Identification and characterization of the rlp1+, the novel Rad51 paralog in the fission yeast Schizosaccharomyces pombe

DNA Repair 3 (2004) 1363–1374

Identification and characterization of the rlp1+, the novel Rad51 paralog in the fission yeast Schizosaccharomyces pombe Fuat K. Khasanov a , Albina F. Salakhova a , Olga V. Chepurnaja b , Vladimir G. Korolev b , Vladimir I. Bashkirov a,∗ a

Institute of Gene Biology, Russian Academy of Sciences, Molecular Biology of DNA Repair, Vavilov Street 34/5, 119334 Moscow, Russia b St. Petersburg Nuclear Physics Institute, Russian Academy of Sciences, 188350 Gatchina, Russia Received 17 May 2004; received in revised form 18 May 2004; accepted 18 May 2004 Available online 9 June 2004

Abstract A new DNA repair gene from fission yeast Schizosaccharomyces pombe rlp1+ (RecA-like protein) has been identified. Rlp1 shows homology to RecA-like proteins, and is the third S. pombe Rad51 paralog besides Rhp55 and Rhp57. The new gene encodes a 363 aa protein with predicted Mr of 41,700 and has NTP-binding motif. The rlp1∆ mutant is sensitive to methyl methanesulfonate (MMS), ionizing radiation (IR), and camptothecin (CPT), although to a lesser extent than the deletion mutants of rhp55+ and rhp51+ genes. In contrast to other recombinational repair mutants, the rlp1∆ mutant does not exhibit sensitivity to UV light and mitomycin C (MMC). Mitotic recombination is moderately reduced in rlp1 mutant. Epistatic analysis of MMS and IR-sensitivity of rlp1∆ mutant indicates that rlp1+ acts in the recombinational pathway of double-strand break (DSB) repair together with rhp51+ , rhp55+ , and rad22+ genes. Yeast two-hybrid analysis suggests that Rlp1 may interact with Rhp57 protein. We propose that Rlp1 have an accessory role in repair of a subset of DNA damage induced by MMS and IR, and is required for the full extent of DNA recombination and cell survival under condition of a replication fork collapse. © 2004 Elsevier B.V. All rights reserved. Keywords: Rlp1; Double strand breaks; DNA damage; DNA repair; MMS; CPT; Ionizing radiation; DNA recombination

1. Introduction DNA double-strand breaks (DSB) are the most severe damage to DNA caused by environmental factors and occur spontaneously during normal cellular metabolism. DSBs are also the initiating events of meiotic recombination, and in yeast, of mating type switching. In eukaryotes DSBs are repaired either by homologous recombination (HR), or non-homologous end-joining (NHEJ) mechanisms. In yeast, both Schizosaccharomyces pombe and Saccharomyces cerevisiae, HR is the preferred pathway for DSB repair [1,2]. It was established that in the budding yeast S. cerevisiae DSB repair by homologous recombination is under the control of the RAD52 epistasis group of genes. These genes are involved in DSB repair, mating-type switching, and in meiotic and/or mitotic recombination. There are at least 10 genes ∗ Corresponding author. Tel.: +7 95 1359815; fax: +7 95 1354105. E-mail addresses: [email protected], [email protected] (V.I. Bashkirov).

1568-7864/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.dnarep.2004.05.010

that have been identified in this group: RAD50, RAD51, RAD52, RAD54, RDH54/TID1, RAD55, RAD57, RAD59, MRE11, and XRS2 (reviewed in [3]). MRE11/RAD50/XRS2 are thought to act in HR during the initial steps of DSB processing and also play a role in NHEJ [4,5]. RAD51, RAD52 and RAD54 are involved in the presynaptic, synaptic and postsynaptic steps of the recombination mechanism, and are the most important genes of the group (reviewed in [3]). Rad51 protein is an eukaryotic homolog of bacterial RecA and can form a nucleoprotein filament, active in homology search and strand exchange in vitro [6,7]. Rad55 and Rad57 are Rad51 paralogs and together with Rad51 comprise a subgroup of RecA-like proteins. Rad55 and Rad57 form a heterodimer, playing a stimulatory role to Rad51 in HR; however, they cannot promote on their own DNA pairing and strand-exchange in vitro in the presence of RPA [8]. Mutations in RAD52 group genes result in sensitivity to genotoxic agents including methyl methanesulfonate (MMS), ionizing radiation (IR), and UV, and lead to defects in meiotic and/or mitotic recombination ([9] and reviewed in [3]).

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The recombinational repair pathway of DSB repair is evolutionarily conserved, as homologs of virtually all RAD52 group genes have been found in other eukaryotes (reviewed in [10,11]). The fission yeast S. pombe proved to be a successful model to study DNA repair, including the repair by HR. Homologs of all S. cerevisiae Rad52 group proteins (except Rdh54) were identified in S. pombe, including the Rad51 homolog, Rhp51, and a heterodimer of two Rad51 paralogs, Rhp55/Rhp57 [11–15]. Recently, the recombinational repair group of genes in S. pombe has been expanded by the discovery of rad60+ gene, with the possible role in regulation of recombination at stalled replication forks [16,17]. Here, we report the identification in fission yeast of another new member of the recombinational repair group, rlp1+ . This gene encodes a protein with similarity to RecA-family members, and, thus, represents the third Rad51 paralog in S. pombe besides Rhp55 and Rhp57. Five Rad51 paralogs have been identified in vertebrates: Rad51B, Rad51C, Rad51D, XRCC2 and XRCC3 (reviewed in [3,18]). Identification of Rlp1 places the fission yeast with three Rad51 paralogs between S. cerevisiae and mammals in terms of functional diversification of RecA-like proteins in evolution. Sensitivity of rlp1 mutant to IR and MMS, but not to UV and cross-linking agents suggests that Rlp1 may act in the repair of a subset of damage induced by IR and alkylating agent MMS. Our data indicates that, unlike Rhp55 and Rhp57, Rlp1 has no role in mating type switching, and only a minor role in recombinational rescue of stalled or collapsed replication forks. Our data suggest the functional specialization among the three Rad51 paralogs in fission yeast.

