Control of DNA rereplication via Cdc2 phosphorylation sites in the origin recognition complex

Abstract

Cdc2 kinase is a master regulator of cell cycle progression in the fission yeast Schizosaccharomyces pombe. Our data indicate that Cdc2 phosphorylates replication factor Orp2, a subunit of the origin recognition complex (ORC). Cdc2 phosphorylation of Orp2 appears to be one of multiple mechanisms by which Cdc2 prevents DNA rereplication in a single cell cycle. Cdc2 phosphorylation of Orp2 is not required for Cdc2 to activate DNA replication initiation. Phosphorylation of Orp2 appears first in S phase and becomes maximal in G(2) and M when Cdc2 kinase activity is required to prevent reinitiation of DNA replication. A mutant lacking Cdc2 phosphorylation sites in Orp2 (orp2-T4A) allowed greater rereplication of DNA than congenic orp2 wild-type strains when the limiting replication initiation factor Cdc18 was deregulated. Thus, Cdc2 phosphorylation of Orp2 may be redundant with regulation of Cdc18 for preventing reinitiation of DNA synthesis. Since Cdc2 phosphorylation sites are present in Orp2 (also known as Orc2) from yeasts to metazoans, we propose that cell cycle-regulated phosphorylation of the ORC provides a safety net to prevent DNA rereplication and resulting genetic instability.

FIG. 1

Cdc2 phosphorylation sites in Orc2 homologs. The N-terminal region shown in black is least conserved. The beginning of the conserved region is indicated (amino acid 193 in Orp2). Bars are drawn to scale. Percent amino acid identity (ID) of the various C-terminal domains compared with Orp2 is shown. P represents consensus Cdc2 phosphorylation sites [(S/T)PX(K/R)]; SP and TP sites that are not perfect matches to the consensus are not shown. The amino acid sequences of Orp2 wild type, Orp2-T4A, and Orp2-T3D show the mutations made, and the single consensus Cdc2 phosphorylation site in Orp2 is underlined. Abbreviations for homologs: h, human; X, Xenopus; Dm, D. melanogaster; Sc, S. cerevisiae; Sp, S. pombe.

FIG. 2

In vitro phosphorylation of Orp2 by Cdc2 kinase. (A) GST-Orp2⁺ (wt) and GST-Orp2-T4A (T4A) were incubated with S. pombe Cdc2 and [γ-³²P]ATP and analyzed by SDS-PAGE. Incorporation of ³²P into GST-Orp2 was visualized by autoradiography (left). Purity and relative amounts of GST-Orp2 and GST-Orp2-T4A added to the reactions were seen by Coomassie staining (right). (B) Phosphopeptide maps of proteins from kinase reactions analyzed in panel A. The GST-Orp2 or GST-Orp2-T4A bands were cut out of the gel, digested with trypsin, and analyzed. To visualize phosphopeptides, 10-fold more material (10×) was analyzed for the low-level reactions with GST-Orp2 (no kinase) and GST-Orp2-T4A (site mutant) than for GST-Orp2 plus Cdc2.

FIG. 3

In vivo modification of Orp2 requires sites needed for Orp2 phosphorylation in vitro. Endogenous Orp2 protein in whole-cell extract was analyzed by SDS-PAGE and immunoblotting using Orp2 antibodies. Several mobility forms of Orp2 were observed. Orp2-specific bands are indicated. A cross-reacting band that is not Orp2 is present in all cells including those lacking Orp2 (orp2Δ) and is marked (*). Lanes are as follows: M, nuc2 mutant cells arrested in mitosis; G1, cdc10 mutant cells arrested in G₁; orp2Δ, selective spore germination and growth of cells lacking orp2; orp2⁺, wild type; orp2-T4A, mutant in which orp2-T4A is the only orp2 gene in the cells, strain JLP238.

FIG. 4

Cell cycle-regulated phosphorylation of Orp2. (A) Orp2 protein throughout the cell cycle. Cells were synchronized by block and release of a cdc25-22 mutant. Samples were harvested for analysis every 20 min through two consecutive cell cycles. Whole-cell extracts were analyzed by SDS-PAGE and immunoblotting using affinity-purified Orp2 antibodies. Synchrony and cell cycle progression were determined by septation index (above). Asynchronous cells (WT) and an orp2 deletion control (Δ) on the left show which bands are Orp2 specific; the orp2Δ was made by selective spore germination (see Materials and Methods). (B) Orp2 protein during cell cycle arrest. Mutants were arrested by shift to 36.5°C for 4 h, the mutant is indicated above each lane, and the stage of cell cycle arrest is shown at the top. Orp2 bands are indicated. Cross-reacting bands that are not Orp2 are marked (*). Abbreviations: asyn, asynchronous wild type shifted to 36.5°C for 4 h; HU, wild type incubated with 12 mM HU for 4 h at 32°C; orp2Δ, cells with orp2 deletions from selective spore germination of an orp2Δ/orp2⁺ heterozygote lack Orp2-specific bands. (C) Orp2 protein in cells lacking essential genes. Immunoblot analysis of Orp2 from cells obtained by selective germination of spores with deletions of essential genes orp2, cdc13, and hsk1 as indicated. wild-type, selective spore germination of a wild-type diploid heterozygous at ura4 (ura4-D18/ura4⁺, JLP52).

FIG. 5

Sensitivity of orp2-T4A mutants to rereplication. Haploid isogenic strains JLP524 orp2⁺ and JLP519 orp2-T4A were compared upon induction of GST-Cdc18* from the nmt1 promoter. (A) DNA content of cells after removal of thiamine to induce the nmt1-driven GST-cdc18*. 2C DNA content is indicated. “Re-replication” marks DNA content greater than approximately 4C. (Top) orp2⁺ GST-Cdc18* (JLP524); (bottom) orp2-T4A GST-Cdc18* (JLP519). (B)