http://orcid.org/0000-0002-7249-4420
Figures
Abstract
Thymidine kinase converts 5-fluorodeoxyuridine to 5-fluorodeoxyuridine monophosphate, which causes disruption of deoxynucleotide triphosphate ratios. The fission yeast Schizosaccharomyces pombe does not express endogenous thymidine kinase but 5-fluorodeoxyuridine inhibits growth when exogenous thymidine kinase is expressed. Unexpectedly, we found that 5-fluorodeoxyuridine causes S phase arrest even without thymidine kinase expression. DNA damage checkpoint proteins such as the 9-1-1 complex were required for viability in the presence of 5-fluorodeoxyuridine. We also found that strains with circular chromosomes, due to loss of pot1+, which have higher levels of replication stress, were more sensitive to loss of the 9-1-1 complex in the presence of 5-fluorodeoxyuridine. Thus, our results suggest that strains carrying circular chromosomes exhibit a greater dependence on DNA damage checkpoints to ensure viability in the presence of 5-fluorodeoxyuridine compared to stains that have linear chromosomes.
Citation: Shamim HM, Minami Y, Tanaka D, Ukimori S, Murray JM, Ueno M (2017) Fission yeast strains with circular chromosomes require the 9-1-1 checkpoint complex for the viability in response to the anti-cancer drug 5-fluorodeoxyuridine. PLoS ONE 12(11): e0187775. https://doi.org/10.1371/journal.pone.0187775
Editor: Robert S. Weiss, Cornell University, UNITED STATES
Received: April 26, 2017; Accepted: October 25, 2017; Published: November 9, 2017
Copyright: © 2017 Shamim et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper.
Funding: MU is supported by: MEXT/JSPS KAKENHI Grant Number 17K07502 and Platform Project for Supporting in Drug Discovery and Life Science Research (Platform for Dynamic Approaches to Living System) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) and Japan Agency for Medical Research and Development (AMED). JMM is supported by: MRC Project Grants G1001668 and G0901011 and BBSRC project grant BB/K019805/1. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
DNA replication relies on the availability of deoxyribonucleoside triphosphates and replication fidelity is dependent on their balanced ratios. Deoxyuridine monophosphate (dUMP) is converted to deoxythiamine monophosphate (dTMP) in the presence of thymidylate synthase (TS) [1]. 5-fluorodeoxyuridine (Fudr) is phosphorylated to FdUMP by thymidine kinase (TK) (Fig 1A). FdUMP acts as an inhibitor of TS thereby hampering the synthesis of dTMP and dTTP (Fig 1B). The imbalanced dNTP synthesis consequently negatively impacts on DNA replication and induces DNA damage [2,3]. Accordingly, Fudr and 5-FU are used as a cancer chemotherapy agent to induce double stranded DNA breaks [4] (Fig 1C).
(A) Fudr conversion to FdUMP by thymidine kinase (TK) [3]. (B) In cell, dUMP is converted to dTMP by thymidilate synthase (TS) but FdUMP inhibits the thymidilate synthase resulting in no or very low amounts of dTMP and dTTP production that hamper the DNA replication process [3]. (C) Chemical structure of 5-FU. (D) The telomeres of wild-type (WT), hus1Δ, rad1Δ, pot1Δ hus1Δ and pot1Δ rad1Δ cells were analyzed using Southern hybridization at 30°C. Genomic DNA was digested with EcoRI and separated by 1.5% agarose gel electrophoresis. A DNA fragment containing telomeric DNA was used as a probe [18]. The Ethidium bromide (EtBr) image shows approximately the same amount of DNA is loaded into the all lanes. Bands with strong telomere signal are denoted ‘Telomere signal’. The weak bands above the telomere signal are either non-specific bands or telomere bands that are not fully digested by EcoRI. Sizes of marker are shown. (E) Diagram of restriction enzyme sites around the telomere and telomere associated sequences (TAS1 and TAS2) of a chromosome arm cloned in the plasmid pNSU70 [18]. The scale bar corresponds to 1 kb. (F) (Left) EtBr stained PFGE agarose gel. (Middle) NotI-digested S. pombe chromosomal DNA from the wild-type (WT), a pot1Δ isolate, a pot1Δ hus1Δ isolate, and a pot1Δ rad1Δ isolate were analyzed by PFGE. Probes for the telomeric NotI fragments (M, L, I, and C) were used [19]. The asterisk indicates a non-specific band present in all lanes. The arrow indicates a non-specific band present only in lanes 2, 3, and 4. The weak band corresponding to the size similar to C+M signal in lane 1 is a non-specific band. The size of chromosome end fragments digested by NotI, M, L, I, C, I+L, and C+M, are shown [19]. (Right) Probes for the telomeric NotI fragments (C, M, I+L) were used separately to show that the C+M signal in pot1Δ rad1Δ double mutant overlaps with L+I signal. (G) NotI restriction site map of S. pombe chromosomes. Chromosomes I, II, and III (Ch. I, Ch. II, and Ch. III) are shown. The scale corresponds to 1Mpb.
