Role of swi7H4 Mutant Allele of DNA Polymerase α in the DNA Damage Checkpoint Response

Besides being a mediator of initiation of DNA replication, DNA polymerase α plays a key role in chromosome maintenance. Swi7H4, a novel temperature sensitive mutant of DNA polymerase α was shown to be defective in transcriptional silencing at the mating type centromere and telomere loci. It is also required for the establishment of chromatin state that can recruit the components of the heterochromatin machinery at these regions. Recently the role of DNA polymerase α in the S-phase alkylation damage response in S. pombe has also been studied. Here we investigate whether defects generated by swi7H4, a mutant allele of DNA polymerase α can activate a checkpoint response. We show that swi7H4 exhibit conditional synthetic lethality with chk1 null mutant and the double mutant of swi7H4 with chk1 deletion aggravate the chromosome segregation defects. More importantly swi7H4 mutant cells delay the mitotic progression at non permissive temperature that is mediated by checkpoint protein kinase Chk1. In addition we show that, in the swi7H4 mutant background, cells accumulate DNA damage at non permissive temperature activating the checkpoint kinase protein Chk1. Further, we observed synthetic lethality between swi7H4 and a number of genes involved in DNA repair pathway at semi permissive temperature. We summarize that defects in swi7H4 mutant results in DNA damage that delay mitosis in a Chk1 dependent manner that also require the damage repair pathway for proper recovery.


Introduction
Faithful chromosomal segregation is precisely monitored by the array of proteins involved in cell cycle events, like DNA replication, repair, mitosis and cytokinesis. Checkpoints monitor key events during the cell cycle and block subsequent event if earlier ones are incomplete and thus ensure the accuracy of each step in the cell cycle [1]. The cell maximizes its efforts of survival by coordinating with these proteins. DNA replication is the process through which a cell passes its genetic information from one generation to another. Hence the DNA replication machinery must be properly governed and protected from any genotoxic agent. In response to genotoxic assault the cell cycle abort, DNA repair machinery takes over and if the defects are still not repaired, then the cells undergo apoptosis. DNA damage response (DDR) senses different types of DNA damage and transmit the signal to activate the cell cycle checkpoint response [2]. Checkpoint pathways consist of various sensor, transducer and effector molecules. Sensors protein includes MRN complex (mre11, rad 50 and nbs1) that recognises DNA double strand break [3] while Rad17 and Rad9-Hus1-rad1 (9-1-1) complex recognises replication stress [4]. Sensor molecules further activate transducer kinases, ATM (Ataxia-telangiectasia mutated) and ATR (ATM and Rad3-related). The Tel1/Rad3 in S. pombe and Tel1/Mec1 in S. cerevisiae are responsible for the activation of the effector kinases, Chk1 and Chk2 [5].
DNA polymerases are a family of enzymes that are responsible for all forms of DNA replication [6]. DNA polymerases ensure high fidelity of DNA replication through a balance between polymerase and the proof reading activities. In vitro reconstitution studies have demonstrated that three proteins are required for the initiation of DNA synthesis on duplex DNA molecules. These include the DNA polymerase α-primase complex, replication protein A, and T antigen [7,8]. DNA polymerase α was the first polymerase identified in eukaryotic cells and for several years it was thought to be the only polymerase required for chromosomal DNA replication [9]. Mutation in the polα gene affects several cellular processes such as DNA repair and recombination [10,11], transcriptional silencing [12,13], checkpoint activation [14,15] and telomere length maintenance [16][17][18][19]. Recently it is also reported that DNA polymerase α (swi7) and the flap endonuclease Fen1 (rad2) act together in the S-phase alkylation damage response in S. pombe [20].
A temperature sensitive mutant allele of DNA polymerase α, swi7H4 is defective in silencing at centromere, telomere and mating type loci [12,13]. Further, DNA polymerase α was shown to be required for replication mediated recruitment of Swi6 to heterochromatin [13]. Fission yeast Cds1, a critical transducer of the replication checkpoint was isolated as multi-copy suppressor of swi7H4 [21] indicating a connection between replication machinery and checkpoint response.
In this study, we elucidated the effect of swi7H4 mutant on cell cycle progression. A conditional synthetic lethality of swi7H4 mutant with chk1 deletion was observed and the double mutant of swi7H4 with chk1 deletion aggravates the chromosome segregation defects. Further swi7H4 mutant cells delay the mitotic progression at non permissive temperature in a Chk1 dependent manner. More importantly, swi7H4 mutant cells accumulate DNA damage at non permissive temperature and hence activate the checkpoint kinase protein Chk1. The genetic interaction of swi7H4 with a number of genes involved in DNA repair pathway was also studied.

