DNA Repair 24 (2014) 98–106

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DNA Repair journal homepage: www.elsevier.com/locate/dnarepair

Fission yeast Drp1 is an essential protein required for recovery from DNA damage and chromosome segregation Rajeev Ranjan, Nafees Ahamad, Shakil Ahmed ∗ Molecular and Structural Biology Division, CSIR – Central Drug Research Institute, Sector 10, Jankipuram Extension, Sitapur Road, Lucknow 226031, India

a r t i c l e

i n f o

Article history: Received 28 May 2014 Received in revised form 8 August 2014 Accepted 16 September 2014 Available online 28 September 2014 Keywords: S. pombe Rint1 Drp1 rad50 DNA repair Chromosomes

a b s t r a c t DNA double strand breaks (DSBs) are the most critical types of DNA damage that can leads to chromosomal aberrations, genomic instability and cancer. Several genetic disorders such as Xeroderma pigmentosum are linked with defects in DNA repair. Human Rint1, a TIP1 domain containing protein is involved in membrane trafficking but its role in DNA damage response is elusive. In this study we characterized the role of Drp1 (damage responsive protein 1), a Rint1 family protein during DNA damage response in fission yeast. We identified that Drp1 is an essential protein and indispensable for survival and growth. Using in vitro random mutagenesis approach we isolated a temperature sensitive mutant allele of drp1 gene (drp1-654) that exhibits sensitivity to DNA damaging agents, in particular to alkylation damage and UV associated DNA damage. The drp1-654 mutant cells are also sensitive to double strand break inducing agent bleomycin. Genetic interaction studies identified that Rad50 and Drp1 act in the same pathway during DNA damage response and the physical interaction of Drp1 with Rad50 was unaffected in drp1654 mutant at permissive as well as non permissive temperature. Furthermore Drp1 was found to be required for the recovery from MMS induced DNA damage. We also demonstrated that the Drp1 protein localized to nucleus and was required to maintain the chromosome stability. © 2014 Elsevier B.V. All rights reserved.

1. Introduction The fission yeast Schizosaccharomyces pombe is a useful model system for studies of cell cycle events, like DNA replication, repair, mitosis and cytokinesis. All these events must be performed with high accuracy to ensure proper genomic integrity [1]. Genomic instability can arise due to the defects in DNA replication, repair or chromosome segregation [2]. If these defects are not repaired, damaged DNA may be replicated and segregated into daughter cells that can lead to chromosome instability [3] which is a major reason for tumour development [4]. DNA double strand breaks (DSBs) can also leads to chromosomal aberrations, disruption of genome integrity and cancer. Several genetic disorders such as Xeroderma pigmentosum are linked with defects in DNA repair [5]. The mechanisms of DNA repair have been investigated in the lower eukaryotes, especially in Saccharomyces cerevisiae and S. pombe and have been shown to be highly conserved among eukaryotes [5]. DSBs are generated not only by exogenous sources such as ionizing irradiation but also by endogenous factors such as free radicals generated during cellular metabolism [6]. DSBs can also arise due to

∗ Corresponding author. Tel.: +91 522 2771940x4453; fax: +91 522 2771941. E-mail address: shakil [email protected] (S. Ahmed). http://dx.doi.org/10.1016/j.dnarep.2014.09.006 1568-7864/© 2014 Elsevier B.V. All rights reserved.

the replication fork collapse. However, programmed DSBs are created at recombination sites during meiosis [7] they can also activate DNA damage checkpoints leading to cell cycle arrest and induction of appropriate repair machinery [8]. DSBs in eukaryotes are repaired by two major DNA repair pathways: homologous recombination (HR) and nonhomologous end joining (NHEJ) [9–11]. In S. cerevisiae recombination repair genes belongs to the Rad52 epistasis group [9] that include RAD50, MRE11, XRS2, RAD51, RAD52, RAD54, RAD55, and RAD57 [12]. Mutants of these genes are sensitive to ionizing radiation and other DNA damaging agents [13–15]. The S. pombe rad50 gene was isolated on the basis of its sequence similarity to its homologues from S. cerevisiae, Caenorhabditis elegans, and humans [15], The Rad50 deletion mutant is sensitive to ionizing radiation and produce inviable spores during meiosis [16]. Rad50 belongs to the structural maintenance of chromosome (SMC) family of proteins. This protein family has its role in chromosome condensation and segregation, transcriptional repression and recombination [17]. The Rad50 protein contains an N-terminal Walker A and C-terminal Walker B NTP binding domains, linked by two extensive coiled-coil regions [18,19]. Rad50 acts as the binding sites for Mre11 at the N- and C-terminal ends of the coiled-coil region [20,21]. The Mre11 protein is the catalytic subunit of the Mre11/Rad50/ Nbs1 complex that dimerizes through its phosphodiesterase

