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DNA Repair (Amst). Author manuscript; available in PMC May 1, 2009.
Published in final edited form as:
PMCID: PMC2675035

Screening a genome wide S. pombe deletion library identifies novel genes and pathways involved in the DNA damage response


The maintenance of genome stability is essential for an organism to avoid cell death and cancer. Based on screens for mutant sensitivity against DNA damaging agents a large number of DNA repair and DNA damage checkpoint genes have previously been identified in genetically amenable model organisms. These screens have however not been exhaustive and various genes have been, and remain to be, identified by other means. We therefore screened a genome wide Schizosaccharomyces pombe deletion library for mutants sensitive against various DNA damaging agents. Screening the library on different concentrations of these genotoxins allowed us to assign a semi-quantitative score to each mutant expressing the degree of sensitivity. We isolated a total of 229 mutants which show sensitivity to one or more of the DNA damaging agents used. This set of mutants was significantly enriched for processes involved in DNA replication, DNA repair, DNA damage checkpoint, response to UV, mating type switching, telomere length maintenance and meiosis, and also for processes involved in the establishment and maintenance of chromatin architecture (notably members of the SAGA complex), transcription (members of the CCr4-Not complex) and microtubule related processes (members of the DASH complex). We also identified 23 sensitive mutants which had previously been classified as “sequence orphan” or as “conserved hypothetical”. Among these, we identified genes showing extensive homology to CtIP, Stra13, Ybp1/Ybp2, Human Fragile X mental retardation interacting protein NUFIP1, and Aprataxin. The identification of these homologues will provide a basis for the further characterisation of the role of these conserved proteins in the genetically amenable model organism S. pombe.


DNA is susceptible to various kinds of damage. DNA lesions can be spontaneous, as a result of physiological processes, or as a result of endogenous or exogenous DNA damaging agents. These lesions include chemical modification of the bases, and breaks in the DNA backbone. Failure to accurately repair these lesions can lead to deleterious mutations, cell death, and ultimately cancer (in multicellular organisms). To avoid these detrimental consequences cells have developed a range of specialised repair mechanisms which deal with various DNA lesions and a complex network of cellular responses that together maintain genome stability.

Several pathways deal with base lesions by excising the damaged (or mismatched) base and filling the remaining gap through DNA polymerisation and ligation. Base excision repair (BER) deals with various kinds of damaged bases, which are removed by specialised glycosylases. Nucleotide excision repair (NER) deals predominantly with bulky DNA adducts, such as those resulting from UV radiation. Mismatch repair (MMR) removes base mismatches, that can result from erroneous replication [1].

Other pathways are responsible for repairing breaks in the DNA backbone. Single strand DNA breaks (SSBs) are repaired using a subset of proteins involved in BER [2]. Double strand DNA breaks (DSBs) are one of the most deleterious DNA lesions, a single DSB can lead to the loss of large chromosomal fragments distal from the break and subsequent loss of heterozygosity (and cancer) and is often lethal. DSBs can arise spontaneously during replication (when the replication fork encounters a SSB), result from ionising radiation, oxidative damage or abortive topoisomerase II action. DSBs are also formed as physiological intermediates in different recombination-dependent processes like meiotic recombination or V(D)J recombination and class switch recombination [3]. DSB repair mechanisms can employ various general strategies. The non-homologous end-joining (NHEJ) pathway joins the ends of a DSB together, without the need for extensive homology. As DSB formation is often associated with the loss of one or more base pairs, NHEJ frequently leads to small deletions. In homologous recombination (HR) pathways a single DNA strand invades a homologous sister molecule (often a sister chromatid) to copy the missing information, which is used to accurately repair the DSB (see [4] for a detailed review of several variations of HR repair pathways).

If damaged DNA remains unrepaired when cells enter S-phase, several DNA damage tolerance mechanisms allow the replication fork to bypass the lesion. These mechanisms consist of pathways that employ specialised (error prone) polymerases that can bypass and allow replication past the damage (translesion synthesis), as well as error free mechanisms that employ HR to either fill in the gaps that remain after replication fork stalling, or to switch template and use the undamaged strand as a copy template for DNA synthesis [5].