Strains and growth condition
Schizosaccharomyces pombe strains used in this study are listed in Table 1. Standard genetic methods were utilized for making strains as described in [22]. Standard yeast protocols were used for strain construction, growth and medium preparation as described in [23]. For temperature-shift experiments, cells were grown to mid log phase at 25°C, 10 7 cells were serially diluted and spotted on YEA plates and incubated at the indicated temperature. For survival assay, the mid log phase culture was shifted at 36°C, samples were collected at different time intervals, 1000 cells from each sample were plated on YEA plates and incubated at 25°C until colonies appear. Colonies were counted and graph was plotted. For passing mitosis experiments, cells were synchronized in S phase by growing them in 12 mM hydroxyurea (HU) for 4 h at 25°C, washed and released at 36°C. The samples were collected at a 15 min interval and the number of cells having two nuclei was counted as an indication of cells entering into mitosis as described previously [24].

Fluorescence Microscopy
For staining nuclei, cells were grown to mid-log phase at 25°C then shifted to 36°C, samples were collected at the indicated times, fixed with 70% ethanol and stained with DAPI(4',6diamidino-2-phenylindole), visualized using a fluorescence microscope. About 200 cells were counted for aberrant nuclei and their percentage was plotted.

Preparation of lysates and Western-blot analysis
Cells containing Chk1 tagged with triple HA were grown to mid-log phase at 25°C then shifted at 36°C in water bath. Cells were harvested at different time intervals by centrifugation and lysed using glass beads and fast prep cell disruptor (MP Biosciences). Lysate was prepared as described in [23]. For Western-blot analysis, about 200 mg of total cell lysate was run on 8% SDS-PAGE, transferred to nitrocellulose membrane and probed with anti HA antibody (F7).

Immunofluorescence
Immunofluorescence studies were performed using exponentially growing cells, essentially as described earlier [25]. The cells were harvested, washed and re-suspended in PEMS containing zymolyase. The samples were washed gently with 1X PEMS containing 1% triton and the cell pellet was re-suspended in 1 ml PEMBAL and further incubated at room temperature for 1hr. Rad22-YFP was detected using anti GFP antibody at 1:50 dilution and Alexa fluor 488 (Life Technologies) secondary antibody. Approximately 200 cells were analyzed using fluorescence microscope and the number of cells containing Rad22 YFP foci was counted.

Flow Cytometry
Strains were grown till mid log phase, synchronized in early S phase by treating with 12mM hydroxyurea (HU) for 4 hr at 25°C, washed and released at 36°C. Aliquots of 10 6 cells were collected, fixed with 70% ethanol before. For flow cytometry, rehydrated samples were re- suspended in 0.5 ml of 50mM sodium citrate containing 0.1 mg/ml RNase A and incubated at 37°C for 2h. Cells were stained by adding 0.5 ml of sodium citrate solution containing 10μg/ml Propidium Iodide and subjected to flow cytometry as described earlier [26]. Just prior to flow cytometry, samples were sonicated to avoid inaccurate readings resulting from the clumping of cells. Samples were analyzed with a Becton-Dickinson FACS Calibur.