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domains [21]. It has 3 –5 dsDNA exonuclease and ssDNA endonuclease activities [22]. The Nbs1/Xrs2 protein is the least conserved protein of the MRN(X) complex and interacts with different proteins during DNA repair [23,24]. The Mre11-Rad50-Xrs2 protein complex (MRX complex) from S. cerevisiae has been reported to be involved in the homologous recombination and non homologous end joining [25,26]. Fission yeast Drp1 is an essential protein [27] that belongs to RINT1/TIP-1 family. Human Rint1, a Drp1 homologous protein was first identified as a Rad50-interacting protein that participates in radiation-induced G2/M checkpoint control [19]. Moreover cells expressing N-terminally truncated Rint1 protein display defective radiation-induced G2/M checkpoint [19]. The homozygous deletions of rint1 alleles in mice resulted in early embryonic death [28] with defects in the positioning of the Golgi body, centrosome amplification and chromosome mis-segregation [29]. Earlier study in human cell line suggested that Rint1 is also involved in telomere length control through Rad50 dependent recombination mechanism involving p130 which interacts specifically with the Rint1 protein [30]. In this study we report the functional characterization of fission yeast Drp1, a functional homolog of human Rint1 which is known to be involved in membrane trafficking between the ER and Golgi [31]. The fission yeast Drp1 (SPBC691.02c) is an essential protein and indispensible for its growth and survival. A temperature sensitive mutant allele of drp1 causes genetic instability and sensitivity to DNA damaging agents. Furthermore, we showed that Rad50 and Drp1 act in the same pathway during DNA damage response. A physical interaction of Drp1 with Rad50 was observed and mutant Drp1 protein retains its ability to interact with Rad50 under non permissive conditions as well as during DNA damage response.

Table 1 Strains used in this study.

2. Materials and methods

gene with drp1 overhangs was transformed in a diploid strain. Transformants were selected by replica plating on plates containing 100 ␮g/ml of G418. Heterozygous diploid strain with drp1 deletion was isolated. Deletion was confirmed by PCR using wild type drp1 gene as a negative control.

2.1. Strains and growth condition S. pombe strains used in this study are listed in Table 1. Standard genetic methods were utilized for making strains as described earlier [32]. For temperature shift experiments, cells were grown to mid-log phase at 25 ◦ C and then shifted to restrictive temperature 36◦ C in a water bath. To measure MMS and bleomycin sensitivity, cells were grown at 25 ◦ C up to mid log phase, 107 cells were serially diluted and spotted on plates containing drugs. For UV sensitivity assay the plates were irradiated with the indicated doses of UV light and incubated at 25 ◦ C for 3–4 days. For survival analysis cells were grown to mid-log phase, cells were further transferred to rich medium containing indicated dosage of MMS and were allowed to grow at permissive temperature for 5 h. Samples were collected, 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. To facilitate detection of Drp1 and Rad50, strains carrying Drp1-HA and Rad50-FLAG were constructed using PCR based tagging as described earlier [33,34]. All tagged strains were viable at temperatures ranging from 25 ◦ C to 36 ◦ C and behave exactly like the wild type cells. 2.2. Gene disruption For drp1 gene disruption two step gene replacement method as described by [34] was used. Plasmid containing kanamycin module was digested with SmaI/SpeI and was ligated at SwaI/AvrII giving rise plasmid pS1which completely replaces the Drp1 ORF with kanamycin resistant gene but contains upstream and downstream sequences of drp1 gene. The pS1 plasmid was further digested with SacII/XhoI and a 2.2 kb fragment containing kanamycin resistant