DNA damage activates cell cycle checkpoint signaling pathways, which are crucial for maintaining the cellular integrity by arresting the cell cycle, inducing apoptosis, and repairing DNA. Specifically, in response to DNA damage or replication inhibition (intra S phase checkpoint), unique sensor proteins including the ataxia-telangiectasia mutated-Rad3-related kinase (ATR), ataxia telangiectasia-related-interacting protein complex (ATRIP), and the Rad9-Hus1-Rad1 (9-1-1) complex recognize and bind to the damaged DNA [5] and block cell cycle progression [6,7]. The 9-1-1 complex is a heterotrimeric DNA clamp conserved in human, Schizosaccharomyces pombe, and Saccharomyces cerevisiae, where the functional analogs are called Rad17, Ddc1 and Mec3 respectively [8]. The 9-1-1 complex structurally resemblance to a sliding clamp, required for replication, proliferating cell nuclear antigen (PCNA) and is loaded onto DNA analogously to PCNA by a specialized clamp loader complex, Rad17-replication factor C (RFC) [9]. Eukaryotic RFC complexes consist of five subunits, which for replication are RFC1-5. For the DNA damage response the large subunit RFC1 is replaced by the cell cycle checkpoint protein Rad17 forming the Rad17-RFC2-5 complex [10]. After DNA damage, single-strand (ss) DNA generated either via resection of the DNA double-strand break or replication-fork stalling, becomes bound by replication protein A (RPA). RPA stimulates Rad17 to bind ssDNA, resulting in the loading of the 9-1-1 complex to this site [11]. The Rad17-RFC2-5 complex then binds to the 3´end of the DNA and uses ATP to open the ring of the 9-1-1 complex so that it can encircle the DNA [8]. TOPBP1 (S. pombe Rad4/Cut5) bridges between the 9-1-1 complex and the independently loaded ATR-ATRIP complex to promote checkpoint signaling.
DNA integrity is also maintained by telomeres, which comprise DNA-protein complexes located at the ends of eukaryotic chromosomes. Pot1, which is conserved from yeasts to humans, is essential for telomere protection. In the fission yeast S. pombe, deletion of pot1+ causes immediate telomere loss and chromosome circularization [12]. Circular chromosomes are found in many eukaryotes and circular chromosomes in human have been linked to some genetic disorders and cancers [13]. S. pombe cells that have circular chromosomes are sensitive to MMS, an alkylating agent that leads to damage in S phase [14]. However, the reasons for this sensitivity to replication stress are not well understood. Here, we investigated the effect of Fudr on fission yeast strains that exhibit defects in DNA damage checkpoints and/or have circular chromosomes. We show that, even though fission yeast does not express endogenous thymidine kinase, the checkpoint-defective hus1Δ single mutant is sensitive to Fudr. Notably, a hus1Δ strain with circular chromosomes (pot1Δ hus1Δ double mutant), exhibits greater sensitivity to Fudr than each single mutant. Our findings reveal that Fudr causes DNA replication arrest and induces DNA damage and that the 9-1-1 complex is required for viability upon exposure to Fudr and especially in strains with circular chromosomes.
