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

Ring chromosomes are circular chromosomal abnormalities that have been reported in association with some genetic disorders and cancers. In Schizosaccharomyces pombe, 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 pot1Δ pic1-T269 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 pot1Δ sgo2Δ and pot1Δ swi6Δ 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 CPC pot1Δ 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.


Measurement of telomere length
Telomere length was measured using Southern hybridization as previously described protocol [38] with an AlkPhos Direct Kit (GE Healthcare). For probing, the telomere-associated sequence plus telomere fragment digested with EcoRI derived from pNSU70 was used.

Pulsed-field gel electrophoresis (PFGE)
PFGE was performed as previously described [39]. For the detection of NotI-digested chromosomes, S. pombe NotI-digested chromosomal DNA was fractionated in a 1% agarose gel with 0.5 × TBE (50 mM Tris-HCl, 5 mM boric acid, and 1 mM EDTA [pH 8.0]) buffer using the CHEF Mapper PFGE system at 6 V/cm (200 V) and a pulse time of 60-120 s for 24 h. DNA was visualized by staining with ethidium bromide (1 μg/ml) for 30 min.

Microscopy
Microscope images of living cells were obtained using an AxioCam digital camera (Zeiss) connected to an Axio Observer Z1 microscope (Zeiss) with a plan-Apochromat 63 × objective lens (numerical aperture, 1.4). Pictures were captured and analyzed using AxioVision Rel. 4.8.2 software (Zeiss).

Lactose gradient synchronization
One hundred milliliters of cell culture were grown in YEA to mid-log phase (5×10 6 cells/ml) at 25˚C. Ten milliliters of 20% lactose solution was prepared in a 15 ml Falcon tube, frozen at -80˚C for 4 h, and then thawed without disturbance for 3 h at 30˚C to generate a 10-30% gradient. Cells were harvested by centrifugation at 3000 rpm for 3 min and the cell pellet was resuspended in 750 μl of sterile water. The cell suspension was layered on top of the lactose gradient using cut-off blue tips and centrifuged at 1000 rpm for 8 min, during which the cells formed a smear about half-way down the gradient. Fractions of about 0.1-0.4 ml were quickly removed from just below the top of the smear using cut-off blue tips. The cells were harvested by centrifugation at 4000 rpm for 30 sec in an Eppendorf tube, re-suspended in YEA medium, and examined under the microscope for the uniformly small early G2 cells.

Pot1 is synthetically lethal with CPC components
Since pot1Δ cells can survive only through chromosome circularization, we used the pot1Δ strain to identify the gene that is involved in the maintenance of the circular chromosome. To investigate whether CPC is required for the survival of cells with circular chromosomes, we constructed double mutants between Pot1 and CPC subunits (Cut17/Bir1, Ark1, and Pic1) and examined the ability of the double mutants to survive. Knowing that CPC is essential for cell viability, we used temperature-sensitive mutant alleles of CPC subunits as follows: cut17-275 (A990T), bir1-T1, which displays a better growth phenotype than cut17-275 at the permissive temperature [40], ark1-T7, ark1-T8, and pic1-T269. The permissive temperature of these temperature-sensitive mutants was 25˚C. The restrictive temperature for cut17-275, bir1-T1, and pic1-T269 was 36˚C, and for ark1-T7 and ark1-T8 was 33˚C (S1 Fig). The pot1Δ cut17-275, pot1Δ bir1-T1, pot1Δ pic1-T269, pot1Δ ark1-T7 and pot1Δ ark1-T8 constructed strains carry a plasmid containing pot1 + in addition to the gene for thymidine kinase (tk + ), which is used as a negative selection marker. The expression of tk + in the presence of FUDR is lethal to cells. Therefore, FUDR-containing plates were used as to counter-select cells able to grow after the loss of the plasmid. We found that all double mutants retaining the Pot1 plasmid could grow. However, the pot1Δ cut17-275, pot1Δ bir1-T1, pot1Δ ark1-T7, and pot1Δ ark1-T8 double mutants failed to grow after the loss of the Pot1 plasmid even at 25˚C (Fig 1). In the case of the pot1Δ pic1-T269 double mutant, some colonies could grow at 25˚C after the loss of Pot1 plasmid, however, the cells lost the viability at 30˚C (Fig 1). These results indicate that Pot1 is synthetically lethal with CPC and point out the importance of CPC for the survival of cells with ring chromosomes.