Strain

Genotype

Source

SP6 NW158

h− leu1-32 h+ leu1-32 ura4D18 chk1::ura4 ade6-216 h− /h+ leu1-32/leu1-32 ura4D18/ura4D18 drp1::kanR /drp1+ ade6-210/ade6-216 h+ leu1-32 ura4D18 rad50::ura4 ade6-216 h leu1-32 ura4D18 drp1::kanR ade6-210 (pSP1 Drp1-HA) h leu1-32 ura4D18 rad50::ura4 drp1::kanR ade6− (pSP1 Drp1-654-HA) h− leu1-32 Drp1-HA::kanR h− leu1-32 Rad50-FLAG::KanR h leu1-32 Drp1-HA::kanR Rad50-FLAG::KanR h− leu1-32 ura4D18 drp1::kanR ade6− (pSP1 Drp1-654-HA) h leu1-32 ura4D18 drp1::kanR rad50-FLAG::KanR ade6-216 (pSP1 Drp1-HA) h leu1-32 ura4D18 drp1::kanR Rad50-FLAG::KanR ade6-216 (pSP1 Drp1-654-HA) h− ura4D18 Chr16[ade6-216] ade6-210 h leu1-32 ura4D18 drp1::kanR Chr16[ade6-216] ade6-210 (pUR19 drp1-654) h− leu1-32 ura4D18 Rad22-YFP::KanR h+ leu1-32 ura4D18 drp1::kanR ade6-216 Rad22-YFP::kanR (pUR19 drp1-654)

Lab stock Nancy Walworth

SH271

SH 318 SH428 SH 457

SH295 SH393 SH398 SH441 SH477

SH456

SH73 SH451

NW1497 SH603

This study

Jagmohan Singh This study This study

This study This study This study This study This study

This study

This study This study

Nancy Walworth This study

2.3. Construction of drp1-654 mutant strain by plasmid shuffling A library of drp1− mutants on a ura based plasmid (Drp1-pUR19) was constructed by in vitro mutagenesis using hydroxylamine. In short 10 ␮g of drp1-pUR19 plasmid was incubated with 500 ␮l hydroxylamine solution for 20 h at 37 ◦ C in an eppendorf tube. Plasmid shuffling was performed essentially as described by [35]. DNA was purified and the mutagenized DNA was introduced in a drp1::kanR haploid strain containing drp1+ gene on a leucine based plasmid (pSP1-Drp1-HA). G418 resistant, leu+ and ura+ transformants, were selected. The wild type plasmid (pSP1-Drp1-HA) was shuffled out and G418 resistant ura+ colonies were selected. Selected strains were checked for temperature sensitivity by replica plating. The strains that were unable to form colonies at 36 ◦ C were selected as containing drp1 mutant gene. For identification of mutation, DNA was isolated from temperature sensitive strain, complete gene coding for drp1 mutant was sequenced using appropriate primers and was compared with the wild-type sequence of drp1 gene. 2.4. Mini chromosome loss assay The minichromosome Ch16 [36] was introduced in the wild type and drp1 deleted cells carrying mutant drp1-654 gene on a plasmid by standard genetic techniques. To determine the rate of minichromosome loss, the cells were allowed to grow up to mid log phase in rich medium lacking adenine at 25 ◦ C. An aliquot was removed

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Fig. 1. Drp1 is essential for survival. (A) Heterozygous diploid strain was sporulated and tetrads were dissected. Spores were allowed to grow on rich media at 25 ◦ C until colonies become visible and photographed. (B) Temperature sensitive phenotype of drp1-654 mutant allele. Indicated strains were grown at 25 ◦ C, serially diluted and spotted on YEA plates. Plates were incubated at indicated temperature for 3–4 days before taking photographs. (C) Percent survival of drp1-654 mutant cells at non permissive temperature: Indicated strains were grown till mid log phase at 25 ◦ C then shifted at 36 ◦ C. Samples were collected at 2 h intervals; equal number of cells were plated on YEA plates and incubated at 25 ◦ C. Number of surviving colonies was calculated as the percentage of colonies appearing at permissive temperature. Values shown are the average of three independent experiments with standard deviation. (D) ESPript generated sequence alignment of the human (hs) Rint1 and fission yeast (sp) Drp1 protein showing the conserved residues and position of mutation.