smt-0 or mat1P∆17::LEU2, which are deletions of DSB site in mat1 locus. The reason was to avoid the potential effect of a DSB at mat1 on the repair of genotoxin-induced DSBs by the rlp1∆ mutant. Genetic manipulations and S. pombe growth media—yeast extract agar (YEA) and liquid (YEL), malt extract agar (MEA), minimal medium agar (MMA)—have been described elsewhere [19,20]. When necessary 0.01% (w/v) of amino acids and nucleosides were added to the media. Methylmethane sulfonate (MMS), camptothecin (CPT) and mitomycin C (MMC) were added to media cooled to 50◦ before pouring plates. Crosses have been performed at 25 ◦ C, while 30 ◦ C was used for growing S. pombe cells. The lithium acetate procedure was used for S. pombe transformation [21]. 2.2. Construction of rlp1 deletion strain To construct the rlp1∆::KanMX deletion mutant, the complete ORF of rlp1+ was replaced by the KanMX marker conferring resistance to geneticin (Gibco) using the strategy described in [22]. The gene replacement cassette was generated by PCR using plasmid pFA6a-kanMX6 [23] as a template, and two 99-mer oligonucleotides: both with 78 nucleotide homology to either 5 -, or 3 -flanking sequences surrounding rlp1+ ORF: 5 -TACACTAAGGAAAGTAGAGTGTAAATGTTAAAATTTTTTGCAAGAGCGTTAATAATAATCATCTTTACAGTCTAAATGACGGATCCCCGGGTTAATTAA-3 and 5 -CGTTCTTGACGCATCGCATTAGATCTACAATATATGAATCTTCATGCCTTGTATTAATTATTCCATTACGAATCTTTCGAATTCGAGCTCGTTTAAAC-3 . The gene disruption was confirmed by Southern blot hybridization.

2. Materials and methods 2.3. Tests for sensitivity to genotoxic agents 2.1. Strains, media and growth conditions The genotypes of S. pombe strains used in this study are listed in Table 1. Majority of these strains has the mutation

MMS, CPT and MMC sensitivity was tested in drop-assay: exponentially growing cells were diluted serially from an OD600 = 1.0 suspension by 10-fold or 4-fold,

Table 1 S. pombe strains used in this study Name

Relevant genotype

Source

BVY5 BVY12 BVY19 BVY35 BVY109 BVY111 BVY112 BVY116 BVY119 BVY129 BVY130 BVY167 IBGY605 WDHY1253 EH118

h− smt-0 leu1-32 ura4-D18 h+ mat1P∆17::LEU2 leu1-32 ade7-150 h− smt-0 rhp55∆::ura4+ ura4-D18 leu1-32 h− smt-0 rhp51∆::ura4+ ura4-D18 leu1-32 h− smt-0 rlp1∆::KanMX ura4-D18 leu1-32 h+ mat1P∆17::LEU2 rlp1∆::KanMX leu1-32 ura4-D18 ade7-150 h− smt-0 rlp1∆::KanMX rhp55∆::ura4+ ura4-D18 leu1-32 h− smt-0 rlp1∆::KanMX rad22∆::ura4+ ura4-D18 leu1-32 h− smt-0 rlp1∆::KanMX rhp51∆::ura4+ ura4-D18 leu1-32 h90 ura4-D18 leu1-32 arg3-D4 h− smt-0 rad22∆::ura4+ ura4-D18 h+ rlp1∆::KanMX ura4-D18 leu1-32 arg3-D4 smt0 ura4-D18 ade6-M375 int::pUC8/ura4+ /ade6-469 rlp1∆::KanMX h+ rad2::ura4+ ura4-D18 leu1-32 ade6-704 smt-0 ura4-D18 ade6-M375 int::pUC8/ura4+ /ade6-469

This study This study This study This study This study This study This study This study This study This study This study This study This study W.-D. Heyer [13], [48]

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and 4 ␮l was spotted on YEA plates with or without the drug. Plates were incubated at 30 ◦ C for 3–4 days, or when necessary at 23 ◦ C for 8 days. MMS and MMC sensitivity tests were performed at least twice and included independent clones of each strain to avoid phenotypic variations among clones. Alternatively, acute exposure experiment was performed as follow: MMS at 0.1% was added to exponentially growing cells; at time points indicated aliquots have been withdrawn and equal volume of ice-cold 10% sodium thiosulfate was added to neutralize the MMS; appropriate dilutions were plated on complete media to determine the survival after incubating plates for 3–4 days at 30 ◦ C. To test UV sensitivity, exponentially growing cells were plated on YEA from appropriate dilutions and irradiated with the given doses of UV light. The plates were incubated at 30 ◦ C for 3–4 days, and counted to assess survival. To examine IR survival, exponentially growing cells were washed, resuspended in saline and irradiated with ␥-rays using 60 Co source with a dose rate of 35 Gy/min. Appropriate dilutions were plated on YEA plates to determine the survival at 30 ◦ C. The experiment on acute exposure to MMS and all irradiation experiments were repeated at least twice. 2.4. Recombination assay To assay for spontaneous mitotic intrachromosomal recombination (intra-sister and inter-sister recombination) 20 independent single colonies from strains EH118 and IBGY605 were grown to stationary phase in 10 ml of YEL + supplements. Appropriate dilutions were plated on supplemented YEA to determine the titer of viable cells and on MMA + uracil to score for ade+ recombinants. Colonies were counted after 5 days of growth at 30 ◦ C. The method of the median [24] was used to determine the frequency of recombination events per division. Statistical significance of the observed differences in recombination assay was detected using the Mann–Whitney U-test. Repetition of experiment gave similar result.