Materials and methods
Strain construction and growth media
The strains used in this study are listed in Table 1. The pot1Δ hus1Δ double mutant, which carries a plasmid encoding pot1 (pPC27-pot1+-HA, ura4+, SH001), was created by deleting hus1+ in strain YI002 by replacement with the hus1::LEU2 DNA cassette amplified from strain SW794. The pot1Δ rad1Δ double mutant, which carries a plasmid encoding pot1+ (SH003), was constructed by deleting rad1+ in strain YI002 by replacement with the rad1::LEU2 cassette amplified from strain KT108. YEA plates containing 2 mg/ml 5-Fluoroorotic acid (FOA) at 25°C were used to select for loss of ura4+ and removal of the pot1+ plasmid to obtain pot1Δ hus1Δ and pot1Δ rad1Δ double mutants (SH002 and SH004). The pot1Δ rad9Δ double mutant, which carries the pot1 plasmid (pPC27-Leu-pot1+-HA, tk, SH009), was generated by deleting rad9+ from SH007 by integration of the rad9::ura4 DNA cassette amplified from SH008. This was plated on YEA plates containing 100 μM Fudr and incubated at 36°C to remove the pot1 plasmid to obtain the pot1Δ rad9Δ double mutant (SH010). To detect RPA in hus1Δ (FY18394) and pot1Δ hus1Δ (SH002) cells the large subunit, encoded by rad11, was tagged with monomeric red fluorescent protein (mRFP) at the C terminus, the pFA6a-mRFPnatMX6-rad11 plasmid was linearized with NspV and transformed into (FY18394) and (SH002) to create SH005 and SH006 respectively [15].
Yeast growth and flow cytometry
After overnight culture of S. pombe cells in YEA media, one sample of cells was immediately stained with propidium iodide (PI) (0 h) and the remaining samples were taken after 1, 2 and 3 h incubation with Fudr (300 μM). Cells were fixed in cold 70% ethanol in 50 mM sodium citrate, and then the samples were treated with 10 μg/ml RNase A and stained using 2.5 mg/ml PI [16]. Samples were sonicated and then analyzed by fluorescence activated cell sorting (FACS) using a Becton Dickinson FACS Calibur.
Analysis of telomeres
Telomeric sequences were detected by Southern hybridization using an AlkPhos direct kit module (GE Healthcare), according to a previously described procedure [17,18].
Pulsed-field gel electrophoresis (PFGE)
PFGE was conducted as in [17,19]. For the observation of NotI-digested chromosomes, (NotI-digested S. pombe chromosomal DNA) was fractionated on a 1% agarose gel with 0.5% TBE (50 mM Tris-HCl, 5 mM boric acid, and 1 mM EDTA [pH 8.0]) buffer at 14°C utilizing the CHEF Mapper PFGE system at 6 V/cm (200 V) and a pulse time of 60 to 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, plated on a glass-bottom dish (Iwaki) coated with 5 mg/ml lectin from Bandeiraeasimplicifolia BS-I (Sigma), were acquired using an AxioCamdigital camera (Zeiss) connected to an AxioObserverZ1microscope (Zeiss) with a Plan-Apochromat 63% objective lens (numerical aperture, 1.4). Pictures were analyzed using AxioVision Rel.4.8.2 software (Zeiss).
Lactose gradient cell cycle analysis
100 ml cultures of S. pombe cells were grown overnight to mid log phase (5x106cells/ml) in YEA media. Lactose gradients were made by freezing 10 ml aliquots of a 20% lactose solution in a clear 15 ml Falcon tube and thawing for 1 h before use. Cells were harvested at 3000 rpm for 3 min and resuspended in 750 μl water before slowly adding to the top of the gradient using a cut off blue tip. The gradients were then centrifuged at 1000 rpm for 8 min. Small G2 phase cells were collected by taking out about 0.1–0.4 ml from just below the top of smear of cells using a cut off blue tip. Cells were pelleted at 13000 rpm for 30 sec spin in an microtube and resuspended in 500 μl media, and incubated in YEA liquid medium with Fudr 300 μM at 30°C. Cell cycle progression was monitored by sampling every 20 min from 0 to 300 min, staining with diamidino 2- phenylindole (DAPI) and scoring the percentage of septated and mitotic cells under fluorescence.
