Smc5p promotes faithful chromosome transmission and DNA repair in Saccharomyces cerevisiae

Abstract

Heterodimers of structural maintenance of chromosomes (SMC) proteins form the core of several protein complexes involved in the organization of DNA, including condensation and cohesion of the chromosomes at metaphase. The functions of the complexes with a heterodimer of Smc5p and Smc6p are less clear. To better understand them, we created two S. cerevisiae strains bearing temperature-sensitive alleles of SMC5. When shifted to the restrictive temperature, both mutants lose viability gradually, concomitant with the appearance of nuclear abnormalities and phosphorylation of the Rad53p DNA damage checkpoint protein. Removal of Rad52p or overexpression of the SUMO ligase Mms21p partially suppresses the temperature sensitivity of smc5 strains and increases their survival at the restrictive temperature. At the permissive temperature, smc5-31 but not smc5-33 cells exhibit hypersensitivity to several DNA-damaging agents despite induction of the DNA damage checkpoint. Similarly, smc5-31 but not smc5-33 cells are killed by overexpression of the SUMO ligase-defective Mms21-SAp but not by overexpression of wild-type Mms21p. Both smc5 alleles are synthetically lethal with mms21-SA and exhibit Rad52p-independent chromosome fragmentation and loss at semipermissive temperatures. Our data indicate a critical role for the S. cerevisiae Smc5/6-containing complexes in both DNA repair and chromosome segregation.

Figure 1.

Characterization of temperature-sensitive alleles of SMC5. (a) Thirty-five thousand cells from log-phase cultures were deposited onto YPAD plates prewarmed to the indicated temperature and grown at that temperature for 2 days. (b) Long-term viability of smc5-31 (yGC231) and smc5-33 (yGC233) cells at 36°. The dashed line is an exponential curve fit to the data. The half-life of both strains at 36° is 1.1 days, R² = 0.74 and 0.97 for smc5-31 and smc5-33, respectively. (c) Twenty thousand cells and fivefold dilutions thereof were deposited onto prewarmed YPAD plates and grown at the indicated temperature for 2 days. (d and e) Liquid culture growth of SMC5 (yGC141), smc5-31 (yGC231), and smc5-33 (yGC233) cells at 22° and 36°. (f and g) Viability of SMC5 (yGC141), smc5-31 (yGC231), and smc5-33 (yGC233) cells grown at 22° and 36°. The experiment in d–g was repeated, and representative data are shown. The offset between viability data from liquid culture vs. that from solid medium arises from the fact that 100% of the cells plated in b are viable. The slow death of smc5 cells allows for some cell division, effectively increasing the number of cells assayed.

Figure 2.

Cell-cycle distribution and nuclear morphology of smc5 cells. The percentages of cells that were unbudded, single budded, large budded, and anomalous were assayed microscopically at 22° (a) and at 36° (b). More than 300–700 cells per strain at both time points were counted while blind with respect to the genotype and temperature. (c) Asynchronous cultures of SMC5 (yGC141), smc5-31 (yGC231), and smc5-33 (yGC233) cells grown at 22° or at 36° for 6 hr were stained with propidium iodide and analyzed by FACS for DNA content. (d) DNA in cells from a were stained with 4′,6-diamidino-2-phenylindole (DAPI) and examined by fluorescence or differential interference contrast (DIC) microscopy.

Figure 3.

DNA-damage checkpoint induction in smc5 cells and synthetic viability with rad52Δ0. (a) Lysates from SMC5 (yGC141), smc5-31 (yGC231), and smc5-33 (yGC233) cells grown at 22° and at 36° for 0–6 hr were probed with anti-Rad53p antibody. (b) Lysates from a were assayed for Rad53p auto-kinase activity in situ after refolding on the membrane. SMC5 yGC141 cells exposed to 0.033% MMS for 2 hr served as a positive control for Rad53p activation. An unidentified lower-molecular-weight kinase (std) serves as a loading control. (c) Fifteen thousand, 3000, 600, 120, and 24 cells from log-phase SMC5 RAD52 (yGC141), SMC5 rad52Δ0 (yGC177), smc5-31 RAD52 (yGC231), smc5-31 rad52Δ0 (yGC182), smc5-33 RAD52 (yGC233), and smc5-33 rad52Δ0 (yGC185) cultures were grown on YPAD for 2 days at 22° or 36°. After 3 days at 36°, the plate was placed at 22° and allowed to grow for a further 3 days.

Figure 4.

The DNA-damage response is different in smc5-31 and smc5-33. (a) Ten thousand, 1000, 100, and 10 cells from log-phase SMC5 RAD52 (yGC141), SMC5 rad52Δ0 (yGC177), smc5-31 RAD52 (yGC231), and smc5-33 RAD52 (yGC233) cultures were plated onto YPAD medium containing the indicated concentrations of drug or exposed to UV-C at 60 J/m² and allowed to grow for 3 days at 22°. (b) One-hundred-twenty thousand, 12,000, 1200, 120, and 12 cells were plated on YPAD medium containing the indicated concentration of MMS. Similar results were obtained with plates containing 0.04% MMS, but growth of even wild-type strains on this medium is poor. (c) Lysates from SMC5 (yGC141), smc5-31 (yGC231), and smc5-33 (yGC233) cells grown for 3 hr at 22° in YPAD, YPAD + 25 mm HU, or YPAD + 0.03% MMS were probed with anti-Rad53p antibody. (d) As in a, but with SMC5 RAD52 (yGC141), SMC5 rad52Δ0 (yGC177), smc5-31 rad52Δ0 (yGC182), and smc5-31 RAD52 (yGC231). The above assays were performed multiple times.