pot1Δ pic1-T269 double mutant survivors have lost telomeric DNA and harbor circular chromosomes
To determine whether the pot1Δ pic1-T269 double mutant that survives at 25˚C maintains the circular chromosome phenotype, genomic DNA from the pot1Δ pic1-T269 double mutant was analyzed by Southern blotting at 25˚C. DNA was digested with EcoRI and the telomeric repeats were examined utilizing a probe containing telomere and telomere-associated sequence 1 (TAS1). pot1Δ and pic1-T269 single mutants were used as control strains for cells with circular and linear chromosomes, respectively. We found that the pot1Δ pic1-T269 double mutant completely lost the telomeric hybridization signal, similar to the pot1Δ single mutant (Fig 2A  and 2B). To further confirm that the pot1Δ pic1-T269 double mutant lacked linear chromosomes and harbored circular chromosomes, the genomic DNA was digested with NotI and analyzed by PFGE at 25˚C. We found that the NotI-digested fragments M, L, I, and C, which are located at the end of chromosome I and II, were lost and bands corresponding to C+M and L+I were detected (Fig 2C and 2D). These results mirrored the behavior of cells having

Pic1 is required for the viability of trt1Δ cells having circular chromosomes
Our observation that Pot1 is synthetically lethal with CPC raised the question of whether this lethality is peculiar to Pot1 or is a generic phenotype for other cells with circular chromosomes. To address this question, we investigated the synthetic lethality between CPC and another mutant that displays the circular chromosome phenotype. In fission yeast, deletion of trt1 + encoding the catalytic subunit of telomerase results in gradual attrition of the telomere and progressive loss of viability, producing cell progeny with circular chromosomes [23]. Given that only the pot1Δ pic1-T269 double mutant was able to survive at 25˚C, we examined the synthetic lethality between Trt1 and Pic1. We constructed the trt1Δ pic1-T269 double mutant that harbors a plasmid expressing trt1 + and tk + , and examined the ability of the cells to grow after the loss of the plasmid using FUDR-containing plates. We found that the trt1Δ pic1-T269 double mutants produced colonies at 25˚C (Fig 3A). We next examined the loss of telomeric DNA by Southern blotting and chromosome circularization by PFGE in trt1Δ pic1-T269 cells, as described for the pot1Δ pic1-T269 double mutant (see Fig 2). We found that some of trt1Δ pic1-T269 double mutant cells completely lost telomeric DNA and harbored circular chromosomes (Fig 3B and 3C). Using these cells, we examined the ability of the trt1Δ pic1-T269 double mutant having circular chromosomes to grow on YEA at the semi-permissive temperature of 33˚C, the temperature at which Pic1 partially loses its function. We found that the trt1Δ pic1-T269 double mutant completely lost the ability to grow at 33˚C (Fig 3D), indicating that the trt1Δ pic1-T269 double mutant is also synthetically lethal, and implying that functional Pic1 is required to sustain the viability of trt1Δ cells having circular chromosomes. This result affirms the importance of CPC for the survival of cells with circular chromosomes and supports the notion that the genetic interaction between CPC and pot1 + is not specific, but it is a prevailing phenotype of cells with circular chromosomes. pot1Δ pic1-T269 double mutant loses viability with time and displays elevated rates of chromosome segregation defects and DNA damage foci at 33˚C As the pot1Δ pic1-T269 double mutant failed to grow on YEA at 33˚C, while the pic1-T269 single mutant grew (Fig 