Involvement of RAD9-dependent damage checkpoint control in arrest of cell cycle, induction of cell death, and chromosome instability caused by defects in origin recognition complex in Saccharomyces cerevisiae

Abstract

Perturbation of origin firing in chromosome replication is a possible cause of spontaneous chromosome instability in multireplicon organisms. Here, we show that chromosomal abnormalities, including aneuploidy and chromosome rearrangement, were significantly increased in yeast diploid cells with defects in the origin recognition complex. The cell cycle of orc1-4/orc1-4 temperature-sensitive mutant was arrested at the G2/M boundary, after several rounds of cell division at the restrictive temperature. However, prolonged incubation of the mutant cells at 37 degrees C led to abrogation of G2 arrest, and simultaneously the cells started to lose viability. A sharp increase in chromosome instability followed the abrogation of G2 arrest. In orc1-4/orc1-4 rad9delta/rad9delta diploid cells grown at 37 degrees C, G2 arrest and induction of cell death were suppressed, while chromosome instability was synergistically augmented. These findings indicated that DNA lesions caused by a defect in Orc1p function trigger the RAD9-dependent checkpoint control, which ensures genomic integrity either by stopping the cell cycle progress until lesion repair, or by inducing cell death when the lesion is not properly repaired. At semirestrictive temperatures, orc2-1/orc2-1 diploid cells demonstrated G2 arrest and loss of cell viability, both of which require RAD9-dependent checkpoint control. However, chromosome instability was not induced in orc2-1/orc2-1 cells, even in the absence of the checkpoint control. These data suggest that once cells lose the damage checkpoint control, perturbation of origin firing can be tolerated by the cells. Furthermore, although a reduction in origin-firing capacity does not necessarily initiate chromosome instability, the Orc1p possesses a unique function, the loss of which induces instability in the chromosome.

FIG. 1.

Induction of growth arrest and lethality in orc1-4/orc1-4 cells at nonpermissive temperature. Cells were precultured at 23°C, diluted with fresh medium as described in Materials and Methods, and grown with vigorous shaking at 23 or 37°C for 24 h. (A) Diploid strains, RD301 (wild-type) and RD603 (orc1-4/orc1-4), were grown at 23 and 37°C. At indicated time points, aliquots were withdrawn, appropriately diluted with YPD medium, and cultured on YPD plates at 23°C for 5 to 7 days. The relative density of viable cells was calculated by normalizing with the density of viable cells at the zero time point. (B) Cell cycle progression in diploid strains, RD301 (wild-type) and RD603 (orc1-4/orc1-4), grown at 37°C was analyzed by flow cytometry.

FIG. 2.

Cell morphology and nucleus of orc1-4/orc1-4 diploid strain grown at nonpermissive temperature. Diploid strains, RD301 (wild-type) and RD603 (orc1-4/orc1-4), were grown as described in Materials and Methods. Cells were withdrawn at indicated times after temperature shift to 37°C, stained with DAPI, and photographed with the same magnification of a microscope. (A) Wild-type cells grown for at 37°C 8 h. (B through D) orc1-4/orc1-4 cells grown at 37°C for indicated time.

FIG. 3.

Induction of cell death and patterns of cell cycle arrest in orc2-1/orc2-1 diploid cells at different temperatures. RD605 (orc2-1/orc2-1) cells were precultured at 23°C, diluted with fresh medium as described in Materials and Methods, and grown with vigorous shaking at indicated temperatures (zero time) for 24 h. (A) At indicated time points, aliquots were withdrawn, appropriately diluted with YPD medium, and cultured on YPD plates at 23°C for 5 to 7 days. The relative density of viable cells was calculated by normalizing with the density of viable cells at the zero time point. (B) Cell cycle progression in RD605 (orc2-1/orc2-1) grown at indicated temperatures was analyzed by flow cytometry.

FIG. 4.