Statistical analysis
Data from two independent experiments were subjected to one-way analysis of variance (ANOVA) followed by Duncan’s multiple range tests. Analyses were performed using statistical applications and differences were considered significant at an alpha level of 0.05. The statistical program used was Stat-ViewR 5.0 (Mind Vision Software, Abaccus, Concepts, Inc. Berkeley, CA, USA).
Results
Both pot1Δ hus1Δ and pot1Δ rad1Δ double mutants completely lose telomeric DNA and exhibit circularized chromosomes
To examine the role of 9-1-1 complex in the maintenance of circular chromosomes, we created a pot1Δ hus1Δ double mutant. The pot1+ deletion leads to a complete loss of telomeric DNA, thus survival relies on the circularization of chromosomes [20]. In contrast, a pot1 null mutant expressing pot1+ from a plasmid has linear chromosomes and behaves like wild-type cells. If the 9-1-1 complex is required for the maintenance of circular chromosomes, a pot1Δ hus1Δ double mutant would not be viable. Therefore, we created a pot1Δ hus1Δ double mutant with linear chromosomes, due to expression of pot1+ from a plasmid. We then selected for loss of the pot1+ expressing plasmid on YEA containing FOA. ura4+ cells accumulate a toxic intermediate on FOA so only cells that had lost the pot1+ expressing plasmid, which carries the ura4+ gene, can survive. We successfully obtained viable pot1Δ hus1Δ double mutants that had lost the pot1+ expressing plasmid. We also obtained the pot1Δ rad1Δ double mutant using same strategy. These results demonstrate that loss of 9-1-1 components Hus1 and Rad1 is not lethal with pot1Δ.
The pot1+ disruptant that has circular chromosomes loses telomeric DNA completely [20]. We next performed Southern blotting to confirm the loss of telomere signal in the pot1Δ hus1Δ and pot1Δ rad1Δ double mutants, indicative of chromosome circularization. We probed for telomere sequences in wild-type, hus1Δ and rad1Δ single mutant and pot1Δ hus1Δ and pot1Δ rad1Δ double mutant strains. Only linear chromosomes show the telomere signal and hus1Δ and rad1Δ single mutants showed very strong telomere signals consistent with having linear chromosomes, as previously reported [21]. In contrast, we observed no telomeric signal in both the pot1Δ hus1Δ and pot1Δ rad1Δ double mutants consistent with complete loss of telomeric DNA (Fig 1D and 1E). These results suggest that the pot1Δ hus1Δ and pot1Δ rad1Δ double mutants have circular chromosomes.
For the further confirmation of the circular chromosomes we examined the chromosome structure by pulse field gel electrophoresis (PFGE) of chromosome fragments generated by NotI digestion. Fragments M, L, I, and C, which map to the ends of chromosomes I and II, were seen in the wild-type cells but not in a pot1Δ strain with circular chromosomes or in the pot1Δ hus1Δ and pot1Δ rad1Δ double mutants. Instead, C+M and L+I bands were detected in the pot1Δ control strain and the pot1Δ hus1Δ double mutant (Fig 1F and 1G). Thus, this result indicates that the chromosomes of the pot1Δ hus1Δ double mutant were circularized. We also detected the L+I band in the pot1Δ rad1Δ double mutant. However, we did not detect any C+M signal for pot1Δ rad1Δ double mutant in the expected position. To test the possibility that C+M band overlaps with L+I signal in pot1Δ rad1Δ double mutant, we performed probing using C, M and I+L probe separately and observed strong signal for both probes in the same position. This result suggests that the size of C+M signal is same as that of L+I signal (Fig 1F). We suggest that the novel size of the C+M band is due to chromosome rearrangement which occurred during breakage-fusion-bridge cycles after telomere uncapping [22, 23]. Overall, results are consistent with both pot1Δ hus1Δ and pot1Δ rad1Δ double mutants carrying circular chromosomes.