to determine the percentage of ade− cells. Remaining cells were shifted to 36 ◦ C for 24 h in rich medium containing adenine, samples were taken and plated on plates with limiting adenine. The number of red colonies were counted and the percent chromosome loss was calculated. 2.5. Preparation of whole cell lysate and co-immunoprecipitation Desired strains were grown up to mid-log phase at 25 ◦ C or shifted to non permissive temperature for 6 h. Cells were harvested by centrifugation and lysed using glass beads and a Fast Prep (Bio 101) vortex machine as described earlier [37]. Lysate

was prepared in lysis buffer (PBS) containing 50 mM sodium fluoride, 1 mM PMSF and 1× protease inhibitor as described by [38]. Lysates were precleared with sepharose A beads. Precleared lysate was incubated with anti-FLAG antibody. The antigen–antibody complex was incubated with sepharose beads for 2 h. Beads were centrifuged and washed four times with 1 ml of cold lysis buffer. After the final wash, beads were re-suspended in 1× sample buffer and boiled. Samples were separated on 8% SDS-PAGE, transferred to nitrocellulose membrane (Amersham). Immunoblotting was performed using (F7) anti-HA (1:1000; SantaCruz) or anti-FLAG (1:1000; Sigma) monoclonal antibody. A peroxidase-coupled antimouse secondary antibody and the enhanced chemiluminescence

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detection system (Millipore) were used to detect the immune complexes. 2.6. Microscopy and indirect immunofluorescence For visualization of nuclei, cells fixed with 70% ethanol were rehydrated and stained with 4,6 diamidino-2-phenylindole (DAPI). Stained cells were observed under an epifluorescence microscope and photographed. Immunofluorescence studies were performed using exponentially growing cells essentially as described earlier [39]. Drp1 was detected using anti-HA antibody at 1:50 dilution, incubated overnight at room temperature with rotation, washed, and then detected with secondary antibody coupled to Alexa fluor488 (Life Technologies) at a dilution of 1:100 and incubated at room temperature for 4 h. The cells were analyzed using a fluorescence microscope and processed by using Adobe Photoshop. Rad22-YFP was detected by indirect immunofluorescence using anti GFP antibody at 1:50 dilution, complex was detected with secondary antibody coupled to Alexa fluor 488 (Life Technologies). About 200 cells were analyzed using fluorescence microscope and the number of cells containing Rad22 YFP foci was counted. 3. Results 3.1. Drp1 is an essential gene Fission yeast Drp1 (SPBC691.02c) belongs to RINT1 family protein that contains TIP1 domain. In order to explore the role of fission yeast Drp1 during DNA damage response we constructed a drp1 knockout by two-step gene replacement. The 2.2 kb AvrIISwaI region containing the complete ORF of the drp1 coding region was replaced with the DNA fragment containing the kanMX6 gene in a diploid strain as described earlier [34]. The heterozygous diploids were allowed to sporulate and tetrad analysis was performed. Twelve asci were dissected and allowed to germinate on YEA medium at 25 ◦ C. Only two viable spores were able to grow from each asci (Fig. 1A). Further examinations showed that all viable segregants were unable to grow on YEA plates containing G418, indicating that these segregants were carrying a wild type copy of the drp1gene. The non viability of G418 resistant spores in each tetrad suggests that deletion of drp1 gene is lethal and there is absolute requirement of drp1 gene for growth of fission yeast cells. To further examine the lethal phenotype of a drp1 gene disruption, we attempted the bulk spore germination. The spores were cultured in medium containing 100 ␮g/ml of G418 at 25 ◦ C to induce preferential germination of drp1 delete cells. We cultured the spores up to 48 h but were unable to detect any spore germination. DAPI staining and microscopic observation revealed that drp1 spores could not germinate and might be arrested in interphase without undergoing any cell division (data not shown). 3.2. Isolation of temperature sensitive mutant allele of drp1 To characterize the essential functions of drp1 gene, we constructed a temperature sensitive allele of drp1 by in vitro mutagenesis with hydroxyl amine as described in Section 2. Mutagenized drp1 gene was used to transform a strain, in which the drp1 gene was deleted from the chromosomes but carries a multicopy plasmid containing the drp1+ gene under its native promoter. After plasmid shuffling, about 1000 transformants carrying the mutagenized drp1 gene were examined for temperature sensitivity. We isolated a temperature sensitive mutant allele of drp1 (drp1-654) that was able to grow normally at 25 ◦ C but fail to form colonies at non permissive temperature 36 ◦ C (Fig. 1B). Transient exposure of drp1-654 mutant cells at non permissive temperature leads to 80% loss of viability after 10 h (Fig. 1C) suggesting that drp1-654