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formed into the reporter strain, and three colonies grown on selective medium were used for quantitative ␤-galactosidase assays in each case [25]. ␤–Galactosidase activity was expressed in Miller units. The yeast two-hybrid system consisting of LexA DBD fusion plasmid pEG202, AD fusion plasmid pJG4-5, and S. cerevisiae strain EGY48 carrying the reporter plasmid pSH18-34 was employed [26,27]. Construction of plasmids with DBD and AD fusions of the rhp51+ , rhp55+ , rhp57+ , and rad22+ genes have been described [28]. Plasmids with the rlp1+ gene were constructed by in-frame cloning of the coding sequence (cds) of the gene generated by Pfu polymerase-mediated PCR on S. pombe genomic DNA. The rlp1+ cds was amplified using 5 -GGCGAATTCATGAACGCAGCTGAATGGG-3 and 5 -GGCCTCGAGCTAAACTTTTTCCATTTCTTGC-3 primers and cloned into EcoRI-XhoI to produce pEG202rlp1+ and pJG4-5-rlp1+ . To construct the plasmids with truncated rhp51+ alleles two pairs of primers, 5 -GGCCTCGAGATGGCAGATACAGAGGTGG-3 and 5 - GGCCTCGAGTTATCGAATATGGTACTCAGTGGCGG-3 , and 5 -GGCCTCGAGGAGTACCATATTCGAAGAAGTC-3 and 5 GGCCTCGAGTTAGACAGGTGCGATAATTTCC-3 , and chromosomal DNA as a template were used to amplify the coding region covering aa 1-117 and 114-365 of the Rhp51 protein correspondingly. The PCR products were cloned into XhoI site of the yeast two-hybrid vectors to generate two pairs of plasmids, pEG202-rhp51∆C and pJG4-5-rhp51∆C, and pEG202-rhp51∆N and pJG4-5-rhp51∆N. The cellular levels of the overexpressed fusion proteins have been checked by immunoblotting of total protein extracts after SDS-polyacrilamide gel electrophoresis (PAGE) using anti-HA and anti-LexA antibodies for detection of AD- and DBD-fusions, correspondingly.

3. Results 3.1. rlp1+ encodes a new RecA-like S. pombe protein with highest homology to human XRCC2

2.5. Sporulation efficiency and spore viability The sporulation efficiency was evaluated by scoring microscopically the number of spores, asci and vegetative cells. The sporulation efficiency (% sporulation) was calculated as (0.25 S + A)/(0.25 S + A +0.5 C), where S is the number of spores, A, the number of asci, and C, the number of vegetative cells. To determine the spore viability, tetrads were dissected on YEA plates and the number of colony forming spores was expressed as the percentage to the number obtained in wild type cross. Totally 30 tetrads from wild type and rlp1∆ cross were dissected. 2.6. Yeast two-hybrid analysis Pairwise combinations of DNA-binding domain (DBD) and activator domain (AD) fusion plasmids were trans-

Previously four RecA-like proteins, including meiotic Dmc1, Rhp51 (Rad51 homolog) and Rhp55 and Rhp57 (Rad51 paralogs) have been found in S. pombe. We analyzed the S. pombe database by TBLASTN 1.4.11 [29] using the Rhp55 protein as a query and identified an ORF SPBC1685.11 on cosmid SPBC1685 (mapped on chromosome II) potentially encoding a new polypeptide with significant homology to proteins belonging to Rad51/RecA family. The gene was named rlp1+ , for RecA-like protein 1. The rlp1+ ORF comprises 1094 bp, which can be translated into a predicted protein of 363 aa with molecular mass of 41,700. Three in frame TAA stop codons precede the ATG codon, which makes the use of this codon for initiation of translation highly likely. Rlp1 has the highest homology to human Rad51 paralog XRCC2 [30], especially in N-terminal part (see Fig. 1A). Amino acid sequence of

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HsXrcc2 Rlp1 SpRhp51

1 ----MCSAFHRAESGTELLARLEGRSSLKEIEPNLFADEDSPVHGDILEF 1 MNAAEWVAELKRKSQIESFKEQKGLVYDDHIEKVLYSG---SVNGTLLVI 100 ASKLVPMGFTTATEYHIRRSELITITTGSKQLDTLLQG--GVETGSITEL Box A

Xrcc2 Rlp1 SpRhp51

47 HGPEGTGKTEMLYHLTARCILPKSEGGLEVEVLFIDTDYHFDMLRLVTIL 48 EGNSCSGKTELLYHLASNVLLRSS----QELVLIVSSEWDWSIKRLTFIL 148 FGEFRTGKSQICHTLAVTCQLPIDMGGGEGKCLYIDTEGTFRPVRLLAVA

Xrcc2 Rlp1 SpRhp51

97 EHRLSQSSEEIIKYCLGRFFLVYCSSSTHLLLTLYSLESMFCSHPSLCLL 94 HERLISSRG-VSQTCKCNCAVKLENESTVHLQNAAREDGTDEDINSNSVN 198 DRYGLNGEE-----VLDNVAYARAYNADHQLELLQQAANMMS-ESRFSLL Box B

Xrcc2 Rlp1 SpRhp51

147 ILDSLSAFYWIDRVNGGESVNLQESTLRKCSQCLEKLVNDYRLVLFATTQ 143 DVSLSSGETMLQFPEHEEQHECNR-EMEQLYESASSTVYPCSFWEDAERK 242 VVDSCTALYRTDFSGRGE-LSARQMHLARFMRTLQRLADEFGIAVVITNQ

