Chromosome passenger complex is required for the survival of cells with ring chromosomes in fission yeast

<div><p>Ring chromosomes are circular chromosomal abnormalities that have been reported in association with some genetic disorders and cancers. In <i>Schizosaccharomyces pombe</i>, lack of function of protection of telomere 1 (Pot1) or telomerase catalytic subunit (Trt1) results in survivors with circular chromosomes. Hitherto, it is poorly understood how cells with circular chromosomes survive and how circular chromosomes are maintained. Fission yeast Cut17/Bir1, Ark1, Pic1, and Nbl1 is a conserved chromosome passenger complex (CPC) functioning mainly throughout mitosis. Here, using a temperature-sensitive mutant of CPC subunits, we determined that CPC is synthetically lethal in combination with either Pot1 or Trt1. The <i>pot1Δ pic1-T269</i> double mutant, which has circular chromosomes, showed a high percentage of chromosome mis-segregation and DNA damage foci at 33°C. We furthermore found that neither Shugoshin Sgo2 nor heterochromatin protein Swi6, which contribute to the centromeric localization of CPC, were required for the survival in the absence of Pot1. Both the <i>pot1Δ sgo2Δ</i> and <i>pot1Δ swi6Δ</i> double mutants displayed a high percentage of DNA damage foci, but a low percentage of chromosome mis-segregation, suggesting the link between the high percentage of chromosome mis-segregation and the lethality of the <i>CPC pot1Δ</i> double mutant. Our results suggest that CPC is required for the survival of cells with circular chromosomes and sheds light on the possible roles of CPC in the maintenance of circular chromosomes.</p></div

Pic1 is required for the survival of <i>trt1Δ</i> cells having circular chromosomes.

<p>(A) <i>trt1Δ pic1-T269</i> cells were streaked on YEA+FUDR plates to select for cells that could grow after the loss of plasmid expressing <i>trt1</i>^<i>+</i> and <i>tk</i>^<i>+</i>. The plasmid was retained on EMM plates supplemented with leucine and uracil (EMM+LU). (B) <i>trt1Δ pic1-T269</i> double mutants lost telomeric DNA. The loss of telomeric DNA in <i>trt1Δ pic1-T269</i> double mutant survivors was analyzed by Southern hybridization at 25°C. (C) NotI-digested chromosomal DNA from <i>pic1-T269</i>, <i>trt1Δ</i> and <i>trt1Δ pic1-T269</i> cells were analyzed by PFGE at 25°C. (D) Lack of function of Pic1 results in loss of the viability of <i>trt1Δ</i> with circular chromosome. <i>trt1Δ pic1-T269</i> double mutant cells having circular chromosomes were streaked on YEA plates at 33°C to examine the ability of the cells to grow. <i>trt1Δ</i> with circular chromosomes and <i>pic1-T269</i> were used as controls.</p

Survival of the double mutants before and after loss of Pot1 plasmid.

<p>The <i>pot1Δ cut17-275</i>, <i>pot1Δ bir1-T1</i>, <i>pot1Δ ark1-T7</i>, <i>pot1Δ ark1-T8</i> and <i>pot1Δ pic1-T269</i> double mutants carrying plasmid-borne <i>pot1</i>^<i>+</i> and <i>tk</i>^<i>+</i> were streaked on selective and counter-selective media at the indicated temperatures. Pot1 plasmid was retained on EMM plates with adenine and uracil (EMM+AU). FUDR-containing plates were used as a counter selection to examine the ability of cells to grow after loss of the Pot1 plasmid.</p

Analysis of the synthetic lethality phenotypes associated with the lack of function of Pic1 in <i>pot1Δ</i> cells.

