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Redundant control of rereplication in fission yeast - PubMed

  • ️Mon Jan 01 2001

Redundant control of rereplication in fission yeast

V Gopalakrishnan et al. Proc Natl Acad Sci U S A. 2001.

Abstract

The initiation of DNA replication at replication origins in eukaryotic cells is tightly controlled to ensure that the genome is duplicated only once each cell cycle. We present evidence that in fission yeast, independent regulation of two essential components of the initiation complex, Cdc18 and Cdt1, contributes to the prevention of reinitiation of DNA replication. Cdc18 is negatively controlled by cyclin-dependent kinase (CDK) phosphorylation, but low level expression of a mutant form of Cdc18 lacking CDK phosphorylation sites (Cdc18(CDK)) is not sufficient to induce rereplication. Similar to Cdc18, Cdt1 is expressed periodically in the cell cycle, accumulating in the nucleus in G(1) and declining in G(2). When Cdt1 is expressed constitutively from an ectopic promoter, it accumulates in the nucleus throughout the cell cycle but does not promote reinitiation. However, constitutive expression of Cdt1, together with Cdc18(CDK), is sufficient to induce extra rounds of DNA replication in the absence of mitosis. Significantly greater levels of rereplication can be induced by coexpression of Cdc18(CDK) and a Cdt1 mutant lacking a conserved C-terminal motif. In contrast, uncontrolled DNA replication does not occur when either mutant protein is expressed in the absence of the other. Constitutive expression of wild-type or mutant Cdt1 also leads to an increase in the levels of Cdc18(CDK), possibly as a result of increased protein stability. Our data are consistent with the hypothesis that control of rereplication depends on a redundant mechanism in which negative regulation of Cdt1 functions in parallel with the negative regulation of Cdc18.

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Figures

Figure 1
Figure 1

Regulation of Cdt1 expression in the cell cycle. (A) Cells expressing Cdt1-HA under the control of its endogenous promoter were fixed and stained with 4′,6-diamidino-2-phenylindole (dapi, left) or 16B12 anti-HA antibodies (right). The stained images are superimposed on Nomarski images of the fixed cells. (B) cdc25-22 cells were arrested in G2 by shifting to the nonpermissive temperature (35.5°C) for 4 h and released into synchronous culture at the permissive temperature (25°C). Cell extracts were prepared at the indicated times and fractionated into chromatin and soluble fractions. The Mcm6 and Cdt1 proteins were detected by Western blotting. A nonspecific band detected with anti-Mcm6 antibody (c) served as a loading control. (C) Extracts prepared from cells expressing Cdt1-HA under the control of the nmt1 promoter (pREP41X vector) were incubated with anti-HA antibody or nonspecific anti-glutathione S-transferase (anti-GST) antibody on agarose beads. The immunoprecipitates were analyzed by Western blotting to detect Cdt1, Mcm6, or Mcm7 proteins. A 5-fold excess of the immunoprecipitate was loaded compared with the starting material. The band marked with an asterisk is Ig heavy chain. (D) Cells expressing Cdt1-HA under the control of the 41X-nmt1 promoter were fixed and stained with 4′,6-diamidino-2-phenylindole (left) or anti-HA antibodies (right). (E) cdc25-22 cells expressing Cdt1 under the control of the 41X-nmt1 promoter were arrested in S phase (S) by incubation for 4 h in hydroxyurea (25 mM) or in G2 phase by incubation for 4 h at the nonpermissive temperature. Extracts prepared from the arrested cells and from a control population of asynchronous (asyn) cells were fractionated into detergent insoluble (chromatin) and soluble fractions. Cdt1 was detected by Western blotting with anti-Cdt1 antibodies.

Figure 2
Figure 2

Constitutive expression of Cdt1 induces rereplication in cells expressing nonphosphorylatable Cdc18. Three strains of S. pombe were constructed. The first strain (left panels) contained a plasmid expressing wild-type Cdt1 under the control of the nmt1 promoter (pREP3X vector). The second strain (center panels) contained a plasmid expressing a mutant form of Cdc18 (Cdc18CDK) under the control of the cdc18+ promoter. Cdc18CDK lacks five CDK phosphorylation sites in the N-terminal region of Cdc18. The third strain contained both plasmids (right panels). Cells were incubated for 20 h in the presence (+T) or absence (−T) of thiamine to repress or derepress the nmt1 promoter, and the DNA contents were measured by flow cytometry.

Figure 3
Figure 3

Rereplication induced by coexpression of Cdt1 and Cdc18 mutants. (A) S. pombe strains expressing the Cdt1S382A mutant alone (left panels) or coexpressing Cdt1S382A and Cdc18CDK (right panels) were analyzed for DNA content by flow cytometry after 20 h under inducing (−T) or repressing (+T) conditions. As shown in Fig. 2, the Cdt1S382A mutant was expressed under the control of the nmt1 promoter by using a pREP3X vector, and the Cdc18CDK mutant was expressed under the control of the cdc18+ promoter on a separate plasmid. (B) Cells expressing Cdt1S382A (upper) or both Cdt1S382A and Cdc18CDK (lower) were fixed and stained with 4′,6-diamidino-2-phenylindole. (C) Extracts were prepared from cells transformed with plasmids encoding wild-type Cdt1 or the mutant Cdt1 (Cdt1S382A) or vector alone under the control of the 3X-nmt1 promoter after 20 h under inducing conditions. Cdt1 protein was detected by Western blotting. Tubulin represents a loading control. (D) Wild-type Cdt1 (left panels) or Cdt1S382A (right panels) under the control of the weak 81X-nmt1 promoter was coexpressed with Cdc18CDK under the control of the cdc18+ promoter. Cells were incubated for 20 h under inducing (−T) or repressing (+T) conditions. The DNA contents of the cells were determined by flow cytometry.

Figure 4
Figure 4

Stabilization of Cdc18CDK by overexpression of Cdt1. S. pombe strains expressing the Cdc18CDK alone (lane 1) or coexpressing wild-type (WT) Cdt1 and Cdc18CDK (lanes 2, 3, and 4) or coexpressing Cdt1S382A and Cdc18CDK (lanes 5, 6, and 7) were derepressed for 20 h. Extracts were prepared as described previously (15) and analyzed by SDS/PAGE followed by immunoblotting with 16B12 anti-HA monoclonal antibodies (Babco, Richmond, CA) to detect Cdc18CDK. The blot was stained with Ponceau S to confirm equal loading.

Figure 5
Figure 5

Model for control of rereplication in fission yeast. See Discussion for details.

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