4A), we measured the change in cell number of the double mutant with the passage of time after a temperature shift to 33˚C in liquid culture. An advantage of monitoring growth in liquid culture is the ability to quantitatively detect subtle changes in the growth profile of the cells. By increasing the temperature from 25˚C to 33˚C, Pic1p in pic1-T269 cells partially loses its function. Therefore, if Pic1 is important for the survival of cells with circular chromosomes, a subtle decrease in its function after temperature shift would result in loss of the viability of pot1Δ cells. To test this, wild type (WT), pot1Δ, pic1-T269 and pot1Δ pic1-T269 strains were cultured overnight at 25˚C and then the cells were shifted to 33˚C for 3 h. The change in the cell number after temperature shift was determined and compared. We found that the pot1Δ pic1-T269 double mutant experienced notably growth defect tk + 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.
https://doi.org/10.1371/journal.pone.0190523.g001 The telomere length of pot1Δ pic1-T269 double mutants was analyzed by Southern hybridization at 25˚C. pot1Δ and pic1-T269 single mutants were used as a control for strains that lost and retained the telomeric DNA, respectively. Genomic DNA was digested by EcoRI and fractionated by 1.5% agarose gel electrophoresis. Telomere plus telomere associated sequence (TSA1) derived from pNSU70 was used as a probe for hybridization. To assess the total amount of DNA, the gel was stained with ethidium bromide (EtBr) before blotting onto the membrane.  compared to pot1Δ and pic1-T269 single mutants after temperature shift (Fig 4B), indicating that functional CPC is important for the survival of cells with circular chromosomes.
The existence of pot1Δ pic1-T269 double mutant survivors that could grow at 25˚C prompted us to investigate the phenotypes associated with the depletion of CPC subunits, in this case Pic1, in pot1Δ cells. Since chromosome mis-segregation is a remarkable phenotype associated with CPC dysfunction, one possible phenotype to be examined is the accumulation of chromosome mis-segregation events. Moreover, some studies linked chromosome segregation errors and the occurrence of DNA damage [41,42]. Therefore, we investigated whether the synthetic lethality of the pot1Δ pic1-T269 double mutant is associated with elevated rates of chromosome segregation defects and DNA damage. To this end, Rad11, which encodes for the large subunit of replication protein A (RPA), was tagged with monomeric red fluorescent protein (mRFP) and used as a marker for chromosome segregation and DNA damage foci simultaneously. RPA is a known marker of single-stranded DNA that accumulates during DNA replication, damage, and repair processes. Examples of the chromosome mis-segregation events we examined are cut-phenotype, uncoupling of nuclear and cellular division resulting in septum tearing segregated chromosomes, and chromosome non-disjunction. WT, pot1Δ, pic1-T269, and pot1Δ pic1-T269 cells expressing Rad11-mRFP were incubated overnight at 25˚C then shifted to 33˚C for 3 h. The percentage of chromosome segregation defects and DNA damage foci were scored at both 25˚C and 33˚C. We observed an increase in both chromosome segregation defects and DNA damage foci in pot1Δ pic1-T269 double mutants compared to pot1Δ and pic1-T269 single mutants even at 25˚C (Fig 4C and 4D). These results imply that the elevated rates of chromosome segregation defects and the accumulation of DNA damage may be the cause of the synthetic lethality phenotype of the pot1Δ pic1-T269 double mutant.