Xrcc2 Rlp1 SpRhp51

197 TIMQKASSSSEEPSHASRRLCDVDIDYRPYLCKAWQQLVKHRMFFSKQDD 192 IDAQCAILWPMEFSGVVESIPQSTKDLLRIWKEAKIQMHEDFRCNKTEFN 291 VVAQVDGISFNP---DPKKPIGGNILAHSSTTRLSLRKGRGEQRICKIYD

Xrcc2 Rlp1 (A) SpRhp51

247 SQSSNQFSLVSRCLKSNSLKKHFFIIGESGVEFC 242 EACFDASSSRLGCILMDGLSTFYWQLRLERGYTQ 338 SPCLPESEAIF-AINSDGVGDPKEIIAPV-----

ScRad55 SpRhp55 Rlp1 Xrcc2 RecA hRad51C hRad51B ScRad51 SpRhp51 hRad51 SpRhp57 ScRad57 Xrcc3

(B)

hRad51D

Fig. 1. Protein sequence alignment of S. pombe Rlp1p with yeast Rhp51 and human XRCC2, and the relationship of Rlp1p to other RecA-like proteins. (A) The alignment was generated using Clustal X and viewed with Boxshade (Bioinformatics Group, ISREC). Black and gray shading indicates identical and similar residues, correspondingly. (B) Dendrogram representing relationship between eukaryotic RecA-like proteins was generated by ClustalX. Sp, S. pombe; Sc, S. cerevisiae, Hs, Homo sapiens.

S. pombe Rhp51 is included in alignment. This comparison shows that Rlp1 protein is structurally distinct, but retains a number of conserved residues. Pairwise global alignment of protein sequences [31] indicates that Rlp1 has the

highest homology to human XRCC2 protein (28% identity and 44% similarity), while it is less homologous to other human paralogs (XRCC3—23 and 36%, RAD51B—21 and 38%, RAD51C—23 and 38%, RAD51D—26 and 37%),

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and to S. pombe Rhp51 (22 and 38%) and Rhp55 (20 and 34%). Thus, Rlp1 might represent the third S. pombe Rad51 paralog. Multiple sequence alignment and molecular phylogenetic analysis using ClustalX version 1.81 [32] also shows closer similarity of Rlp1 to XRCC2 (Fig. 1B). All RecA-like family members have two NTP binding motifs: Walker A box and B box. The analysis of Rlp1 protein sequence revealed the Walker A box (GxxxxGKT/S), however, no clearly defined Walker B box with the characteristic hydrophobic ␤-sheet is present. The sequence similarity of Rlp1p to RecA-like proteins implicated the protein in recombinational repair of DNA damage. 3.2. rlp1+ is a DNA damage repair gene acting in one pathway with rhp51+ , rhp54+ and rhp55+ To test the suggestion that rlp1+ gene is involved in repair of DNA damage, a null allele was constructed by substitution of entire ORF of the gene by the KanMX marker conferring resistance to geneticin. The deletion mutant was viable in haploid cells indicating that rlp1+ gene is not essential for mitotic growth. Deletion mutants of RecA-like genes involved in recombinational repair of DSBs in fission yeast are sensitive to alkylating drug MMS. Among them mutants of the Rad51 paralogs, rhp55 and rhp57, exhibit an unusual phenotype: increased sensitivity to damaging agents at low temperatures [28,33,34]. Thus, we tested the sensitivity of rlp1∆::KanMX strain to MMS at 30 and 23 ◦ C in drop-assays. As shown in Fig. 2A, the rlp1 mutant showed sensitivity to 0.005% and 0.0075% MMS at 30◦ and was not further sensitized when the drop-assay was performed at 23 ◦ C. As MMS is known to slow down DNA replication affecting the results of drop-assay, we also demonstrated in acute exposure experiments that rlp1 mutant is sensitive to MMS (Fig. 2B). However, the survival of rlp1 cells is less affected compared to the rhp55 mutant. Mutants of recombinational repair genes in budding yeast are sensitive to cross-linking agent MMC [35]. Rad51 paralog mutants in vertebrates, including irs1 cells defective in XRCC2, also exhibit high sensitivity to MMC (reviewed in [36]). Thus, we tested by drop-assay the sensitivity of rlp1 mutant to this drug. Fig. 2C indicates that the rlp1 cells are not sensitive to MMC at 200 ␮g/ml, while rhp51 and rhp55 mutants show hypersensitivity to MMC. Increasing the dose of MMC up to 400 ␮g/ml, which is clearly toxic for the wild type cells, did not sensitized the rlp1 mutant cells compared to the wild type. The apparent resistance of rlp1 mutant to MMC was not specific to particular clone (data not shown). We concluded that rlp1 mutant is not sensitive to MMC unlike mutants of other Rad51 paralogs in yeast and vertebrates. In S. pombe rhp51+ , rhp54+ , rhp55+ , and rad22+ have been assigned by double-mutant analysis to one epistasis group, the recombinational repair group ([33] Khasanov et al., unpublished observation). Since Rlp1p shows similarity with RecA-like proteins, we surmised that Rlp1p could