To avoid catastrophic consequences of damaged DNA going into mitosis or S-phase, DNA damage checkpoints halt the cell cycle to allow ample time for the completion of DNA repair. These checkpoints have also been associated with damage detection, and several other aspects of the regulation of repair and replication [6].

The importance of the various DNA repair pathways and checkpoint mechanisms is illustrated by the high degree of evolutionary conservation of the majority of these pathways from bacteria to humans. Also, several genetic diseases have been associated with mutations in DNA repair genes. These diseases are associated with a wide variety of symptoms, including cancer predisposition and neurodegeneration [7]. As various DNA damaging agents are routinely used in cancer therapy, an in depth understanding of DNA repair mechanisms is also important for improving therapeutic efficiency.

Mutations of genes involved in the maintenance of genome stability often confer sensitivity to DNA damaging agents, and this characteristic has been extensively and successfully used to identify genes and proteins involved in various genome maintenance pathways. These studies have mainly been performed in genetically amenable organisms. The analysis of radiation sensitive mutants in bacteria has contributed significantly to our understanding of DNA repair pathways in prokaryotes (reviewed in [8]).

As higher eukaryotic organisms are refractive to large scale genetic screens, and many DNA repair genes are essential for viability in higher eukaryotes, identification and analysis of DNA repair genes in the lower eukaryotic model organisms Saccharomyces cerevisiae and Schizosaccharomyces pombe has proven to be essential for our understanding of eukaryotic DNA repair. As these model organisms are as distantly related to each other as to animals [9], detailed studies of similarities and differences between DNA repair in these yeasts are informative as to which mechanisms are conserved in higher eukaryotes. Initial screens in S. cerevisiae (e.g. [10], [11]) of random mutants for sensitivity against various DNA damaging agents have identified a large number of the core DNA repair genes. Screening of an S. cerevisiae ordered genome-wide deletion library has led to the identification of several more genes involved in the maintenance of genome stability (e.g. [12], [13]).

Also in S. pombe, various DNA repair and checkpoint genes have been identified from collections of random mutants on the basis of their sensitivity to DNA damaging agents, and the identity of several mutated genes has been determined using complementation of this sensitivity [14], [15]. These mutant sensitivity screens, however, have not been exhaustive in their identification of genes involved in the maintenance of genome stability, and various DNA repair genes have been identified in screens for mutants defective in mating type switching [16] or screens looking for mutants which are synthetically lethal with rad2[increment] [17], [18], [19], [20], [21], [22]. However, several novel genes have been (e.g. [23], [24]), and probably remain to be, identified by other means, e.g. based on sequence homology with repair genes in other organisms (especially since the completion of the S. pombe genomic sequence [25]). To try and identify novel genes involved in the maintenance of genome stability, we screened an ordered genome wide S. pombe haploid deletion library (which has recently become available) for mutants sensitive to various DNA damaging agents.

Materials and Methods

Bioneer deletion library

A genome wide haploid deletion library was constructed and supplied by the Bioneer corporation and the Korea Research Institute of Biotechnology and Bioscience (http://pombe.bioneer.co.kr/). This library contains 2662 haploid deletions strains. As an estimated 17.5 % of the genes is essential [26], and the total number of protein encoding genes is currently estimated to be 4941 (http://www.sanger.ac.uk/Projects/S_pombe/genome_stats.shtml), this represents approximately 65 % of the non-essential S. pombe genes.

Growth and storage conditions

The library was frozen at −80 °C in 96-well microtitre plates in 30 % glycerol in liquid yeast extract. Before an experiment, the library was transferred using a 48 pin replicator to 96-well microtitre plates containing 200 μl liquid yeast extract and incubated at 30 °C for at least 48 hours or until saturation.