Formation of RPA foci in pot1Δ pic1-T269 does not directly link to chromosome mis-segregation events
The high percentage of RPA foci and chromosome mis-segregation patterns observed in the pot1Δ pic1-T269 double mutant prompted us to ask whether there is a link between the chromosome mis-segregation and the accumulation of DNA damage foci. To investigate this, we monitored the percentage of RPA foci and chromosome segregation defects at each stage of the cell cycle by utilizing the lactose gradient synchronization method that synchronizes the cells at early G2, marked with mono-nucleated small size cells. During the synchronization steps, pot1Δ pic1-T269 cells were cultured at 25˚C. Then, the synchronized cells were shifted to 33˚C and sampled every 20 min. We found that the RPA foci were detected in G2 cells and the percentage of RPA foci did not increase at the time points corresponding to an increase in the percentage of M and S-phase cells with chromosome mis-segregation (Fig 5A). This result suggests that chromosome mis-segregation does not directly induce RPA foci in S-phase. Moreover, we scored the percentage of mitotic cells with chromosome mis-segregation displaying RPA foci. We found that a very small fraction (~5%) of cells with chromosome mis-segregation displayed RPA foci; in other words, the majority of cells with chromosome mis-segregation had no evidence of DNA damage (Fig 5B). This result ruled out the possibility that DNA supplemented with leucine and uracil (EMM+LU). (B) trt1Δ pic1-T269 double mutants lost telomeric DNA. The loss of telomeric DNA in trt1Δ pic1-T269 double mutant survivors was analyzed by Southern hybridization at 25˚C. (C) NotI-digested chromosomal DNA from pic1-T269, trt1Δ and trt1Δ pic1-T269 cells were analyzed by PFGE at 25˚C. (D) Lack of function of Pic1 results in loss of the viability of trt1Δ with circular chromosome. trt1Δ pic1-T269 double mutant cells having circular chromosomes were streaked on YEA plates at 33˚C to examine the ability of the cells to grow. trt1Δ with circular chromosomes and pic1-T269 were used as controls.
https://doi.org/10.1371/journal.pone.0190523.g003  WT, pot1Δ, pic1-T269, and pot1Δ pic1-T269 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 damage in G2 phase induces chromosome mis-segregation. Next, to assess the possibility that the execution of cytokinesis on the mis-segregated chromosomes produces RPA foci, we scored the percentage of septated (S-phase) cells with chromosome mis-segregation displaying RPA foci. We found that a very small proportion (~10%) of septated cells with chromosome mis-segregation had RPA foci (Fig 5C), suggesting that cytokinesis does not induce RPA foci in S-phase, where the DNA damage response is active. Taken together, our results suggest that the formation of RPA foci in pot1Δ pic1-T269 cells is not directly linked to the chromosome mis-segregation events.

Loss of function of Shugoshin (Sgo2) or heterochromatin protein (Swi6) is not synthetically lethal with Pot1
The fission yeast S. pombe has two members of the Shugoshin family, Sgo1 and Sgo2. While Sgo1 has only meiotic functions, Sgo2 has meiotic and mitotic roles [43]. It was previously reported that the deletion of sgo2 + results in a remarkable reduction in the centromeric localization of the Aurora kinase complex [40,44]. In a like manner, the fission yeast heterochromatin protein Swi6 plays an important role in the centromeric localization of the Aurora kinase complex, and deletion of swi6 + results in a reduction in the centromeric localization of Aurora kinase complex [40]. This prompted the question of whether the deletion of either swi6 + or sgo2 + might exhibit a synthetic lethal interaction with pot1Δ. To test this possibility, we constructed pot1Δ sgo2Δ and pot1Δ swi6Δ double mutants carrying a plasmid containing pot1 + and tk + , and examined the ability of cells to grow after the loss of the Pot1 plasmid on FUDR-containing plates using a spot assay. We found that both double mutants were able to grow after the loss of the Pot1 plasmid. The colony formation efficiency of both pot1Δ sgo2Δ and pot1Δ swi6Δ cells was almost comparable to that of pot1Δ cells (Fig 6A), suggesting that deletion of sgo2 + or swi6 + does not influence the survival of pot1 disruptant. We further examined the loss of telomeric DNA and chromosome circularization in pot1Δ sgo2Δ and pot1Δ swi6Δ cells using Southern blotting and PFGE as described for pot1Δ pic1-T269 cells (see Fig 2). We found that both the pot1Δ sgo2Δ and pot1Δ swi6Δ double mutants completely lost the telomeric hybridization signal and that the chromosomes were circularized (Fig 6B and 6C). These results indicate that the pot1Δ sgo2Δ and pot1Δ swi6Δ double mutants are not synthetically lethal and imply that the residual accumulation of CPC is sufficient for the survival of cells with circular chromosomes.
Percentage of RPA foci, but not aberrant chromosome segregation, increases in pot1Δ sgo2Δ and pot1Δ swi6Δ double mutants To determine the reason behind the lack of synthetic lethality in the pot1Δ sgo2Δ and pot1Δ swi6Δ double mutants, we explored the phenotype of these double mutants in greater detail. If the high percentage of chromosome segregation defects and the RPA foci are the reasons for the synthetic lethality phenotype observed in the pot1Δ pic1-T269 double mutant at 33˚C, then we might expect that both the pot1Δ sgo2Δ and pot1Δ swi6Δ double mutants would display lower levels of chromosome segregation defects and RPA foci. To investigate this, we analyzed the percentage of chromosome segregation defects and the RPA foci using the pot1Δ sgo2Δ and pot1Δ swi6Δ double mutants harboring Rad11 endogenously tagged with mRFP. Both the pot1Δ sgo2Δ and pot1Δ swi6Δ double mutants displayed a high percentage of RPA foci, but 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.
https://doi.org/10.1371/journal.pone.0190523.g004  both showed a low percentage of chromosome mis-segregation compared to the pot1Δ pic1-T269 double mutant (Fig 6D). These results suggest that the low percentage of aberrant chromosome segregation would be the reason for the lack of synthetic lethality phenotype observed in the pot1Δ sgo2Δ and pot1Δ swi6Δ double mutants.