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mutant cells were unable to recover from restrictive temperature. We also tried to integrate the drp1-654 mutant allele at chromosomal level by replacing the endogenous drp1+ gene using marker switch approach [40] without success. To determine the nature of the mutation in drp1-654, the mutated plasmid was isolated from S. pombe cells. Sequencing and comparison with wild type sequence of drp1+ gene (SPBC691.02c) indicated a mutation from nucleotide G to A, at position 654 changing the amino acid from methionine to isoleucine at position 218. Multiple sequence alignment studies showed that this region lies in the vicinity of conserved residues (Fig. 1D) indicating that this region of protein might be having important role in Drp1 function. 3.3. drp1-654 mutant cells are sensitive to DNA damaging agents Earlier studies have suggested physical interaction of human Rint1 with Rad50 [19] which indicate that this protein may contribute to repair of the double strand DNA breaks. To investigate the role of fission yeast Drp1 in the DNA damage response, we analyzed the sensitivity of drp1-654 mutant to DNA alkylating agent methyl methane sulfonate (MMS) that generates a double strand break during DNA replication [41]. We observed that even at permissive temperature drp1-654 mutant cells were sensitive to MMS (Fig. 2A) and also exhibit sensitivity towards UV light (Fig. 2B) indicating that though the drp1-654 mutant allele is competent for the essential function of drp1 at permissive temperature but is defective for the DNA damage responsive function of drp1. 3.4. drp1-654 mutant cells are hypersensitive to double strand break inducing agent bleomycin In order to explore the role of drp1 in response to DNA double strand break we checked the bleomycin sensitivity of the drp1654 mutant along with rad50 strain. Cells were grown till mid log phase, serially diluted and spotted on the plates containing 0.25 and 0.5 ␮g/ml of bleomycin. The drp1-654 mutant cells were mildly sensitive to bleomycin as compare to rad50 cells that was highly sensitive to double strand break inducing agent, bleomycin at permissive temperature (Fig. 2C). The bleomycin sensitivity of drp1-654 rad50 double mutant was similar to that of the rad50 single mutant (Fig. 2C) suggesting that Drp1 and Rad50 participate in the same pathway. 3.5. Genetic interaction of drp1-654 mutation with rad50 deletion mutant Rad50, a structural maintenance of chromosomes (SMC) protein family member, is required for homologous recombination and has been shown to interact with human Rint1 [19]. In order to study the genetic interaction of drp1-654 mutant allele with rad50 knockout, a double mutant was constructed and their sensitivity towards MMS was analyzed at permissive temperature. The rad50 mutant cells were more sensitive to MMS than drp1-654 single mutant (Fig. 3A), however, the MMS sensitivity of drp1-654 rad50 double mutant was similar to that of the rad50 single mutant (Fig. 3A) suggesting that Drp1 and Rad50 work in the same pathway in the alkylation damage response. 3.6. Drp1 is required for recovery from MMS induced DNA damage Since the drp1-654 mutant were also losing viability after transient exposure to MMS (Fig. 3B), we tested whether drp1 is needed for the recovery from MMS induced DNA damage. We performed the DAPI staining of the drp1-654 strain following MMS treatment and after recovery from MMS induced DNA damage. The normal

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Fig. 2. drp1-654 mutant cells exhibit sensitive to DNA damaging agents. (A) Indicated strains were serially diluted and spotted on YEA plates containing indicated concentrations of MMS, incubated at 25 ◦ C for 3–4 days. (B) Strains were processed as above and cells were exposed to UV light at indicated dosage. The plates were incubated at 25 ◦ C for 3–4 days before taking photograph. (C) Cells were grown at 25 ◦ C, serially diluted and spotted on plates containing indicated concentration of bleomycin. Plates were incubated at 25 ◦ C for 3–4 days.