Exposure to DNA damaging agents

Yeast extract plates containing phloxin (2.5 mg/l) and different amounts of Camptothecin (CPT), Methyl Methane Sulphonate (MMS), 4-nitroquinoline 1-oxide (4-NQO) or hydroxyurea (HU) where prepared the day before the experiment. Deletion strains were transferred from the 96-well microtitre plates onto drug containing media or control plates using a 48 pin replicator. Gamma radiation was administered using a 137Cs source after transfer onto yeast extract plates.

Analysis of GO term enrichment

Cytoscape [27] with the BiNGO plugin [28] was used for the GO term enrichment analysis. The Hypergeometric Test with Benjamini and Hochberg False Discovery Rate Correction was chosen for the analysis [28].

Homology searches and alignments

We used psi-Blast ([29]; http://www.ncbi.nlm.nih.gov/blast/Blast.cgi) to detect potential orthologues of the mutant sequence orphans or conserved hypothetical genes which we identified as sensitive to DNA damaging agents. Alignments presented in Fig. 4 are made using MUSCLE ([30]; http://www.ebi.ac.uk/Tools/muscle/index.html) and BOXSHADE (http://www.ch.embnet.org/software/BOX_form.html).

Figure 4
Alignments between a) S. pombe SPCC576.12c, S. cerevisiae Ydl160c-a and mouse Stra13. b) Alignments between S. pombe SPCC1442.02 and S. cerevisiae YBP2 and YBP1. c) alignments between S. pombe SPBC16c6.03c, Candida albicans SC5314 and the human fragile ...

Results and Discussion

For the screen we chose to use a variety of DNA damaging agents to cause different DNA lesions. Camptothecin (CPT) locks topoisomerase I covalently onto the DNA, and the resulting single strand break interferes with replication and transcription [31]. Methyl Methane Sulfonate (MMS) is a methylating agent which forms adducts with DNA, but also with RNA and protein [32]. The UV mimetic drug 4-nitroquinoline 1-oxide (4-NQO) is a precursor of the metabolite 4-hydroxyaminoquinoline 1-oxide, which forms DNA base adducts [33]. Hydroxyurea inhibits ribonucleotide reductase and thus depletes the nucleotide pool and inhibits DNA replication [34]. Ionising radiation causes a wide range of DNA lesions: base damage, cross links, single and double strand DNA breaks, the latter being the main lesions responsible for cell killing [35].

To determine optimal concentrations for screening the deletion library, WT and rad50[increment] cells were exposed to various concentrations of the different DNA damaging agents, and a concentration was selected where WT showed a weak sensitivity, apparent by a slight darkening of the colony colour (due to the presence of Phloxin, which stains dead cells dark). The suitability of these concentrations was confirmed in a pilot screen where one plate of the library (96 mutants) was exposed to the various agents (data not shown).

In a first screen, the whole library was exposed to the selected high concentrations of the different DNA damaging agents: 16 μM CPT, 0.01 % MMS, 0.8 μM 4-NQO, 10 mM HU and 1000 Gy of Gamma radiation. After 48 hours at 30 °C, the sensitivity of the deletion mutants to the different DNA damaging agents was compared to that of the mutants on the control plate. This screen was repeated once. Using these results, a sub-library was created that contained 564 deletion mutants showing sensitivity against any of the drugs used (including the strains that only showed a slight sensitivity in only one of the screens). As a control, WT and rad50[increment] [36] strains were incorporated in each microtitre plate. This sub-library was then screened against various concentrations of the DNA damaging agents (CPT: 1, 2, 4, 8 16 μM; MMS: 0.001, 0.002, 0.005, 0.01 %; 4-NQO: 0.1, 0.2, 0.4, 0.8 μM; HU: 1, 2, 5,10 mM; Gamma: 200, 500, 1000 Gy). This allowed us to assign a semi-quantitative score expressing the sensitivity of the different mutants against the various DNA damaging agents (see Fig. 1). This semi-quantitative screen of the sub-library was repeated 5 times for both CPT and MMS, and 4 times for 4-NQO, HU and Gamma radiation. Mutants that showed sensitivity to a particular agent in less than half of the screens where labelled as non-sensitive for that agent. This left us with a list of 229 deletion mutants that showed sensitivity against at least one of the agents. A list of these mutants, ordered according to their average sensitivity score against the various agents, can be found in supplementary Table 1.