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function in the same DNA repair pathway. To check this assumption we performed epistatic analysis of rlp1+ gene. The double-mutants rlp1 rhp51, rlp1 rhp55, and rlp1 rad22 were tested for sensitivity to MMS along with single mutants (Fig. 2D). All reference mutants, rhp51, rhp55 and rad22, were sensitive to MMS at lower doses than the rlp1 mutant, which suggest that Rlp1 has a less important role in the repair of MMS damage. Double-mutants rlp1 rhp51, rlp1 rhp55, and rlp1 rad22 were as sensitive to MMS as the single rhp51, rhp55 and rad22 mutants, indicating that rlp1+ is epistatic to recombinational repair genes. It is known that damage caused by MMS is rather complex and DSBs constitute only a portion of various lesions produced by the drug [37]. Thus, we extended the epistasis analysis of rlp1+ gene to the IR-induced DNA damage. Fig. 3 shows that the rlp1 mutant is moderately sensitive to ␥-rays in comparison to rhp51 and rhp55 mutants. This data corroborate our finding on the rlp1 mutant sensitivity to MMS and indicates that Rlp1 has a function in DSB repair, though its role is not as crucial, as that of Rhp51p and Rhp55p. The double mutants rlp1 rhp51 and rlp1 rhp55 showed the same ␥ ray sensitivity, as the rhp51 and rhp55 single mutants. This indicates that rlp1 is epistatic to rhp51 and rhp55 and functions in the same DNA repair pathway, namely recombinational repair of DSBs. 3.3. UV damage repair and replication stress response in rlp1 mutant UV damage produces pyrimidine dimers and pyrimidine 6–4 photoproducts [37], which stall replication forks. They are repaired in S. pombe by two mechanisms: nucleotide excision repair and UVDE repair [38]. Recombinational repair genes also have an important role in cellular survival upon UV damage: in addition to UVDE pathway they are involved in UV damage tolerance pathway controlled by the Cds1 checkpoint kinase [39]. The UV damage tolerance mechanism is thought to involve the replication fork restart by recombinational repair machinery. Consequently, S. pombe mutants in DSB repair genes show significant hypersensitivity to UV light [33,34,39–41]. They also exhibit considerable sensitivity to camptothecin (CPT), a DNA topoisomerase I inhibitor, which produces single-strand nicks leading finally to replication fork collapse [42]. Since rlp1+ belongs to recombinational repair epistasis group (see Fig. 2D and Fig. 3), we tested if the rlp1 mutant is also sensitive to UV and CPT. UV survival curves for rlp1 and rhp55 mutants are shown on Fig. 4A. While the rhp55 mutant showed the expected significant UV sensitivity, the rlp1 mutant was as resistant to UV damage as the wild type strain. Similarly, in a drop-assay the deletion of rlp1+ gene did not confer sensitivity to 5 ␮M CPT, the dose lethal for rhp51 and rhp55 mutants (Fig. 4B). However, at high CPT concentration, 20 ␮M, the rlp1 mutant exhibited sensitivity to the drug compared to wild type strain, indicating that Rlp1 contributes to the rescue of replication forks under conditions of

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Fig. 2. MMS sensitivity and epistatic analysis of rlp1+ gene. (A) MMS survival test of the rlp1 mutant at two different temperatures. MMS sensitivity was tested by drop-assay after sequential 10-fold dilutions. Plates were incubated for 3 days at 30 ◦ C, and for 8 days at 23 ◦ C. (B) Survival of rlp1 mutant under acute exposure to MMS. Survival test was performed as described in Section 2. (C) Test for the sensitivity to cross-linking agent MMC. Drop-assay was performed after sequential 10-fold dilutions of exponential cultures. (D) Epistatic analysis of rlp1+ gene for repair of MMS-induced damage. Drop-assay with five-fold sequential dilutions of cultures was performed. Isogenic strains used were wild type (BVY5), rlp1∆ (BVY109), rhp51∆ (BVY35), rhp55∆ (BVY19), rad22∆ (BVY130), rlp1∆ rhp51∆ (BVY119), rlp1∆ rhp55∆ (BVY112), and rlp1∆ rad22∆ (BVY116).

massive collapse. The lack of UV sensitivity phenotype of rlp1 mutant set it apart from the other recombinational repair mutants. Mutations in the S. pombe recombinational repair genes rhp51+ , rhp54+ , rhp57+ , rad32+ are synthetically lethal with a mutation in the rad2+ gene, encoding the FEN-1 flap-structure-specific endonuclease implicated in Okazaki fragment maturation during replication [34,41,43]. The

reason is that mutants in bacteria and yeast with a defect in processing Okazaki fragments require the recombination function for viability, since they may accumulate DSBs during replication [44,45]. We examined if the rlp1 mutant exhibits lethality when combined with rad2. We crossed a rad2 strain (WDHY1253) with a rlp1 strain (BVY109), dissected tetrads and analyzed the progeny of each ascus for the markers associated with deletion of rad2+ (ura+ ) and

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3.4. Spontaneous mitotic intrachromosomal recombination in rlp1 mutant

Fig. 3. Epistatic analysis of rlp1+ gene for repair of ␥ ray-induced damage. Strains used were the same as indicated in Fig. 2 (䊏), wild type; (䊊), rlp1; (), rhp51; (䊐), rhp55; (䉱), rlp1 rhp51; (䊉), rlp1 rhp55. All survival curves were determined at 30 ◦ C.

rlp1 (kanR ). Among 9 tetrads with 4 viable spores we found, besides 1 tetrad with parental ditype (PD), 4 tetrads with tetratype (TT) and 4 tetrads with non-parental ditype (NPD) configuration containing a recombinant ura+ kanR spores. Thus, the rad2 rlp1 double mutant is viable, unlike the double mutants of other recombinational repair genes with rad2.