Figure 5.

Global, Rad52p-independent loss of heterozygosity in smc5 strains. All assays were performed at 30° unless noted. (a) Appearance of SMC5/SMC5 (BY4743), smc5Δ0/smc5Δ0 pGC251-SMC5 (yGC170), smc5Δ0/smc5Δ0 pGC251-smc5-31 (yGC172), and smc5Δ0/smc5Δ0 pGC251-smc5-33 (yGC173) colonies when plated on medium containing lead ions. (b) Percentage of cell divisions from the strains in a experiencing MET15 LOH at 22° and at 30°. In multiple experiments, 1000–5000 cell divisions were assayed per strain per temperature. For comparisons between wild type and smc5 at both temperatures, χ²-analysis returned values >50. The LOH assay is the only one we have performed to find any difference between episomal and genomic SMC5 (χ² = 23). (c) LOH at 22° at the MAT loci on chromosome III in BY4743, yGC170, yGC172, and yGC173 cells. The rate of LOH at the MAT loci in wild-type cells is comparable to that found by Spencer and Hieter (Spencer et al. 1990). The simple mean of the data gave rates of 0.011, 0.015, 0.12, and 0.28% for BY4743, yGC170, yGC172, and yGC173, respectively. (d) LOH rates at 30° of rad52Δ0 versions of the strains in a (yGC187, yGC188, yGC189, and yGC190). χ²-analysis gave values >1000 for comparisons between wild type and smc5. The difference between yGC187 and yGC188 is not significant. (e) LOH in the arg1∷MET15 strains yGC250, yGC251, and yGC252. From 5000 to 33,000 cell divisions were analyzed. (f) LOH at arg1∷MET15 in rad52 cells. From 4000 to 20,000 cell divisions were analyzed. (g) LOH at ADE2 in rad52 cells. From 14,000 to 40,000 cell divisions were analyzed. (h) LOH at ADE2 in rad52 cells. From 8000 to 22,000 cell divisions were analyzed.

Figure 6.

Loss of heterozygosity in smc5 cells is a result of chromosome fragmentation and loss. (a) Diagram of colorimetric markers on chromosome XV in yGC250, -251, and -252 and yGC280, -281, and -282. The diagram is to scale. (b) Frequency of loss of both the MET15 and ADE2 markers in yGC251 and -252.

Figure 7.

Overexpression of MMS21 suppresses the temperature sensitivity of smc5 cells. (a) SMC5 (yGC141), smc5-31 (yGC231), and smc5-33 (yGC233) cells were transformed with either pRS316 or a 2μ vector with the indicated gene driven by the GAL1 promoter. Fifteen thousand, 3000, 600, 120, and 24 cells were plated on prewarmed SC–ura–leu medium containing 2% dextrose or 2% galactose as indicated. Cells were allowed to grow for 3.5 days. Equal growth was seen on dextrose at 36° (data not shown). Transformants of strains with an intact chromosomal SMC5 locus performed identically to yGC141 transformants under all conditions in these assays (data not shown). Three transformants per strain were tested and the experiment was performed twice. Overexpression of the relevant protein was assayed by Coomassie stain (data not shown). (b) Mms21p suppression of smc5 in liquid culture. smc5-31 and smc5-33 strains with either pRS316 or GAL1-inducible MMS21 were grown in dextrose until midlog and then shifted to 36° SC–ura Gal medium. The doubling times of smc5-31 + Mms21p and smc5-33 + Mms21p strains are 16.3 and 17.7 hr, respectively. (c) Plates from a were grown at 22° for 3 days following 3.5 days of growth at 36°. (d) Overexpression of Nse4p causes DNA damage hypersensitivity. This was done as in a, except dilutions were spotted on SC–ura–leu plates with 25 mm hydroxyurea.

Figure 8.

mms21-SA cannot suppress smc5 and is synthetically lethal with it. (a) Suppression of smc5 temperature sensitivity by Mms21p requires the SUMO ligase activity of Mms21p. SMC5 (yGC141), smc5-31 (yGC231), and smc5-33 (yGC233) cells were transformed with either pRS316 or a 2μ vector with the indicated gene driven by the GAL1 promoter. Twenty-five thousand, 5000, 1000, 200, and 40 cells were plated. The overall level of suppression was lower than that in Figure 7a, as cells were grown for 2.5 days. (b) Mms21p promotes survival of smc5 cells at the restrictive temperature. This was done as in Figure 7c. (c) The smc5 and mms21-SA alleles are synthetically lethal. MMS21 or mms21-SA cells containing SMC5 on a URA3 plasmid and SMC5, smc5-31, or smc5-33 on a LEU2 plasmid (yGC301–306) were grown on 5-FOA medium at 22° for 5 days. This assay was done in triplicate with independent transformants. On one FOA plate there was slight growth of some smc5-33 mms21-SA cells. (d) Hypomorphism of smc5 alleles rescues the nibbled-colony morphology of “wild-type” yGC141. yGC141 (SMC5), yGC231 (smc5-31), and yGC233 (smc5-33) cells were grown for 5 days on YPAD at 22°.