Figure 1
Example illustrating the semi-quantitative scoring system used in the screens. Mutants where assigned a sensitivity score according to the lowest concentration of the DNA damaging agent at which they show clear growth inhibition (numbers in white).

Among the first 30 mutants, the majority of the genes have a confirmed role in maintaining genome stability (see supplementary table 1). The first 16 mutants in the list show sensitivity to all tested agents. The majority of these genes fulfil core functions in homologous recombination (rhp51, rad32) or DNA damage checkpoint pathways (rad1, rad26, rad3, hus1, rad17, rad9, crb2). SPCC338.08 is the homologue of CtIP [23] and discussed below in more detail. Nse5 and Nse6 are non-SMC subunits of the Smc5/Smc6 complex and have been implicated in DNA repair [37]. Also the cohesin-associated protein Pds5 is reportedly involved in DNA repair [38]. Pnk1 is a polynucleotide kinase homologue, and has been shown to be sensitive against CPT and γ irradiation [39]. Caf1 is a component of the CCR4-Not transcription factor complex (see below). Ubi1 is an S. pombe ribosomal-ubiquitin fusion protein, and the sensitivity of the mutant may be associated with ubiquitin disruption (ubiquitin is involved in various aspects of DNA repair regulation; [40]) or with disruption of the large (60S) ribosomal subunit. The proportion of core DNA repair/checkpoint proteins goes down gradually for the mutants which are sensitive to 4, 3, 2 or 1 of the used damaging agents.

As expected, most core homologous recombination repair and checkpoint mutants are sensitive to most DNA damaging agents. The observed sensitivity of these mutants is generally lower than reported in the literature, suggesting that the large scale semi-quantitative screening procedure we used is less sensitive than the quantitative assays normally used in small scale experiments. As expected, among the mutants solely sensitive to 4-NQO several genes involved in NER are found (rad13, rhp14, rhp41 and rhp7; see table S1). Whereas NHEJ mutants have been reported to be not more sensitive to γ-irradiation and MMS than WT [41], pku80[increment] was detected in our screen as slightly sensitive to 4-NQO. Some other mutants exclusively sensitive to 4-NQO are involved in postreplication repair (rad8) and translesion synthesis (rev1, rev3).

A relatively large number of mutants is sensitive only to the highest concentration of 4-NQO (0.8 μM) or HU (10 mM; see table S1). As shown in Fig. 3, even WT cells show a significant sensitivity at these concentrations, while WT cells are less sensitive to the highest concentrations used for CPT (16 μM), MMS (0.01 %) or Gamma (1000 Gy). It is therefore likely that among the mutants which are solely sensitive to the highest 4-NQO or HU concentrations, some sensitivities are a result of defects in pathways unrelated to DNA damage response (e.g., deletion might lead to a subtle growth defect which is exacerbated by the treatment with high concentration of DNA damaging agents). The presence of rev1, rev3 (translesion synthesis) and pku80 (NHEJ) in the group of mutants only sensitive to 0.8 μM 4-NQO and mik1 (DNA damage checkpoint), mfh1 (DNA repair) and SPAC1071.02 (NER) in the group of mutants only sensitive to 10 mM HU suggests however that the sensitivity of the uncharacterised mutants might also be due to defects in genome damage response pathways.

Figure 3
Spot test confirming the sensitivity of 23 mutants isolated in the screen which have previously been classified as “sequence orphan” or “conserved hypothetical”. Yellow boxes indicate sensitivities as detected in the large ...