It has previously been shown that several S. pombe RAD52 group mutants affected intrachromosomal mitotic recombination. It was found that rad50 mutant has a strong decrease in sister chromatid recombination, while rad22B exhibited moderate, and rhp51 and rhp54 strong hyper-recombination phenotype [13,46,47]. Thus, we assayed the spontaneous mitotic recombination in rlp1 mutant by measuring the frequency of sister chromatid recombination as described by [13,48]. This assay employed the haploid strain with ura+ marker flanked by the direct repeats of ade6− heteroalleles (ade6-469 and ade6-M375). The recovery of Ade+ recombinants is the result of homologous recombination between ade− alleles of one sister chromatid (intra-sister) or between alleles on different sister chromatids (inter-sister). We found that the frequency of sister chromatid recombination per 1 × 106 plated cells was decreased 1.7-fold in rlp1 mutant compared to the wild type strain (Table 2), which corresponds to a 2.0-fold decrease in recombination rate as determined with the Lea–Coulson method of the median. The significance of the recombination frequency reduction in rlp1 mutant was confirmed by statistical analysis with

Fig. 4. Sensitivity test of rlp1 mutant to agents impeding replication: (A) UV survival curves of wild type, rlp1∆ and rhp55∆ strains; (B) drop-assay for sensitivity to CPT using five-fold serial dilutions of cultures. Strains used were the same as indicated in Fig. 2.

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Table 2 Sister chromatid recombination frequency in wild type and rlp1∆ mutant Median no. of ade+ recombinants per 1 × 106 plated cellsa

Inter/intra-sister recombination frequency per divisionb

Wild type

rlp1∆

Wild type

rlp1∆

2.7 × 103 (1.7×c ; P < 0.05d )

1.55 × 103

2.77 × 10−4 (2.0×)c

1.42 × 10−4

a

Based on 20 independent cultures. Calculated with the Lea–Coulson method of the median [24]. c Fold decrease compared with wild type. d Probability as determined with the Mann–Whitney U-test. b

Mann–Whitney U-test. This result indicates that rlp1+ has the role in homologous recombination between sister chromatids in vegetative cells. 3.5. Meiosis and mating type switching in rlp1 mutant S. pombe rhp51, rhp55 and rad22 mutants are characterized by meiotic defects, such as reduced sporulation efficiency and decreased spore viability [33,49]. Therefore, we examined the sporulation of rlp1 crosses in comparison with wild type crosses. Strains BVY5 × BVY12 (wild type), and BVY109 (rlp1∆) × BVY111 (rlp1∆) were crossed. The sporulation efficiency and spore viability in wild type cross were 33 and 95%, respectively, while in rlp1 cross sporulation efficiency was reduced 2.3-fold (14.3%) but viability was not changed (95%) compared to wild type cross. Mating type (MT) switching in S. pombe is initiated by a DNA lesion (probably a single strand break or other DNA modification) at the mat1 cassette, which is thought to be converted by the ongoing replication to a DSB repaired by a recombination mechanism [50–52]. The impairment of recombinational repair of DSBs can cause a defect in MT switching. Indeed, the DSB repair mutants, rad22, rhp51 and rhp55, are defective in MT switching ([53]; Khasanov et al., unpublished observation). We crossed a h+ rlp1∆ strain BVY167 with a h90 rlp1+ strain BVY129, and dissected the resulting tetrads in order to obtain homothallic h90 rlp1 mutant. 20 fully viable tetrads were analyzed for the genotype of individual spores. Among them we found four tetrads of non-parental ditype containing two h90 rlp1∆ (iodine-positive) and two h+ rlp1+ (iodine-negative) spores. Isolated h90 rlp1 spore clones when plated on sporulation media gave rise to colonies positively stained with iodine vapors like wild type parental h90 strains, indicative of MT switching and spore formation. Moreover, h90 rlp1 strain did not segregate any iodine-negative (unswitchable) heterothallic colonies, the feature observed in some of the MT switching defective mutants [54]. Taken together these observations indicated that the rlp1 mutant does not have the defect of MT switching.

3.6. Yeast two-hybrid analysis of Rlp1 interaction with other recombinational repair proteins Multiple interactions between recombinational repair proteins in S. pombe have been identified [28]. Among them, two S. pombe Rad51 paralogs, Rhp55 and Rhp57, form a heterodimer, which interacts with N-terminal part of Rhp51 via Rhp57 subunit. Since our data suggest that Rlp1 is the third Rad51 paralog in S. pombe, and belongs to the recombinational pathway of DSB repair, we decided to perform a systematic analysis of the interactions between Rlp1 and the other members of the recombinational repair group in S. pombe, using the yeast two-hybrid system. The N-terminal fusions of the full-length Rlp1 protein with DBD (bait) and AD (prey) of the corresponding vectors were constructed and pairwise interactions of Rlp1 with other proteins were tested (Table 3). To ensure that both AD- and DBD-fusions are produced under our conditions in amounts adequate to detect positive interactions we checked the protein levels in assayed cells by immunoblotting of total protein extracts (Fig. 5). As shown in Table 3, no detectable interaction beTable 3 Yeast two hybrid analysis with rlp1+ ␤-Gal activitya

Fold increaseb

DBD fusion

AD fusion

Rlp1

– Rhp51 Rhp55 Rhp57 Rad22 Rlp1

1.06 1.42 2.40 2.63 2.63 2.16

0.16 0.3 0.65 0.33 0.40 0.71

1.0 1.3 2.3 2.5 2.5 2.0

Rhp51

– Rlp1

1.29 ± 0.15 1.05 ± 0.14

1.0 0.8

Rhp55

– Rlp1

0.60 ± 0.07 0.42 ± 0.06

1.0 0.7

Rhp57

– Rlp1

27.88 ± 2.93 158.63 ± 7.02

1.0 5.7

Rad22

– Rlp1

8.12 ± 0.35 4.09 ± 0.19

1.0 0.5

± ± ± ± ± ±

␤-Gal activity was determined as described in Section 2. Fold increase was determined by expressing the specific activity of strains carrying both DBD and AD fusion plasmids relative to the specific activity of strains with DBD fusion plasmid only and AD empty vector. a

b

F.K. Khasanov et al. / DNA Repair 3 (2004) 1363–1374

1371

Fig. 5. The levels of DBD- and AD-fusion proteins overexpressed under condition of yeast two-hybrid system. The overexpression was driven by inducible GAL (DBD-fisions) and constitutive ADH (AD-fusions) promoters. Protein extracts from cells overexpressing pairs of indicated proteins were subjected to 4–12% SDS-PAGE and immunoblotting under standard conditions. DBD-fusion and AD-fusion proteins were detected using anti-LexA and anti-HA antibodies, correspondingly.