pot1Δ hus1Δ and pot1Δ rad1Δ double mutants are sensitive to Fudr and Fudr treatment leads to replication arrest
We assayed the sensitivity of strains with circular chromosomes (pot1Δ), checkpoint defective (hus1Δ and rad1Δ) and strains with circular chromosomes that were also checkpoint defective (pot1Δ hus1Δ and pot1Δ rad1Δ) to differently types of replication stress, namely hydroxyurea (HU), methyl-methanesulfonate (MMS), Fudr and 5-Fluorouracil (5-FU) treatment by plating serial dilutions of the cells on the selective plates (Fig 2A). HU inhibits the synthesis of class I ribonucleotide reductase, which is responsible for the synthesis of dNTPs. Depletion of dNTP pools via HU treatment causes replication fork arrest and subsequent genomic instability [24]. Similarly, MMS modifies both guanine to 7-methylguanine and adenine to 3-methyladenine, which leads to replication blocks [25,26]. pot1Δ cells were more sensitive to MMS than the checkpoint defective strains with linear chromosomes, showing strains with circular chromosomes to be very sensitive to damage in S phase. The checkpoint-defective circular strains pot1Δ hus1Δ and pot1Δ rad1Δ double mutant cells exhibited greater sensitivity than that shown by the single mutants pot1Δ, hus1Δ, and rad1Δ. This was also the case after HU treatment and, unexpectedly, Fudr treatment. To confirm this we analyzed the effect of loss of the third component of the 9-1-1 checkpoint complex, Rad9. Checkpoint-defective pot1Δ rad9Δ double mutant cells with circular chromosomes were similarly more sensitive to Fudr than the single mutants (Fig 2B). Together these data imply that Fudr causes DNA replication stress, even though fission yeast does not express thymidine kinase. In contrast, pot1Δ hus1Δ and pot1Δ rad1Δ double mutant cells were not more sensitive to 5-FU compared to each single mutant (Fig 2A).
(A) Drug sensitivity of wild-type (WT), pot1Δ, rad1Δ, pot1Δ rad1Δ, hus1Δ, and pot1Δ hus1Δ cells was determined using a spot assay. Logarithmically growing S. pombe were serially diluted 10-fold and spotted onto YEA plates as the control and on YEA plates containing HU, MMS, Fudr, or 5-FU at the indicated concentrations. The plates were incubated at 30°C for four days. (B) Sensitivity of pot1Δ rad9Δ double mutants to Fudr. WT pot1Δ, rad9Δ, and pot1Δ rad9Δ cells were assayed as on YEA plates containing Fudr. (C-D) FACS analysis of cell cycle progression of WT, pot1Δ, hus1Δ, and pot1Δ hus1Δ double mutant cells incubated with 300 μM Fudr and 12 mM HU for 1, 2 and 3 h at 30°C. The data of WT cells arrested in G1 phase by nitrogen starvation are shown above the WT data.
The result that pot1Δ hus1Δ and pot1Δ rad1Δ double mutants were very sensitive to Fudr was intriguing. Fudr is converted to FdUMP by thymidine kinase (TK) but yeast such as S. pombe has no thymidine kinase (tk gene). To further investigate the mechanism of Fudr we performed FACS analysis of cell cycle progression in wild-type, pot1Δ, hus1Δ, and pot1Δ hus1Δ cells prior to (0 h) and following (3 h) treatment with 300 μM Fudr. In fission yeast FACS analysis is complicated. Cells spend about 60% of the cell cycle in G2, G1 is very brief and S phase is coincident with septation so the two daughter cells appear as a single unit. Thus, an asynchronous culture appears as a mainly 2C peak and cells progress from G2 (2C), mitosis (2C), through G1 (2x1C) and in S phase transiently becomes 4C (2x2C) before returning to 2C following separation of the cells in early G2. Cells from all strains (wild-type, pot1Δ, hus1Δ, and pot1Δ hus1Δ) arrested in early S phase after 3 h in Fudr (Fig 2C), similarly to cells treated with HU (Fig 2D). Therefore, we concluded from these data that Fudr induces DNA replication arrest in S. pombe cells.