There is a notable absence of MMR mutants, which are generally not sensitive against the agents used in this study. Whereas some BER mutants show a slight sensitivity against MMS (e.g. [42], [43]), these have not been picked up in our screen.

A total of 181 S. pombe genes have been annotated [44] as being involved in the DNA damage checkpoint or DNA repair (see also below). Of the 36 checkpoint/DNA repair mutants previously reported as sensitive in the literature which were present in the library, only one mutant was not picked up in our screen: taz1[increment], which is moderately MMS sensitive [45]. For 61 checkpoint/DNA repair mutants no significant sensitivity to any of the DNA damaging agents used in our screen has been reported in the literature, consistent with our finding that these mutants did not show sensitivity in our screen. A total of 84 checkpoint/DNA repair mutants (46%) where not available in the library, this percentage is representative of the overall coverage of the library (see Materials and Methods).

Previous studies have determined the genome wide transcriptional response to γ-irradiation [46] and MMS [47] in S. pombe. Of the 22 mutants sensitive to γ-irradiation identified in our study, only 2 (SPCC338.08/ctp1 and SPAC644.14c/rhp51) have been shown to be transcriptionally induced by γ-irradiation, whereas only 1 mutant (SPBC725.10) out of 32 MMS sensitive mutants is transcriptionally induced after MMS. Therefore, as previously concluded for S. cerevisiae [48], the transcriptional response to a mutagen is a poor indicator for mutant sensitivity.

The S. pombe genome is extensively annotated using terms from the Gene Ontology Consortium (http://www.geneontology.org), with 98.3 % of its genes having at least one GO (Gene Ontology) annotation [44]. This allowed us to identify the over-representation of GO terms in our set of sensitive mutants using BiNGO/Cytoscape [28]. Table S2 contains a list with all the over-represented GO-terms at a significance level smaller than 0.005. Fig. 2 shows a graphical representation of these data, showing the relations between the various GO terms, and the genes allocated to the various groups of over-represented GO terms. When a gene is associated with a specific GO term, it is also automatically associated with the parents of that GO term, these associations are therefore also shown in Table S2 (e.g. see rhp41 in table S2). To reduce complexity, we assigned each sensitive mutant to a single, (subjectively) most appropriate GO term (Table S3).

Figure 2
Graph showing the over-represented GO-terms at a significance level smaller than 0.005. Colours of the different GO-terms represent statistical significance (see legend in top right corner). Related GO-terms are connected by a line. Grey boxes include ...

As expected, the annotation groups associated with DNA replication, DNA repair, DNA damage checkpoint and response to UV (Fig. 2) are over-represented. Also, the over-representation of mutants involved in mating type switching does not come as a surprise, as many repair and chromatin architecture genes have been implicated in this process [16]. Similarly, many of these factors have been implicated in telomere (length) maintenance and meiosis (reviewed in [3]).

One of the groups of over-represented annotations is related with the establishment and maintenance of chromatin architecture (Fig 2; see also Table S3), which is involved in regulating chromatin accessibility, important for DNA repair [49] and transcription [50]. Whereas some of these genes might be directly involved in the maintenance of genome stability (e.g. rtt109 [51]), other mutants might confer sensitivity by interfering with transcription of DNA damage response genes. One striking observation is that all mutants annotated as a member of the SAGA complex available in the library (gcn5, kap1, sgf29, SPCC126.04c, spt3, spt8 and ubp8; see Table S3) have been picked up by our screen as being sensitive to one or more agents. The SAGA complex possesses histone acetyltransferase activity and is involved in the regulation of transcription [50]. It remains to be determined if the SAGA complex is involved in the transcription of DNA repair factors, or if it plays some other yet uncharacterised role in the resistance to DNA damaging agents. In a similar screen in S. cerevisiae several several SAGA complex mutants where also found to be sensitive against MMS or 4-NQO [48], suggesting that this might be a conserved eukaryotic feature.

