residue protein that displays significant sequence similarity to the small subunit of mammalian DNA polymerase delta. cdc1+ interacts genetically with pol3+, which encodes the large subunit of DNA polymerase delta in fission yeast, and the Cdc1 protein binds to Pol3 in vitro, strongly suggesting that Cdc1 is likely to be the small subunit of Pol delta. In addition, we show that cdc1+ overexpression is sufficient to rescue cells carrying temperature‐sensitive cdc27 alleles and that the Cdc1 and Cdc27 proteins interact in vivo and in vitro. Deletion of either cdc1+ or cdc27+ results in cell cycle arrest with the arrested cells having a single nucleus with 2C DNA content. No evidence was obtained for a cut phenotype, indicating that neither cdc1+ nor cdc27+ is required for checkpoint function. cdc1 mutant cells are supersensitive to the DNA synthesis inhibitor hydroxyurea and to the DNA damaging agent MMS, display increased frequency of mini‐chromosome loss and have an extended S phase.

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Structure of the Cdt1 C‐terminal domain: Conservation of the winged helix fold in replication licensing factors

et al. 2009

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In eukaryotic replication licensing, Cdt1 plays a key role by recruiting the MCM2-7 complex onto the origin of chromosome. The C-terminal domain of mouse Cdt1 (mCdt1C), the most conserved region in Cdt1, is essential for licensing and directly interacts with the MCM2-7 complex. We have determined the structures of mCdt1CS (mCdt1C_small; residues 452 to 557) and mCdt1CL (mCdt1C_large; residues 420 to 557) using X-ray crystallography and solution NMR spectroscopy, respectively. While the N-terminal 31 residues of mCdt1CL form a flexible loop with a short helix near the middle, the rest of mCdt1C folds into a winged helix structure. Together with the middle domain of mouse Cdt1 (mCdt1M, residues 172-368), this study reveals that Cdt1 is formed with a tandem repeat of the winged helix domain. The winged helix fold is also conserved in other licensing factors including archaeal ORC and Cdc6, which supports an idea that these replication initiators may have evolved from a common ancestor. Based on the structure of mCdt1C, in conjunction with the biochemical analysis, we propose a binding site for the MCM complex within the mCdt1C.

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Interaction of Cdc2 and CdclS with a fission yeast ORC2-like protein

Cited by 131 publications

References 28 publications

Negative Regulation of Cdc18 DNA Replication Protein by Cdc2

Negative Regulation of Cdc18 DNA Replication Protein by Cdc2

The fission yeast Cdc1 protein, a homologue of the small subunit of DNA polymerase delta, binds to Pol3 and Cdc27.

Structure of the Cdt1 C‐terminal domain: Conservation of the winged helix fold in replication licensing factors

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