Results and Discussion
Swi7H4 exhibit conditional synthetic lethality with Chk1 null mutant swi7H4, a temperature sensitive mutant allele of DNA polymerase α is required for the establishment of silencing in fission yeast [12,13]. Since this mutant allele exhibit elongated phenotype (an indicator of G2/M arrest) at non permissive temperature, we tried to explore its genetic interaction with Chk1, a G2/M checkpoint kinase protein. A double mutant of swi7H4 chk1 knockout was constructed and its ability to grow at different temperatures was assayed. The swi7H4 mutant cells were unable to form colonies at non permissive temperature 36°C. Interestingly the colony forming ability of swi7H4 chk1 delete cells was much lower than swi7H4 single mutant at semi-permissive temperature 32°C (Fig 1A) indicating conditional synthetic lethality of swi7H4 mutant with chk1 null mutant. To further confirm the synthetic lethality with chk1 deletion, we checked the swi7H4 and chk1 delete swi7H4 double mutant strains for their ability to form colonies at permissive temperature after transiently exposing them at non permissive temperature (36°C). The colony forming ability of chk1 delete swi7H4 double mutant was 4-5 time less as compared to swi7H4 single mutant after 6hr exposure of the cells at 36°C (Fig 1B and 1C). The inability of the swi7H4 mutant to form colonies in the chk1 deletion background indicates that in the absence of normal checkpoint response the swi7H4 mutant cells lose viability at a faster rate as compared to the cells having proficient checkpoint response.
The double mutant of swi7H4 with chk1 deletion aggravates the chromosome segregation defects The synthetic lethality of swi7H4 mutant with chk1 null mutant prompted us to look for the mitotic defects in the double mutant. Wild type, chk1 delete, swi7H4 and chk1 delete swi7H4 double mutant strains were grown at permissive temperature then shifted at 36°C. Samples were collected at 6hr and 10hr time points and cells were stained with DAPI. While wild type and chk1 delete cells did not exhibit any chromosome segregation defects, an approximately 10 fold increase in the frequency of aberrant mitosis in chk1 delete swi7H4 double mutant (10%) as compare to swi7H4 single mutant (1%) was observed after 6 hr at restrictive temperature (Fig 2A and 2B). Further incubation at non permissive temperature for 10hr results in about 14% aberrant mitosis in chk1 delete swi7H4 double mutant as compared to swi7H4 single mutant cells that exhibit only 2.5% aberrant mitosis (Fig 2B). These results strengthen our observation that in the absence of a Chk1-dependent checkpoint response, the swi7H4 mutant cells exhibit severe chromosome segregation defects.

Swi7H4 mutant cells delay the mitotic progression at non permissive temperature in a Chk1 dependent manner
In order to investigate the role of swi7H4 mutant in checkpoint function we performed FACS analysis after releasing the cells from HU arrest at non permissive temperature. As shown in Fig 3 the peak of 2C DNA content was observed after 30 minutes in wild type and chk1 delete cells while in swi7H4 mutant cells the peak corresponding to 2C DNA content appear only after 60 minutes of release suggesting a mitotic delay in swi7H4 mutant cells. Interestingly this mitotic delay was absent in chk1 delete swi7H4 double mutant as the peak of 2C DNA content appear 30 minutes after the release from HU arrest (Fig 3) indicating the requirement of Chk1 for the mitotic delay in swi7H4 mutant.
To further investigate the mitotic delay in swi7H4 mutant cells we monitored the appearance of binucleated cells as an indicator of mitotic progression as described earlier [27]. The cells were arrested in early S phase by growing them in 12mM HU for 4 hr then released at non permissive temperature and stained with DAPI to examine the nuclei. The chk1 deletion and wild type cells enter mitosis at 45 minutes at non permissive temperature (Fig 4A) suggesting normal mitotic progression. In contrast, the swi7H4 mutant cells begin to enter mitosis only at 90 minutes, with 13% of the cells bi-nucleating after 105 minutes shift at non permissive temperature ( Fig 4A) suggesting a mitotic delay in swi7H4 mutant cells. More importantly in chk1 deletion background swi7H4 mutant cells fail to delay mitotic entry (Fig 4A), suggesting that the swi7H4 mutant cells generate a Chk1 dependent checkpoint response at non permissive temperature.