nuclei were observed in drp1-654 mutant cells after 5 h exposure to MMS (Fig. 3C). Further these cells were washed and allowed to grow for 4 h in medium lacking MMS. Recovery after transient exposure to MMS leads to aberrant nuclear morphology with diffuse and fragmented nucleus in about 16% of the drp1-654 mutant cells as compare to wild type cells that had normal hemispherical or round nuclear morphology (Fig. 3C) suggesting that Drp1 is required during MMS induced DNA damage recovery. 3.7. The drp1-654 mutant cells accumulate DNA damage at non permissive temperature To visualize DNA damage in drp1-654 mutant cells, we constructed a strain that expresses Rad22-YFP from its endogenous promoter at its genomic locus in a drp1-654 mutant background. Rad22, a homologue of budding yeast Rad52 [42] has been shown to bind to single stranded DNA during homologous recombination that leads to the formation of Rad22-YFP foci [43]. After shifting the cells at non permissive temperature for 6 h we observed about five fold elevated level of Rad22-YFP foci in drp1-654 mutant cells as compare to wild type cells (Fig. 4A and B) suggesting that drp1-654 mutant cells accumulate DNA damage at non permissive temperature even in the absence of exogenous DNA damaging agent. 3.8. The drp1-654 mutation causes genetic instability In the course of examining the phenotype of drp1-654 mutant strain, we observed abnormal nuclear domain at non permissive temperature, suggesting that chromosome stability might be compromised in drp1-654 mutant cells. To monitor the defects in chromosome morphology, drp1-654 cells were grown at 25 ◦ C and shifted to non permissive temperature 36 ◦ C for 6 h, fixed and

stained with DAPI to visualize chromosomal DNA. After shifting the cells to non permissive temperature for 6hr about 34% drp1654 mutant cells exhibit extended or diffuse chromosome and 5% of the cells showed unequal chromosome segregation (Fig. 5A, right panel). In contrast, the wild type cells did not exhibit segregation defects under the same growth conditions (Fig. 5A, left panel) suggesting that drp1 might be also required to maintain chromosome segregation.

3.9. Drp1 is a nuclear protein and required for proper chromosome segregation To determine whether Drp1 is required for chromosome stability, a nonessential mini-chromosome (Ch16), a derivative of chromosome III [36] was incorporated in drp1-654 cells by genetic crosses. This mini chromosome carries the ade6-216 mutation that complements an ade6-210 mutation present on the yeast genome thus making the strain phenotypically ade+ and white in colour. We examined the spontaneous loss of the mini-chromosome by growing cells at restrictive temperature under non selective conditions for several generations (24 h) and then scoring the number of red colonies produced after plating on media with limited concentration of adenine. About 17% of drp1-654 mutant cells exhibited chromosome loss as compared to wild-type cells that showed only 0.2% of chromosome loss under the same growth conditions (Fig. 5B and C) indicating that drp1-654 mutant cells are defective in maintaining chromosome stability. It was previously reported that mouse Rint1 is located in the ER and Golgi apparatus as well as in the centrosome [28]. In order to determine the localization of the fission yeast Drp1 protein, a HA tag Drp1 strain was used for indirect immunofluorescence study. The

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Fig. 3. Genetic interaction of drp1-654 mutation with rad50 deletion. (A) Strains were grown at 25 ◦ C, serially diluted and spotted on YEA plates containing indicated concentrations of MMS, incubated at 25 ◦ C for 3–4 days before taking photographs. (B) Strains were grown up to log phase; cells were transferred to rich medium containing indicated dosage of MMS and were allowed to grow at 25 ◦ C. Samples were collected, 1000 cells from each sample were plated on YEA plates and incubated at 25 ◦ C. Number of surviving colonies was calculated as the percentage of colonies appearing without MMS treatment. Values shown are the average of three independent experiments with standard deviation. (C) Wild type and drp1-654 mutant cells were treated with 0.03% MMS for 5 h, washed, re-inoculated into medium lacking MMS and further allowed to grow at 25 ◦ C for 4 h. Samples were collected, fixed with 70% ethanol and stained with DAPI. Arrows indicate diffuse and crescent nuclei.