Transcription related annotations are also over-represented (Fig. 2, Table S3). Caf1, Rcd1, Ccr4 and SPCC4G3.15c are members of the CCR4-Not transcription factor complex. Of the 7 CCR4-Not transcription factors available in the library, 4 where detected as sensitive to one or more DNA damaging agents. S. pombe Caf1 and Ccr4 have been implicated in the regulation of ribonucleotide reductase activation [52], possibly explaining the sensitivity of these mutants to various DNA damaging agents. Also in S. cerevisiae, Ccr4 has been implicated indirectly in the regulation of ribonucleotide reductase [53]. The transcription factor Pap1 has been implicated in the response to oxidative stress and various cytotoxic agents [54]. SPAC1071.02 shows homology to S. cerevisiae MET18/MMS19 (www.genedb.org/pombe), which is involved in DNA repair as well as RNA polymerase II transcription [55].

Another over-represented group is associated with microtubule related processes (Fig. 2). Among these mutants are 3 genes (ask1, dad5, dad2) which are part of the DASH complex [56], a kinetochore complex which is thought to play a role in attaching spindle microtubules to the kinetochore (reviewed in [57]). From the 5 DASH complex proteins available in the library, 3 have been picked up as sensitive to DNA damaging agents in our screen. Although microtubules and microtubule organising centres (centrosomes and spindle pole bodies) have previously been associated with the maintenance of genome stability (e.g. [58], [59], [60], [61]), this relation is not well understood and, to our knowledge, a role for the DASH complex in maintaining resistance against DNA damaging agents has not been previously reported.

One of the goals of the screen was to identify novel genes with a function in the maintenance of genome stability. Several (23) sensitive mutants identified have been classified as “sequence orphan” or “conserved hypothetical” (http://www.genedb.org/pombe/; [62]). Supplementary Fig. 1 shows examples of the primary screen data for these mutants. Apparent sensitivities to DNA damaging agents could result from growth reduction and/or increased cell killing. As most spots appear darker (due to increased phloxin levels in dead cells) after exposure to CPT, MMS or HU, whereas after 4-NQO and gamma exposure there is a reduction in the number of colonies, these apparent sensitivities are at least partially due to an increase in cell killing.

To confirm that the observed sensitivities were indeed due to deletion of the corresponding gene in these 23 mutants, we performed PCR with gene-specific primers and found that all the deletions were correct (see supplementary Fig. 2). To exclude that any of the observed sensitivities in these mutants was due to second site mutations, we crossed all the 23 mutants back to WT and showed that the sensitivities were coupled to the G418 marker (used for deletion) for all these mutants (see supplementary Fig. 2). This analysis could not be performed for mutant 15 (SPAC9G1.07), as it was not able to mate with either h+ or h cells (data not shown).

To confirm the sensitivity of these mutants we subjected them to spot tests against the various DNA damaging agents used in the screen. As shown in Fig. 3, this confirmed the (sometimes only slight) sensitivity of the mutants against one or more DNA damaging agents. Next, we used the psi-blast service ([29]; http://www.ncbi.nlm.nih.gov/blast/Blast.cgi) to try and detect (partial) homology of these genes to genes in other organisms. An overview of the detected homologies is found in Table 1. The most significant homologies (to genes in higher eukaryotes with known functions in genome stability maintenance) will be discussed below.

Table 1
Detected homologies of sensitive sequence orphans and hypothetical proteins