pot1Δ hus1Δ cells exhibit a high frequency of chromosome segregation defects in the presence of Fudr or HU
Next we investigated the effect of Fudr on chromosome segregation using Rad11 (large subunit of RPA)-mRFP expressing cells. RPA binds single strand DNA. Rad11-GFP localizes to foci corresponding to single strand regions of DNA and in addition unbound protein is nuclear allowing us to monitor chromosome segregation under the same conditions. After 3 h incubation with 300 μM Fudr, hus1Δ and pot1Δ hus1Δ cells had increased chromosome segregation defects of 8.9 fold and 9.5 fold, respectively, compare to pot1Δ cells and 23.2 fold and 25.7 fold, respectively, compare to wild-type cells (Fig 3B). Similar chromosome segregation defects were seen after exposure to 12 mM HU for 3 h (Fig 3C). These results suggest that Fudr, like HU, causes replication stress and that hus1+, which, as part of the 9-1-1 complex, is required for both the DNA damage and intra S phase checkpoints, is essential for the proper segregation of both linear and circular chromosomes after replication stress.
(A) Representative images of chromosome segregation defects (left and middle panels), RPA foci (upper right panel) and normal fluorescence micrograph (lower right panel) of Rad11-mRFP expressing hus1Δ cells after 3 h incubation with Fudr are shown. Top image RFP, bottom image, RFP and DIC merged image. Examples of cut phenotype, where the septum bisects the nucleus (upper left), and non-disjunction, where the chromosome fail to separate (middle panel), are shown. The bar under the image represents 5 μm. (B-C) Analysis of chromosome segregation defects after Fudr and HU treatment. Wild-type (WT), pot1Δ, hus1Δ and pot1Δ hus1Δ strains that contain Rad11(RPA)-mRFP were incubated with 300 μM Fudr or 12 mM HU for 3 h at 30°C. All types of segregation defects were scored together. Percentages of defects in chromosome segregation in cells at time 0 and 3 h following the exposure to Fudr and HU are shown. Segregation defects were scored in two independent experiments, and the bar charts show the average values ± standard error. The y axis denotes the percentage of cells that showed chromosome segregation defects among the total number of cells. The numbers of cells examined (N) are shown at the top. (D-E) Analysis of RPA foci formation after exposure to Fudr or HU. WT, pot1Δ, hus1Δ and pot1Δ hus1Δ strains that contain Rad11-mRFP were incubated with 300 μM Fudr or 12 mM HU for 3 h at 30°C. % of cells with RPA foci were scored at (0 h) and after incubating (3 h) at 30°C with 300 μM Fudr (D) or 12 mM HU (E) in two independent experiments. The bar charts show the average values ± standard error. Values a and b linked by lines are significantly different at p< 0.05 (Statistical analysis ANOVA single factor followed by Duncan’s multiple ranges for multiple comparison tests). The numbers of cells examined (N) are shown at the top.
pot1Δ hus1Δ cells exhibit increased levels of ssDNA in the presence of Fudr or HU
To further investigate the effect of Fudr we monitored levels of ssDNA. Rad11 (large subunit of RPA) accumulates at the site of DNA damage or replication arrest [17,27]. In untreated conditions the numbers of cells with RPA foci was increased in pot1Δ, hus1Δ single and pot1Δ hus1Δ double mutants, indicating increased levels of DNA damage or replication stress. The percentage of cells containing RPA foci after 3 h incubation with 300 μM Fudr or 12 mM HU increased in all backgrounds but most significantly in the pot1Δ hus1Δ double mutant, which showed a 3 fold and 2 fold increase, respectively, compared to hus1Δ single mutant (Fig 3D and 3E). The increase in cells with RPA foci likely represents an increase in DNA lesions containing ssDNA after stalling of DNA replication forks [28]. We conclude that both Fudr and HU cause S-phase associated DNA damage in checkpoint defective cells and this is increased in the absence of pot1+ when chromosomes are circular.