Swi7H4 mutant cells activates the checkpoint kinase protein Chk1
Activation of Chk1 in response to DNA damage results a decrease in mobility of Chk1 on SDS-PAGE [28]. Aggravated chromosomal segregation defect in the swi7H4 chk1 delete mutant and delay in the mitotic progression in swi7H4 mutant cells prompted us to question whether Chk1 protein get activated at non permissive temperature in swi7H4 mutant background. The HA tagged Chk1 was introduced in swi7H4 background by genetic crosses. Activation of Chk1 was monitored by western blotting using anti HA antibody (F7). We observed a slow migrating band of Chk1-HA in swi7H4 mutant just after 2 hr incubation at non permissive temperature (Fig 4B, right panel). In contrast the wild type cells grown under same conditions do not exhibit this phenomenon (Fig 4B, left panel). These observations suggest that defects generated due to swi7H4 mutation leads to the activation of checkpoint kinase protein Chk1 at non permissive temperature.
The swi7H4 mutant cells accumulate DNA damage at non permissive temperature Rad22, a homologue of budding yeast Rad52 [29] is required for homology-dependent double strand break repair and meiosis. During homologues recombination Rad22 binds to single stranded DNA that leads to the formation of Rad22-YFP foci [30]. We analyzed the appearance of Rad22 foci in swi7H4 mutant at non permissive temperature. In comparison to wild type cells, swi7H4 mutant cells exhibited eight fold increases in the level of Rad22-YFP foci at non permissive temperature (Fig 5A and 5B) suggesting that swi7H4 mutant cells accumulate DNA damage at non permissive temperature even in the absence of exogenous DNA damaging agent. Swi7H4 mutation exhibit synthetic lethality with genes involve in DNA repair pathway Appearance of Rad22-YFP foci in swi7H4 mutant strain suggests accumulation of DNA damage at non permissive temperature. These results prompted us to study the genetic interaction between swi7H4 mutant allele and genes involved in DNA repair pathway. The double mutants of swi7H4 with rhp51, rad50, rqh1/hus2 and mus81 knockout were constructed using standard genetic crosses and their survival was compared at semi and non permissive temperature. As presented in Fig 6A, the colony forming ability of double mutant of swi7H4 with rhp51, rad50, rqh1 and hus2 delete cells was reduced as compared to the single mutants at semi permissive temperature (32°C) indicating the synthetic lethality between the swi7H4 and genes involved in DNA repair pathway. In contrast, the rad2 delete swi7H4 double mutant cells were growing normally under the same condition (Fig 6A) suggesting that the absence of rad2, a Fen1 endonuclease which is required for the removal of RNA primer during DNA replication does not affect the survival of swi7H4 mutant cells.
Cell cycle checkpoints are complex signal transduction pathways that involve sensors, transducers and effectors to block the cell cycle progression. In response to DNA damage the sensor kinase Rad3 and the effector kinase Chk1 play important role for the efficient and timely repair of the genome. DNA polymerase α is a well known component of DNA replication machinery required for lagging strand synthesis. Other than involvement in the initiation of DNA replication it is also found to be associated with the heterochromatin maintenance [13]. Here we tried to widen the characteristic behaviour of the swi7H4, a mutant allele of DNA polymerase α in DNA damage checkpoint response. The defects in swi7H4 mutant results in DNA damage that delay mitosis with about eight fold increase in the Rad22 foci (Fig 4A). To further investigate the mechanism of mitotic delay in swi7H4 mutant, we analyzed the activation of checkpoint kinase, Chk1. The 10 fold increase aberrant mitosis in the double mutant of swi7H4 with chk1 deletion and the activation of Chk1 at non permissive temperature suggests the activation of Chk1 dependent checkpoint response in swi7H4 mutant cells. DNA polymerase α has been shown to play a critical role in coordinating S-phase with mitosis. Temperature sensitive DNA polymerase α mutant has been shown to perturb DNA replication and induces downstream kinase response [14,15]. At semi permissive temperature the swi7H4 mutant allele of DNA polymerase α has been shown to exhibit higher mutation rate as compared to wild type [31,32]. We propose that the replication perturbation by swi7H4 mutation could generate DNA damage that leads to the activation of checkpoint response. Subsequently in absence of DNA damage checkpoint protein (Chk1) or the protein responsible for DNA repair pathway the mutant cells exhibit severe defects that leads to synthetic lethal interaction.