The mutant showing the highest sensitivity against all tested DNA damaging agents is SPCC338.08. Using psi-blast, we could not find any orthologues in other organisms. However, an intron prediction program (http://rulai.cshl.org/tools/genefinder/Pombe/pombe.htm) predicted an intron at position 694-737. Splicing of this intron would lead to an extension of the amino acid sequence. Using this corrected sequence for a psi-blast, we found that SPCC338.08 shows homology to CtIP (NCBI accession nr. AAC14371; E-value 0.001 after 2 iterations). This gene has recently been independently identified and characterised in S. pombe (and was named ctp1), and the presence of the intron has been confirmed experimentally [23]. Another mutant showing high sensitivity against various DNA damaging agents is SPAC3H8.05C. A psi-blast search identifies a range of proteins which contain a “cleavage and polyadenylation specificity factor (CPSF) A region”. One of these proteins is human DDB1/XPE (NCBI accession nr. NP_001914; E value 1×10-117, after 3 iterations), a protein involved in DNA repair [63]. The biological significance of this homology remains to be confirmed. Besides a slow growth phenotype in untreated cells, SPCC576.12c also shows a pronounced sensitivity against all agents tested. A psi-blast detects significant similarity to mouse Stra13 (NCBI accession nr. CAM27115; E value 6×10−8 after 2 iterations; see Fig. 4 for alignment). Stra13 is a basic helix-loop-helix transcription factor and has been implicated in p53-dependent apoptosis in response to DNA damage [64]. SPBC19G7.18c shows homology to human Melanoma Antigen D1 (MAGE-D1; NCBI accession nr NP_008917; E value 1 × 10−21, 2 iterations), this homology is confined to the MHD2 domain, outside of the MAGE homology domain [65]. The significance of this homology remains unknown. MAGE-D1 is part of the MAGE protein family, and is a regulator of apoptosis, transcription and cell cycle progression [65]. The MAGE protein family is represented in S. pombe by a single protein, Nse3, which shows homology to MAGE-G1 [66]. SPCC1442.02c shows homology to both S. cerevisiae YBP2 (NCBI accession nr. EDN62054; E value 2 × 10−7 after 2 iterations) and YBP1 (NCBI accession nr. EDN64825; E value 3 × 10−5 after 2 iterations; see Fig. 5 for alignment). YBP2 has been shown to associate with different protein complexes found at the central kinetochore [67], whereas YBP1 is required for H2O2 specific regulation of the transcription factor Yap1 [68]. SPBC16C6.03c shows homology to the human fragile X mental retardation interacting protein NUFIP1 (NCBI accession nr. AAF15315; E value 2 × 10−4 after 2 iterations; alignment in Fig. 6). NUFIP has been shown to interact with BRCA1 and is involved in activation of transcription by RNA polymerase II [69]. SPCC18.09c shows homology to human Aprataxin (NCBI accession nr. AAP86320; E value 4 × 10−10 after 1 iteration; see Fig. 4 for alignment), which is thought to catalyse the removal of adenylate groups which remain covalently linked to 5′ phosphate DNA ends after abortive DNA ligation [70].

In this study we present the first screen of a genome wide S. pombe deletion library for mutants sensitive to DNA damaging agents. As expected, the group of sensitive mutants was significantly enriched for genes involved in core DNA repair and checkpoint response pathways. Mutants of genes involved in the establishment and maintenance of chromatin architecture were also over-represented (specifically including members of the SAGA complex), as were mutants of genes involved in transcription (including the CCR4-Not transcription factor complex) and mutants of genes involved in microtubule related processes (including the DASH complex). Identification of these mutants is a first step in trying to understand the precise roles of chromatin architecture, transcription and microtubule related processes in the maintenance of genome stability in S. pombe. We also identified 23 as yet uncharacterised genes as being involved in genome stability maintenance. Among those genes, we detected novel orthologues of CtIP, Aprataxin, Stra13 and Human Fragile X mental retardation interacting protein NUFIP1, proteins with an established role in genome stability maintenance in higher eukaryotes. Among others of these 23 genes, various homologies to (domains) of proteins have been detected that might give a clue about their involvement in genome maintenance. This study will likely provide a basis for further studies into the function of these conserved genes in the genetically amenable S. pombe model system and thus contribute to our understanding of genome stability maintenance mechanisms in eukaryotes.

Supplementary Material

Supplementary Tables/Figures


We would like to thank Tony Carr for discussions and critical reading of the manuscript, and members of his lab for discussions. Special thanks go to Valerie Wood for help and discussions. This work was funded by a CRUK grant to EH (CRUK C20600/A6620).


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