Discussion
Some studies reported the association of ring chromosomes with clinical disorders such as epilepsy, mental and developmental defects, as well as cancers [7][8][9][10][11][12][13][14][15][16]. However, little is known regarding the maintenance of circular chromosomes and how cells with circular chromosomes can survive. The unpredicted abnormalities that associate with the ring chromosome formation may stem from the unstable mitosis resulting from abnormal chromosome segregation and sister chromatid exchange [45]. In this study, we characterized the importance of CPC in the survival of cells with ring chromosomes using a synthetic lethality approach.
Throughout this study, we mainly used pot1 disruptant as a model for a strain with circular chromosomes, since pot1Δ has the advantage of producing cells that can survive only through chromosome circularization [22]. Although chromosome circularization in fission yeast has been reported in other mutants [23,46], some of these mutants can alternatively give rise to survivors with linear chromosomes [23].
To examine the importance of CPC for the survival of cells with circular chromosomes, we first generated a double mutant between pot1Δ and a temperature-sensitive mutant allele of cut17/bir1, and tested the ability of the pot1Δ cut17-275 and pot1Δ bir1-T1 double mutants to grow after the loss of plasmid-borne Pot1. We found that the double mutants lost the ability to grow after the loss of the Pot1 plasmid even at 25˚C (Fig 1), indicating that the pot1Δ cut17-275 and pot1Δ bir1-T1 double mutants are synthetically lethal. Cut17/Survivin is detected in a complex with Pic1/INCENP and Ark1/Aurora B in many organisms. This complex is interdependent as the disruption of any of the CPC subunits leads to similar phenotypes [35,47]. In budding yeast, the lack of function of the INCENP homolog (Sli15) and Aurora homolog (Ipl1) has identical phenotypes [37]. These findings are consistent with our observations that the other CPC subunits, Ark1 and Pic1, were also synthetically lethal with Pot1 (Fig 1). We further showed that the lack of function of Pic1 also resulted in death of trt1Δ cells having circular chromosomes (Fig 2D), indicating that CPC is required for survival of cells that have circular chromosomes and that it is not a specific genetic interaction with Pot1.
We further found that the functional inactivation of Pic1 by a temperature shift in pot1Δ cells, which have circular chromosomes, resulted in growth defects, accumulation of high rates of chromosome segregation defects, and increase in the percentage of DNA damage foci ( Fig  4B, 4C and 4D). These results raise the possibility of a link between the chromosome segregation events and the formation of DNA damage foci. One possibility is that the RPA foci are produced earlier and induce chromosome mis-segregation events. It has been reported that the formation of pre-mitotic DNA damage that persists into mitosis can lead to chromosomal instability and segregation errors [48,49]. However, our data suggest that this possibility is unlikely as we found that the majority of mitotic cells with chromosome mis-segregation had 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 pot1Δ sgo2Δ and pot1Δ swi6Δ double mutants was analyzed by Southern hybridization at 30˚C. Both sgo2Δ and swi6Δ were used as a control for strains that retain telomeric DNA and pot1Δ cells as a control for strain that lost telomeric DNA. (C) NotI-digested chromosomal DNA from swi6Δ, pot1Δ, pot1Δ sgo2Δ, and pot1Δ swi6Δ 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 WT, sgo2Δ, swi6Δ, pot1Δ, pot1Δ sgo2Δ, and pot1Δ swi6Δ 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.