<p>(A) <i>pot1Δ pic1-T269</i> double mutant cells lose viability at semi-permissive temperature (33°C). <i>Wild-type</i> (<i>WT</i>), <i>pot1Δ</i>, <i>pic1-T269</i>, and <i>pot1Δ pic1-T269</i> cells were streaked on YEA at 33°C. (B) Change in the cell number with time at 33°C. <i>WT</i>, <i>pot1Δ</i>, <i>pic1-T269</i>, and <i>pot1Δ pic1-T269</i> cells were incubated overnight at 25°C. An equal cell density (2.62×10⁶ cells/ml) of each strain was shifted to 33°C for 3 h. The change in the cell number after 3 h was calculated using a hemocytometer and compared. Error bars represent standard deviation (SD) from three independent experiments (n = 3). (C) Calculation of the percentage of chromosome segregation defects with septum in asynchronous living cells. <i>WT</i>, <i>pot1Δ</i>, <i>pic1-T269</i>, and <i>pot1Δ pic1-T269</i> living cells expressing Rad11 endogenously tagged with mRFP were incubated overnight at 25°C and shifted to 33°C for 3 h. The percentage of chromosome segregation defects at 25°C and 33°C was scored and compared. Representative images of cells that have chromosome segregation defects such as cut phenotype and chromosome non-disjunction are shown. (D) The percentage of RFP foci-containing cells was calculated at 25°C and after the 3-h shift at 33°C using the data from chromosome segregation defects analysis. The arrow indicates RPA foci. N in the top refers to the number of cells examined. Error bars represent SD (n = 3 experiments). The scale bar represents 5 μm.</p

Correlation between DNA damage and chromosome mis-segregation in the <i>pot1Δ pic1-T269</i> double mutant.

<p>(A) The percentage of RPA foci and chromosome mis-segregation in <i>pot1Δ pic1-T269</i> synchronized cells. <i>pot1Δ pic1-T269</i> cells harboring Rad11 endogenously tagged with mRFP were incubated overnight at 25°C. Then, the cells were synchronized by lactose gradient centrifugation producing cells at early G2, shifted to 33°C with sampling every 20 min, and scored for the percentage of RPA foci and chromosome mis-segregation. The time point (0) corresponds to the overnight culture at 25°C before temperature shift. M cells, septated cells (as a marker for S phase), and early G2 cells are shown by bars (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190523#pone.0190523.s002" target="_blank">S2 Fig</a>). (B) The percentage of RPA foci in mitotic cells with chromosome mis-segregation 100 and 120 min after temperature shift to 33°C. The scale bar represents 5 μm. Examples of cells and the total number of cells observed (N) in this experiment are shown on the top. (C) The percentage of RPA foci in septating S-phase cells with chromosome mis-segregation 100, 120, and 140 min after temperature shift to 33°C.</p

Sgo2 and Swi6 are not required for the survival of cells with circular chromosomes.

<p>(A) Spot assay of ten-fold serial dilutions of cells. <i>pot1Δ</i>, <i>pot1Δ sgo2Δ</i> and <i>pot1Δ swi6Δ</i> cells expressing Pot1 from plasmid were spotted on EMM+AU and YEA+FUDR plates at 30°C. The plasmid was retained on EMM+AU plates and cells that could grow after the loss of the plasmid were counter selected on YEA+FUDR at 30°C. (B) The telomere length of the <i>pot1Δ sgo2Δ</i> and <i>pot1Δ swi6Δ</i> double mutants was analyzed by Southern hybridization at 30°C. Both <i>sgo2Δ</i> and <i>swi6Δ</i> were used as a control for strains that retain telomeric DNA and <i>pot1Δ</i> cells as a control for strain that lost telomeric DNA. (C) NotI-digested chromosomal DNA from <i>swi6Δ</i>, <i>pot1Δ</i>, <i>pot1Δ sgo2Δ</i>, and <i>pot1Δ swi6Δ</i> cells were analyzed by PFGE at 30°C. (D) The percentage of RPA foci and chromosome mis-segregation in asynchronous living cells. The percentage of RPA foci and chromosome mis-segregation in <i>WT</i>, <i>sgo2Δ</i>, <i>swi6Δ</i>, <i>pot1Δ</i>, <i>pot1Δ sgo2Δ</i>, and <i>pot1Δ swi6Δ</i> cells harboring Rad11 endogenously-tagged with mRFP were simultaneously scored at 30°C. The total number of cells observed (N) in this experiment are shown on the top.</p

<i>Schizosaccharomyces pombe</i> strains used in this study.

<p><i>Schizosaccharomyces pombe</i> strains used in this study.</p