Regulation of DNA replication machinery by Mrc1 in fission yeast

Abstract

Faithful replication of chromosomes is crucial to genome integrity. In yeast, the ORC binds replication origins throughout the cell cycle. However, Cdc45 binds these before S-phase, and, during replication, it moves along the DNA with MCM helicase. When replication progression is inhibited, checkpoint regulation is believed to stabilize the replication fork; the detailed mechanism, however, remains unclear. To examine the relationship between replication initiation and elongation defects and the response to replication elongation block, we used fission yeast mutants of Orc1 and Cdc45--orp1-4 and sna41-928, respectively--at their respective semipermissive temperatures with regard to BrdU incorporation. Both orp1 and sna41 cells exhibited HU hypersensitivity in the absence of Chk1, a DNA damage checkpoint kinase, and were defective in full activation of Cds1, a replication checkpoint kinase, indicating that normal replication is required for Cds1 activation. Mrc1 is required to activate Cds1 and prevent the replication machinery from uncoupling from DNA synthesis. We observed that, while either the orp1 or the sna41 mutation partially suppressed HU sensitivity of cds1 cells, sna41 specifically suppressed that of mrc1 cells. Interestingly, sna41 alleviated the defect in recovery from HU arrest without increasing Cds1 activity. In addition to sna41, specific mutations of MCM suppressed the HU sensitivity of mrc1 cells. Thus, during elongation, Mrc1 may negatively regulate Cdc45 and MCM helicase to render stalled forks capable of resuming replication.

Figure 1.—

BrdU incorporation in the orp1-4 and sna41-928 mutants. BrdU incorporation followed by density gradient fractionation was carried out to measure the replication efficiency. (A) Positions of the ars2004 and ars2005 replication origins on chromosome II are indicated as open boxes. Positions of the ARS and non-ARS regions used as hybridization probes are indicated as solid boxes. (B) Slot blot analysis to detect the ARS (top) and non-ARS (bottom) regions in the wild-type strain (TNF1055) at 28°. The fraction number of the CsCl density gradient is indicated at the bottom. (C) BrdU incorporation in the ARS region in wild-type and orp1 (TNF1079) cells at 28°. (D) BrdU incorporation in the ARS region in wild-type and sna41 (TNF1065) cells at 30°. The percentage of the total DNA in each fraction in the absence (−BrdU, open circles) or presence (+BrdU, solid circles) of BrdU is indicated. The proportion of the dark shaded area to the total shaded area (dark and light shading) is indicated. Essentially, no BrdU incorporation was observed in the non-ARS region (B, data not shown).

Figure 2.—

HU sensitivity of the orp1-4 and sna41-928 mutants. (A) Serial dilution assay to examine the HU sensitivity of the wild-type (TNF34), chk1Δ (HM350), orp1 (TNF938), and orp1 chk1Δ (TNF1215) strains at 25° and 28°. (B) Serial dilution assay to examine the HU sensitivity of the wild-type, chk1Δ, sna41 (HM328), sna41 chk1Δ (TNF326), cds1Δ (TNF256), and cds1Δ chk1Δ (TNF351) strains at 30°. Log-phase cultures in EMM medium were serially diluted 10-fold with distilled water and plated onto YE medium supplemented with 2 mm HU. The plates were incubated at the temperature indicated above each part.

Figure 3.—

Cds1 kinase activity in the orp1-4 and sna41-928 mutants. (A) Exponentially growing wild-type (TNF34), orp1 (TNF938), and cds1Δ (TNF256) cells in EMM medium were treated with 10 mm HU for 0, 3, or 4 hr at 28°. GST-Wee1⁷⁰ was expressed in Escherichia coli and affinity purified using glutathione Sepharose 4B. Then, 1.5 mg of the total extract prepared from yeast cells and GST-Wee1⁷⁰ beads were mixed and subjected to kinase reactions with [γ-³²P]ATP to probe the phosphorylated proteins (see materials and methods). Reaction products were separated on 12% SDS–PAGE (29:1), the phosphorylated proteins were detected using a phosphorimager (top, ³²P), and the GST-Wee1⁷⁰ was visualized by Coomassie Brilliant Blue staining (bottom, Coomassie). (B) Relative amount of phosphorylated Wee1 in the wild-type, orp1, and cds1Δ cells at 28°. (C) Cds1 kinase activity was measured using wild-type, sna41 (HM328), and cds1Δ cells as in A, except the temperature used was 30°. (D) Relative amount of phosphorylated Wee1 in the wild-type, sna41, and cds1Δ cells at 30°. The value is the mean of two independent experiments, and the error bar shows the standard deviation.