tween Rlp1 and Rhp51 or Rhp55 proteins was found. However, while AD-Rhp51 fusion protein was expressed at the level comparable to other AD-fusions proteins (Fig. 5, upper panel, lane 1), the LexA-Rhp51 fusion was unstable (Fig. 5, lower panel, lane 5) explaining the negative result on interaction at least for one combination of plasmids. We extended our interaction analysis between Rlp1 and Rhp51 by using truncated versions of Rhp51, Rhp51C (aa 1-117) and Rhp51N (aa 114-365), which were found to produce more stable fusion protein [30]. However, even improved stability of the truncated Rhp51 fusions did not result in interaction with Rlp1 protein (data not shown). Similar to Rhp55 and Rhp57, no interaction of Rlp1 with Rad22, was detected, although the expression of Rlp1 and Rad22 fusions was fairly strong (Fig. 5). Interestingly, we consistently saw an almost 6-fold increase in interaction between Rlp1 and Rhp57 paralog. This suggest that Rlp1, like the S. pombe Rad55 paralog, may be not directly associated with Rhp51 recombinase, but rather forms transient complex with Rhp57 subunit of the Rhp55/Rhp57 heterodimer. However, we failed to confirm this by immunoprecipitation approach using chromosomally HA-tagged Rlp1, probably, because of the low abundance of both proteins.

4. Discussion We report here the identification of rlp1+ , a new S. pombe DNA repair gene encoding a protein with homology to the RecA/Rad51-family proteins. Besides Rhp51, Rhp55, Rhp57, and meiotic Dmc1, this is the fifth S. pombe protein with homology to RecA. The sequence similarity of Rlp1 to RecA-like proteins, the DNA repair phenotypes of rlp1 mutant, and epistasis analysis suggest that Rlp1 is involved in recombinational DNA repair. Pairwise and multiple protein sequence alignments (see Fig. 1) shows that Rlp1 is a mem-

ber of RecA/Rad51 family and has the highest homology to the human XRCC2 protein. All RecA-like proteins contain a central core domain encompassing two dNTP-binding folds, the Walker A and B boxes, and less conserved Nand C-terminal extensions of different length. We did not find a well-defined Walker B box in Rlp1, unlike in other RecA-like proteins. At present we do not know if Rlp1 possesses ATP hydrolysis activity and whether this activity is important for its function. The significance of ATP hydrolysis for the function of Rad51 paralogs in different species was not systematically studied. Studies with the fission and budding yeast paralogs, Rhp55/Rhp57 and Rad55/Rad57 respectively, suggest that one active ATP binding site per heterodimer may be sufficient to carry out its function in DNA repair [28,55]. Similarly, the mammalian Rad51 paralogs form and function in vivo as heteromeric complexes and subcomplexes (reviewed in [3]). Substitution of the conserved lysine residue in the Walker box A of XRCC2 did not lead to a defect in XRCC2 function [56]. However, the XRCC2/RAD51D subcomplex possesses DNA-stimulated ATPase activity [57]. Taken together these data may indicate that in both yeast and vertebrates there is functional divergence among subunits of the heteromeric paralog complexes with only one subunit retaining the ATP binding function (discussed in [56]). Similarly to XRCC2 the Rlp1 protein also can be involved in such an asymmetric complex as a partner with a non-active ATP binding site. The rlp1 mutant shows moderate sensitivity to MMS and ␥-rays, but not to UV light (see Fig. 2A and B; Figs. 3 and 4A). The irs1 hamster cell line, as well as chicken DT40 cells, defective in XRCC2 also show moderate sensitivity to IR (2-3-fold) [58,59]. However, unlike irs1 cells, which are extremely sensitive to cross-linking agents (10-60-fold) (reviewed in [36]), the rlp1 mutant is not sensitive to the cross-linking agent MMC (Fig. 2C). This is also in contrast to the high sensitivity to MMC of other RAD51 paralog