Effects of rad9Δ mutation on viability and cell cycle arrest of orc1-4/orc1-4 diploid cells at nonpermissive temperature. Experimental procedures were as described in the legend of Fig. 1. (A) Diploid strains, RD611 (rad9Δ/rad9Δ) and RD613 (orc1-4/orc1-4 rad9Δ/rad9Δ) were grown at 23 and 37°C. As controls, growth curves for RD301 (wild-type) and RD603 (orc1-4/orc1-4) are indicated by broken lines. (B) Cell cycle progression in diploid strains, RD611 (rad9Δ/rad9Δ) and RD613 (orc1-4/orc1-4 rad9Δ/rad9Δ), grown at 37°C was analyzed by flow cytometry.

FIG. 5.

Effects of rad9Δ mutation on temperature-sensitive growth phenotype of orc1-4/orc1-4 mutant strain. Diploid strains, RD611 (rad9Δ/rad9Δ) and RD613 (orc1-4/orc1-4 rad9Δ/rad9Δ), were logarithmically grown in YPD medium at 23°C. Cells were stepwise 10-fold diluted with YPD medium, spotted onto YPD plates, and incubated at 30 or 37°C for 2 days. As controls, growth capabilities of wild-type diploid (RD301) and orc1-4/orc1-4 diploid (RD603) strains are also shown.

FIG. 6.

Effects of rad9Δ mutation on viability and cell cycle arrest of orc2-1/orc2-1 diploid cells at different temperatures. Cells were precultured at 23°C, diluted with fresh medium as described in Materials and Methods, and grown with vigorous shaking at indicated temperatures (zero time) for 24 h. At indicated time points, aliquots were withdrawn, appropriately diluted with YPD medium, and plated onto YPD plates at 23°C for 5 to 7 days. The relative density of viable cells was calculated by normalizing with the density of viable cells at the zero time point. (A) RD301 (wild-type [open circles]), RD605 (orc2-1/orc2-1 [closed circles]), and RD615 (orc2-1/orc2-1 rad9Δ/rad9Δ [closed triangles]) were grown at 26, 30, 32, and 37°C. (B) Cell cycle progression in RD615 (orc2-1/orc2-1 rad9Δ/rad9Δ) grown at the indicated temperatures was analyzed by flow cytometry.

FIG. 7.

Assay system to screen and classify the genetic events leading to LOH. A pair of chromosomes III in parent strain (left of the arrow) and their possible alteration in 5-FOA-resistant (5-FOA^r) convertants (right of the arrow) are illustrated with relative positions of the three markers used for the analysis. The 5-FOA^r convertants are classified according to their indicated phenotypes and the altered patterns of chromosome III detected by PFGE, Southern hybridization, and PCR. The segments of chromosome III originally harboring the markers are shown by white, those of the homologous chromosome III are shown by gray, and those of another chromosome are shown by stripes. The URA3 insert at III-205 is indicated by an open triangle, the ADE2 insert at III-314 is shown by solid triangles, and the positions of intrinsic LEU2 loci are indicated by shaded bars, which are marked with a cross for the leu2 allele. A point mutation inactivating the URA3 insert is shown by a cross on the open triangle.

FIG. 8.

Induction of chromosome instability in orc1-4/orc1-4 and orc2-1/orc2-1 diploid cells in the presence and absence of Rad9p function after the temperature shift. Cells were grown at the indicated temperatures, and aliquots were withdrawn at the indicated time points and examined for frequencies of 5-FOA-resistant (5-FOA^r) cells, as described in Materials and Methods. (A) RD301 (wild-type), RD603 (orc1-4/orc1-4), RD611 (rad9Δ/rad9Δ), and RD613 (orc1-4/orc1-4 rad9Δ/rad9Δ) were grown at 23 and 37°C. (B) RD605 (orc2-1/orc2-1) and RD615 (orc2-1/orc2-1 rad9Δ/rad9Δ) were grown at 26, 30, and 32°C.