https://doi.org/10.1371/journal.pone.0190523.g006 no sign of DNA damage (Fig 5B). Some reports linked aberrant chromosome segregation and the formation of DNA damage, suggesting that the entrapment of mis-segregated chromosome at the cleavage furrow during cytokinesis leads to chromosome breakage and generation of DNA damage [41,42]. Therefore, a second possibility is that the DNA damage foci are produced after chromosome mis-segregation when the septum tears the mis-segregated chromosomes. If this is the case, then we would expect to observe a high percentage of septated Sphase cells with chromosome mis-segregation that display RPA foci. Instead, we found that the majority of the S-phase cells with chromosome mis-segregation that had septa did not have RPA foci (Fig 5C), indicating that this possibility is less likely. Nonetheless, it remains possible that the DNA damage foci are produced in the next G2 as a result of genomic instability arising from chromosome segregation errors. A third possibility is that the RPA foci formation and chromosome mis-segregation events are not directly linked. Indeed, we support this possibility since the first and second possibilities discussed above are less likely. Moreover, as will be discussed below, both the pot1Δ sgo2Δ and pot1Δ swi6Δ double mutants displayed a high percentage of DNA damage foci even though they had low rates of chromosome segregation defects, supporting the idea that DNA damage foci and chromosome segregation defects are not directly linked. In line with this, some studies showed no direct relationship between chromosome mis-segregation and the generation of DNA damage. [50,51]. However, this possibility requires further investigation since there is no direct evidence yet.
We further investigated whether the reduction of the centromeric localization of CPC upon deletion of sgo2 + or swi6 + is lethal to cells with circular chromosomes. In humans, the dual inhibition of hSgo1 and Sgo2 by RNA interference results in reduction in the centromeric localization of the Aurora kinase complex [52]. Similar results have been observed in fission yeast upon deletion of either sgo2 + or swi6 + [40,44]. Interestingly, we found that both the pot1Δ sgo2Δ and pot1Δ swi6Δ double mutants are viable, suggesting that the residual centromeric localization of CPC may be sufficient to sustain the viability of cells with circular chromosomes ( Fig 6A). Consistent with this, both the sgo2Δ and swi6Δ single mutants were viable but deletion of CPC subunits was lethal. Our data also agrees with the finding that Swi6 is dispensable for the telomerase-minus trt1Δ circular survivors [53].
It has been shown that the lack of function of CPC subunits leads to higher rate of chromosome mis-segregation events than either sgo2Δ or swi6Δ single mutants. For instance, deletion of sgo2 + produces a mild effect on chromosome segregation in unperturbed cycling cells [44]. Even though both the pot1Δ sgo2Δ and pot1Δ swi6Δ double mutants displayed a high percentage of DNA damage foci, both consistently displayed a remarkably low percentage of chromosome mis-segregation compared to the pot1Δ pic1-T269 double mutant (Fig 6D), suggesting that the elevated rates of chromosome mis-segregation per se, but not the DNA damage, is the likely reason for the synthetic lethality observed in pot1Δ pic1-T269 cells at 33˚C. This result further supports our suggestion that the link between chromosome mis-segregation and the generation of DNA damage foci in the pot1Δ pic1-T269 double mutant is less likely. Our results also could raise a question regarding the possible roles of CPC, Sgo2, and Swi6 in the prevention of DNA damage when the chromosome is circular, which would be interesting to investigate.  cut17-275, bir1-T1, pic1-T269, ark1-T7 and ark1-T8) on YEA at the indicated temperatures. Note that both ark1-T7 and ark1-T8 cannot form colonies at 33˚C, the temperature at which cut17-275, bir1-T1, and pic1-T269 can still grow. (TIF)