S phase progression in the presence of Fudr causes chromosome segregation defects in the absence of the intra S phase checkpoint
Next, we asked whether S phase progression is necessary for the chromosome segregation defect in hus1Δ and pot1Δ hus1Δ cells in the presence of Fudr. We synchronized cells using lactose gradient to obtain early G2 phase cells, incubated in YEA liquid medium with Fudr 300 μM at 30°C, and analyzed cells in every 20 min from 0 to 300 min. Using diamidino 2- phenylindole (DAPI) staining, we determined that in hus1Δ and pot1Δ hus1Δ cells the percentage of chromosome segregation defects did not increase in the first M phase after release (i.e. before S phase) but increased in second M phase (i.e. after S phase) compared to wild-type and pot1Δ cells (Fig 4). Wild-type, pot1Δ, hus1Δ and pot1Δ hus1Δ cells did not show chromosome segregation defect in the absence of Fudr. These results suggest that Fudr exposure causes problems in S phase in hus1Δ and pot1Δ hus1Δ cells, lacking the intra S phase checkpoint, which induces chromosome segregation defects in the subsequent M phase.
Wild-type (WT), pot1Δ, hus1Δ, and pot1Δ hus1Δ cells were synchronized using lactose gradient and early G2 cells were incubated YEA liquid medium with Fudr 300 μM at 30°C, and cell cycle progression analyzed using DAPI staining and septation index in every 20 min from 0 to 300 min. The y axis denotes the percentage of cells that showed M phase cells, septation index and chromosome segregation defects among the total number of cells.
ssDNA is induced in S phase in the presence of Fudr in pot1Δ hus1Δ cells
Lastly, we addressed when ssDNA increased in pot1Δ hus1Δ cells in the presence of Fudr. Cells were synchronized in early G2 phase and incubated in YEA liquid medium with Fudr 300 μM at 30°C, and analyzed cells in every 20 min from 0 to 300 min. Fudr exposure increased the percentage of cells containing clusters of bright RPA foci at time point 100 min (M or S phase) in pot1Δ hus1Δ cells compared to untreated cells (Fig 5A). To understand exactly when RPA foci increase, we analyzed the percentages of cells with clusters of bright foci in M phase (binucleate, no septum) and S phase (septated binucleate) cells separately at 80 min and 100 min. Clusters of bright foci were observed only in S phase, and Fudr exposure increased the numbers of cells with clusters of bright foci about 4 fold compared to untreated cells (Fig 5C). We therefore concluded that Fudr induces DNA damage in S phase in pot1Δ hus1Δ cells.
(A) The pot1Δ hus1Δ cells that contained Rad11-mRFP were analyzed for RPA foci. Cells synchronized in early G2 using lactose gradients were incubated in YEA liquid medium with Fudr 300 μM at 30°C and samples were taken for RPA foci analysis every 20 min from 0 to 300 min. The y axis indicates the percentage of cells that exhibited no foci, single foci and multi foci among the total number of cells. (B) Representative mitotic (M phase) (binucleates without septum) and S phase (binucleates with septum) cells with no RPA foci, small RPA foci and bright RPA cluster foci in the absence and presence of Fudr at time points 80 min and 100 min after release from early G2 phase. The bar under the image represents 10 μm. (C) Number of no foci, small foci and bright cluster foci counted in M phase and S phase cells at 80 min and 100 min after release from early G2.