Fig. 4. The drp1-654 mutant cells have elevated level of Rad22 YFP foci at non permissive temperature. (A) Indicated strains were grown till mid log phase, shifted at non permissive temperature (36 ◦ C) for 6 h. The cells were processed for indirect immunofluorescence microscopy as described in Section 2. (B) About 200 cells for each sample were counted and the percentage of cells containing Rad22-YFP foci was plotted.

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Fig. 5. The drp1-654 mutation causes genetic instability. (A) Cells were grown at permissive temperature till mid log phase, shifted at non permissive temperature for 6 h, fixed with 70% ethanol and stained with DAPI to visualize the DNA. Arrow indicates extended or diffused chromosome. Scale bars, 10 ␮m. (B) Chromosome loss assay: equal numbers of cells of the indicated strains were plated on plates with limiting concentration of adenine. The number of red and white colonies was counted and percentage of red colonies was calculated. (C) The percentage of ade− (red) colonies as an indicator of chromosome loss was plotted. Values shown are the average obtained from three different experiments with standard deviation. (D) Localization of Drp1 protein in fission yeast cells. Exponentially growing Drp1-HA cells were collected and processed for immunofluorescence microscopy analysis as described in Section 2. The antigen of interest (Drp1-HA) was used for immuno detection, whereas DNA was stained with DAPI and observed under an epifluorescence microscope. Scale bars, 10 ␮m.

indirect immunofluorescence staining using anti HA antibody along with DAPI staining reveals nuclear localization of Drp1 (Fig. 5D). 3.10. Mutation in drp1 gene does not affect its interaction with Rad50 Rint1 was initially identified in yeast two hybrid screening using C-terminal region of human Rad50 as the bait and the physical interaction of human Rint1 with Rad50 has also been extensively studied [19]. In order to understand the physical interaction versus DNA damage response in drp1-654 mutant, we checked the interaction of Rad50 with mutant Drp1. HA tag Drp1 and FLAG tag Rad50 strains were constructed as described in Section 2. Cells were grown at permissive temperature and immunoprecipitation was performed using anti FLAG antibody. As presented in Fig. 6A, Drp1-HA was detected in the immunoprecipitate from yeast cells co-expressing Drp1-HA and Rad50-FLAG suggesting that the two proteins do interact directly. Co-immunoprecipitation with FLAG antibody in a control strain lacking Rad50-FLAG did not give Drp1HA band (Fig. 6A, lane 3). To determine the physical interaction of mutant Drp1 protein with Rad50, drp1 knockout strain containing

wild type and mutant copy of HA tag Drp1 on a plasmid was constructed in a Rad50-FLAG tag background as described in Section 2. Our result establishes that Rad50-FLAG stably bind with HA tag wild type Drp1 expressing on a plasmid at permissive (Fig. 6B, lane 5). Surprisingly Rad50-FLAG was also co-purified with mutant Drp1HA protein at permissive temperature (Fig. 6B, lane 6) suggesting that drp1-654 mutation does not affect its interaction with Rad50 protein. Furthermore this interaction was not affected once the cells were shifted to non permissive temperature (Fig. 6C) or grown in the presence of DNA alkylating agent MMS (data not shown). 4. Discussion DSBs are the most harmful form of DNA damage as they lead to either breaks in chromosome or their rearrangement, which may result in apoptosis or tumorigenesis [8]. Solid tumour formation requires several mutations and chromosomal rearrangements that often lead to missegregation events. Such mutations can leads to defects in mitotic recombination, chromosome stability or nondisjunction [5]. Several human syndromes like Ataxia telangiectasia (A-T) and Nijmegen breakage syndrome (NBS) [44,45] are caused

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Fig. 6. Drp1 interacts with Rad50 in vivo. (A and B) Indicated strains were grown at 25 ◦ C till mid log phase, whole cell extract of was prepared and immunoprecipitation (IP) was performed using anti FLAG antibody as described in Section 2. Samples were run on 8% SDS PAGE, transferred on nitrocellulose membrane and probed with anti HA (F7) or anti FLAG antibody. (C) Indicated strains were grown at 25 ◦ C till mid log phase, shifted to non permissive temperature for 6 h. Immunoprecipitation was performed as described above.