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mutants in vertebrates (reviewed in [36]), and of recombinational repair mutants in fission (see Fig. 2C) and budding [35] yeast. Thus, despite the homology of Rlp1 to XRCC2, it does not play any role in cross-links repair in S. pombe unlike XRCC2 does in mammals. This indicates that Rlp1 is not a fission yeast counterpart of human XRCC2, but rather a Rad51 paralog, which may have a common ancestor with XRCC2, and acquired its unique function in fission yeast DNA repair in the course of evolution. Unlike mutants of other Rad51 paralogs in both S. cerevisiae (rad55, rad57) and S. pombe (rhp55, rhp57), the rlp1 mutant is not sensitive to UV and exhibits some sensitivity to the Topo I inhibitor CPT only at very high doses (see Fig. 4). Moreover, the double rad2 rlp1 mutant is viable, unlike the rad2 double mutant with other S. pombe Rad51 family members. In addition, unlike mutants of other S. pombe mitotic RecA/Rad51-like genes, rlp1 does not exhibit a cell elongation and nuclear aberration phenotype (data not shown) indicative of S-phase progression problem due to impaired repair of stalled or collapsed replication forks by homologous recombination. This suggests that Rlp1 protein is not involved in repair of spontaneous DNA lesions during replication, but plays a minor role in rescue of induced replication fork collapses. However, Rlp1 does have a role in repair of IR- and MMS-induced damage and acts in the same pathway as the other recombinational repair genes (see Figs. 2 and 3). MMS is considered as radio mimetic, however, unlike IR the primary lesion produced by MMS is not a DSB, but methylated bases, mainly 3-methyladenine (3MeA). Alkylation lesions are repaired mainly by the base excision repair mechanism (reviewed in [60]). The consequence of the enzymatic excision of alkylation lesions is the accumulation of single-strand breaks and DSBs in DNA at low and high concentrations of MMS, respectively [61]. As rlp1 mutant is sensitive to rather high doses of MMS, this suggests that Rlp1 may function in repair of a subset of DSBs induced directly by IR or indirectly by the action of base excision repair enzymes on the primary lesions created by MMS. However, we can not exclude that Rlp1 could be involved specifically in the repair of certain lesions, other than DSBs, produced by IR and MMS treatment. Meiotic recombination in S. pombe is initiated by DSBs [62], which is repaired with the help of recombinational repair proteins to result in viable progeny. As rlp1 mutant did not affect spore viability, unlike rhp51, rhp55 and rhp57 mutants [33,34,49], it seems that it is not crucial for completion of meiosis. However, taking in consideration that S. pombe dmc1 mutant is defective in meiotic recombination, but has wild type spore viability [63], the role of Rlp1 in meiosis cannot be ruled out. Indeed, the recent systematic studies of the role of Rlp1 in S. pombe meiosis indicate that rlp1 mutation moderately affects both meiotic intergenic and intragenic recombination and exhibits genetic interaction with other RecA-like proteins (J. Kohli, personal communication).

Several lines of evidences strongly suggest that rlp1+ is a gene involved in recombinational DNA repair in S. pombe. First, the epistasis analysis for MMS and IR sensitivity places rlp1+ gene in the same recombinational repair pathway defined by rhp51+ , rhp55+ , rhp57+ , and rad22+ genes (see Figs. 2 and 3). Second, rlp1 mutant has a moderate defect in spontaneous mitotic intrachromosomal recombination (see Table 2). Third, rlp1 has an effect on meiotic recombination, and is epistatic with rhp51 for spore viability and intergenic recombination (J. Kohli, personal communication). In addition, the yeast two-hybrid analysis indicates an interaction, although not strong, between Rlp1 and Rhp57 proteins (see Table 3). This implies that Rlp1 can form a complex with Rhp57 protein, although, we failed to confirm this by immunoprecipitation approach, probably, because of the low abundance of both proteins. In human cells Rad51 paralogs arranged in two stable subcomplexes: Rad51B–Rad51C–Rad51D–XRCC2 and Rad51C–XRCC3 [64]. At present it is not clear if the three S. pombe Rad51 paralogs exist as a dimeric (Rhp55–Rhp57) and/or trimeric (Rhp55–Rhp57–Rlp1) complexe, or there are two alternative subcomplexes: Rhp55–Rhp57 and Rhp57–Rlp1. Future studies are necessary to differentiate between these possibilities. The phenotypes exhibited by rlp1 mutant compared to those of rhp55 and rhp57 indicate that there is a functional specialization among three S. pombe Rad51 paralogs. First, the rlp1 mutant has no visible role in repair of spontaneous (lack of cell elongation and viability of rad2 rlp1 double mutant) or UV-induced (UV resistance) replication damage in contrast to rhp55 and rhp57 mutants. Second, it has no role in mating-type switching, while rhp55 is strongly defective in this process (Khasanov et al., unpublished observation). Third, the rlp1 mutant has a moderately reduced rate of spontaneous intrachromosomal recombination, while rhp55 mutant shows the hyper-recombination phenotype in this assay (Khasanov and Bashkirov, unpublished observation), similar to rhp51 mutant [46,47], and S. cerevisiae rad51, rad55 and rad57 mutants (reviewed in [3]). In the assay we used, the recombination can occur between sister chromatids as well as within a single chromatid during replication and in G2 phase. As rlp1+ does not have visible role in S-phase, it could be that only spontaneous recombination events in G2 is specifically affected by rlp1 mutation in contrast to rhp55 and rhp57. The exact mechanistic role of Rlp1 in S. pombe recombination and DSB repair is not clear at present and needs to be established. The effects of rlp1 mutation on mitotic and meiotic recombination suggest that it play an auxiliary role in these processes. Similarly, the mild repair phenotypes of rlp1 mutant indicate that Rlp1 protein has an accessory role in DSB repair, or alternatively is involved in repair of a subset of DNA lesions produced by IR and MMS. We propose that, while Rhp55–Rhp57 heterodimer functions in the repair of both damage-induced and spontaneous replication-associated DNA breaks, the Rlp1 has a distinct

F.K. Khasanov et al. / DNA Repair 3 (2004) 1363–1374

role exclusively in the repair of genotoxin-induced damage and spontaneous lesions outside the S-phase.

Acknowledgements We thank A. Grischuk, E. Hagnazari, W.-D. Heyer, and J. Kohli for comments on the manuscript. We are grateful to E. Hartsuiker, A.M. Carr, and W.-D. Heyer for the help with strains and to J. Kohli for communication of unpublished results. V.I. Bashkirov is the International Research Scholar of the Howard Hughes Medical Institute. This work was supported by International Research Scholar’s grant 55000299 from the Howard Hughes Medical Institute, and Research Grant 01-04-48957 from the Russian Fund for Basic Research to V. I. B.

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