Discussion
Circular chromosomes in eukaryotic cells are unstable and this instability induces chromosome lost or rearrangement, which can result in other genomic imbalances and detrimental phenotypes. Examples include ring chromosome 20 in humans that is associated with epilepsy which causes abnormal electrical activity in the brain [29]. Also, the cells from atypical lipomatous tumors contain about 85% circular chromosomes and in dermatofibrosarcoma protuberans tumor cells contain approximately 70% circular chromosomes [30,31]. In these cases, therapies targeting the cells with circular chromosomes may facilitate the selective killing. However, factors affecting the stability of circular chromosomes have not been well studied. Therefore, we generated checkpoint defective strains with circular chromosomes (pot1Δ hus1Δ and pot1Δ rad1Δ double mutant) and found that these strains were more sensitive to HU and MMS than either strains with circular chromosomes (pot1Δ) or checkpoint defective single mutant (hus1Δ). This finding revealed that the DNA damage checkpoint plays important roles in the maintenance of circular chromosomes when DNA replication has been compromised. Notably, we found that Fudr also killed the pot1Δ and 9-1-1 complex double mutant cells with a high frequency compared to single mutant cells. The 9-1-1 complex consists of rad9, rad1 and hus1 and we found pot1Δ rad9Δ double mutants also exhibited synthetic lethality in the presence of Fudr (Fig 2B), demonstrating that the 9-1-1 complex is required to maintain of circular chromosomes when DNA replication has been compromised.
The effects of Fudr in S. pombe are not well understood. As S. pombe does not express thymidine kinase, Fudr may not be converted to FdUMP efficiently. Accordingly, wild-type S. pombe cells show only a slight decrease in growth rate on Fudr, however they cannot grow when thymidine kinase is expressed. Thus, Fudr has been used for counter selection to select strains that have lost the plasmid expressing thymidine kinase [32]. Furthermore, it has been reported that Fudr inhibits DNA synthesis and increases the recombination frequency in S. pombe without ectopic expression of thymidine kinase [33,34]. These findings are consisting with our result showing that Fudr affects S. pombe without thymidine kinase expression.
5-FU and Fudr are both utilized as anticancer drugs. 5-FU is used for colon and breast cancer, and Fudr for colon and ovarian cancer [2,4]. 5-FU is converted intracellularly to the active metabolites FdUMP and fluorouridine triphosphate (FUTP) [35]. FdUMP disrupts the action of TS and hampers DNA metabolism. Fudr follows the same mechanism as FdUMP and impedes the synthesis of DNA [36,37]. Alternatively, FUTP directly disrupts RNA synthesis by inhibiting the processing of pre-rRNA into mature RNA, [38,39] post-transcriptional modification of tRNAs, [40,41] and the assembly and activity of snRNA/protein complexes, thereby inhibiting splicing of pre-mRNA [42,43]. In this study, we found that the pot1Δ and 9-1-1 complex double mutant cells were shown to exhibit severe sensitivity to Fudr but not 5-FU. This might be due to a dependence on the target site of S. pombe cell toxicity and suggests that Fudr affects DNA replication and repair but not RNA metabolism. Fudr induced S phase arrest in all strains tested in this work (Fig 2C), further suggesting that Fudr exposure results in incomplete DNA replication. Incomplete DNA replication also causes chromosome segregation problems because of the physical link between sister chromatids and consistent with this, exposure to Fudr resulted in chromosome segregation defects in both hus1Δ and pot1Δ hus1Δ cells. Fudr exposure induced multiple RPA foci in pot1Δ hus1Δ double mutant cells compare to hus1Δ single mutant cells (Fig 3D) and clusters of bright RPA foci were observed in S phase not M phase (Fig 5C). These results suggest that Fudr induces DNA lesions containing ssDNA in pot1Δ hus1Δ double mutant cells during S phase [28] and leads to increased lethality in checkpoint-defective cells with circular chromosomes compared to checkpoint-proficient cells with circular chromosomes and checkpoint-defective cells with linear chromosomes.
Acknowledgments
We thank P. Baumann, T. Toda, R. Tesin, Shao-Win Wang, and the National Bioresource Project Japan for providing the plasmids and strains.
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