by chromosome instability and characterized by sensitivity to DSBcausative agents [46]. Drp1, a TIP domain containing protein is evolutionary conserved and belongs to RINT1/TIP-1 family protein [47]. This family includes Rint1 and other Rad50 interacting protein which participates in radiation induced checkpoint control [19]. A complete knock out of drp1 gene in haploid renders the fission yeast cells non viable as has also been suggested in a genome wide deletion studies [27]. The lack of any growth in drp1 deleted spores suggests that it might be playing an important role in some cellular process other than the repair of DNA lesions caused by exogenous DNA damaging agents. There are several DNA repair genes like rad4/cut5, rad11, rad15, rad18, and rad21 that are essential for growth in S. pombe. These genes have diverse roles in DNA replication, checkpoint control, nucleotide excision repair and forming cohesin complex [48–50].

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In vitro mutagenesis identified a temperature sensitive mutant allele of drp1 (drp1-654) that changes Met 218 to Ile in the vicinity of highly conserved region. We further attempted to replace the endogenous drp1+ gene with the drp1-654 mutant allele at chromosomal level by marker switch approach [40] without success. After screening of thousands of transformants we were unable to find colonies in which switching of marker took place so we reason that, since in drp1-654, the mutation lies in the vicinity of highly conserved residues hence when integrated under the native promoter, a single copy of drp1-654 might be insufficient for the survival of the cells. Mutant allele of mst1, an essential protein required for DNA damage response and chromosome segregation fails to survive as a single copy when integrated under the native promoter in S. pombe [51]. At non permissive temperature chromosomal domain of drp1-654 becomes extended and there was unequal distribution of nuclear material suggesting that in the absence of functional Drp1 protein, spontaneous DSBs occurs which remain unrepaired, and cause abnormal nuclear architecture. The increase in the Rad22-YFP foci and elevated rate of minichromosome loss at non-permissive temperature suggests that the DSBs generated in drp1-654 mutant may interfere with the compaction of chromosomes which might be responsible for chromosome missegregation event. Several essential genes of S. pombe have been shown to play essential roles in maintenance of genome stability and DNA damage response [15,50,51]. Double mutant analysis is used to define epistasis groups that reflect function in pathways [52]. If a double mutant exhibits sensitivity no greater than the most sensitive single mutant, the two genes are considered epistatic, i.e., being in one pathway. If, however, the double mutant exhibits an additive sensitivity, the two genes are likely to act in different pathways. DNA-damage-repair epistasis groups have been extensively defined in S. cerevisiae [52,53]. Genetic interaction data from our study suggest that Drp1 and Rad50 belong to same epistatic group and work in the same pathway during the DNA damage response. Several rad genes (rad1, rad3, rad9, rad17 and rad26) have been shown to required for checkpoint control and recovery during DNA damage response [54]. Here we have shown that drp1-654 mutant cells are defective for recovery from MMS induced DNA damage. In human cells lines, an N-terminally truncated Rint1 protein has been shown to exhibit defective radiation induced G2/M checkpoint [19]. The drp1-654 mutant cells were not significantly elongated when exposed to DNA damaging agents or when shifted to non permissive temperature, suggesting that G2/M checkpoint was normal in drp1-654 mutant in fission yeast. Consistently Chk1 protein kinase was not activated at non permissive temperature in drp1-654 mutant cells (data not shown). Since the Drp1 mutant protein retains its interaction with Rad50 both at non permissive temperature as well as in presence of DNA damaging agent, we hypothesize that the interaction is not sufficient to rescue the cells from DNA damage response. Contrary to the earlier observation of Rint1 localization to the ER and Golgi compartments in mouse cell lines [28], we demonstrated the nuclear localization of Drp1 in fission yeast, suggesting that fission yeast Drp1 protein might have some different function that affects chromosome segregation. Further work need to be done to strengthen our understanding involving Drp1 protein in DNA damage response. Conflict of interest statement The authors declare that there are no conflicts of interest. Acknowledgements We thank members of our lab for helpful discussions and technical support. We thank Dr. JV Pratap for critical reading of this

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