Figure 4.—

Suppression of the HU sensitivity of the replication checkpoint mutants by the orp1-4 or the sna41-928 mutation. The effect of the orp1-4 (A) and sna41-928 (B) mutations on the HU sensitivity of the mrc1Δ and cds1Δ mutants was examined. HU sensitivity of the isogenic wild-type (TNF34), orp1 (TNF938), sna41 (HM328), mrc1Δ (TNF264), cds1Δ (TNF256), orp1 mrc1Δ (TNF947), orp1 cds1Δ (TNF941), sna41 mrc1Δ (TNF293), and sna41 cds1Δ (TNF283) strains was examined by a 10-fold serial dilution assay. (C) The mrc1Δ leu1 (TNF370) and sna41 mrc1Δ leu1 (TNF1848) transformants harboring the vector plasmid (p.vector; p940XB), which comprised the LEU2 gene of Saccharomyces cerevisiae and ars2004 (Okuno et al. 1997), or the plasmid containing the sna41⁺ gene (p.sna41; pTN520) or the mrc1⁺ gene (p.mrc1; pTN521) were grown to log phase in EMM, 10-fold serially diluted, and spotted onto EMM plates supplemented with HU. The HU concentration and the incubation temperature are indicated above each part.

Figure 5.—

The sna41-928 mutation does not suppress the mrc1 defect in Cds1 activation. (A) Time-course analysis of the HU sensitivity. Exponentially growing cells of the wild-type (TNF34), sna41 (HM328), mrc1Δ (TNF264), cds1Δ (TNF256), sna41 mrc1Δ (TNF293), and sna41 cds1Δ (TNF283) strains that were grown at 30° in EMM medium were treated with an acute dose of HU (10 mm). Aliquots of the cells were collected at the indicated time point, appropriately diluted with distilled water, and plated on YE medium. The viability corresponding to the 0-hr time point is indicated. The value is the mean of three independent experiments, and the error bar shows the standard deviation. (B) Exponentially growing mrc1Δ, sna41 mrc1Δ, and cds1Δ cells in EMM medium were treated with 10 mm HU for 0, 3, or 4 hr at 30°. Using the cell extracts, phosphorylation of GST-Wee1⁷⁰ was examined as depicted in Figure 3. (C) Relative amount of phosphorylated Wee1 in mrc1Δ, sna41 mrc1Δ, and cds1Δ cells.

Figure 6.—

The sna41-928 mutation suppresses the defect in recovery from HU arrest in the mrc1Δ mutant. Exponentially growing cells of the wild-type (TNF34), sna41 (HM328), mrc1Δ (TNF264), and sna41 mrc1Δ (TNF293) strains in EMM medium were treated with 10 mm HU for 3 hr at 30°, washed with distilled water, and then released into HU-free YE medium. Culture aliquots of 1 ml were collected at the indicated time points and stored at 4° in 70% ethanol. (A) Flow cytometry analysis of the DNA content of the cells after release from HU arrest. Solid histograms represent the DNA content at the indicated time points, and shaded histograms represent that at the 0-hr time point. (B) Percentage of binucleate cells indicative of passage through mitosis. At each time point, at least 200 cells were examined by microscopy, and the cells containing two DAPI-stained bodies were counted.

Figure 7.—

Mutations in the MCM helicase suppress the HU sensitivity of the replication checkpoint mutants. (A) HU sensitivity of the isogenic wild-type (TNF34), cdc19 (TNF909), mrc1Δ (TNF264), cds1Δ (TNF256), cdc19 mrc1Δ (TNF929), and cdc19 cds1Δ (TNF899) strains was examined by a 10-fold serial dilution test. (B) HU sensitivity of the isogenic wild-type, nda1 (HM259), mrc1Δ, cds1Δ, nda1 mrc1Δ (TNF912), and nda1 cds1Δ (TNF916) strains was examined. (C) HU sensitivity of the isogenic wild-type, nda4 (TNF344), mrc1Δ, cds1Δ, nda4 mrc1Δ (TNF355), and nda4 cds1Δ (NNF103) strains was examined. The HU concentration and the incubation temperature are indicated above each part. (D) The nda4 mrc1Δ leu1 (TNF374) transformants harboring the vector plasmid (p.vector: pXN289) or the plasmid containing the nda4⁺ gene (p.nda4; pTN774) were grown to log phase in EMM at 35°, serially diluted 10-fold, and spotted onto EMM plates supplemented with 6 mm HU. (E) Exponentially growing cells in EMM medium at 35° were treated with an acute dose of HU (12.5 mm). Aliquots of cells were collected at the indicated time points, appropriately diluted with distilled water, and plated on YE medium. The viability corresponding to the 0-hr time point is indicated. The value is the mean of three independent experiments, and the error bar shows the standard deviation.