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Involvement of Schizosaccharomyces Pombe Rrp1 and Rrp2 in The 8196–8209 Nucleic Acids Research, 2013, Vol. 41, No. 17 Published online 4 July 2013 doi:10.1093/nar/gkt564 Involvement of Schizosaccharomyces pombe rrp1+ and rrp2+ in the Srs2- and Swi5/Sfr1-dependent pathway in response to DNA damage and replication inhibition Dorota Dziadkowiec1,*, Karol Kramarz1, Karolina Kanik1, Piotr Wis´ niewski2 and Antony M. Carr3 1Faculty of Biotechnology, Wrocław University, Przybyszewskiego 63-77, 51-148 Wrocław, Poland, 2Institute of Low Temperature and Structure Research, Polish Academy of Sciences, PO Box 1410, 50-950 Wrocław, Downloaded from Poland and 3Genome Damage and Stability Centre, University of Sussex, Falmer, Brighton, BN1 9RQ, UK Received April 15, 2013; Revised June 1, 2013; Accepted June 4, 2013 ABSTRACT INTRODUCTION http://nar.oxfordjournals.org/ Previously we identified Rrp1 and Rrp2 as two In all organisms, homologous recombination (HR) is a proteins required for the Sfr1/Swi5-dependent high-fidelity DNA repair pathway, essential for the branch of homologous recombination (HR) in repair of DNA double-strand breaks (DSBs) and for Schizosaccharomyces pombe. Here we use a yeast normal DNA replication. The RecA/Rad51 family of proteins forms filaments on single-stranded DNA two-hybrid approach to demonstrate that Rrp1 and (ssDNA), which catalyze homology search DNA strand Rrp2 can interact with each other and with Swi5, an invasion reactions, the hallmark of HR (1). HR mediator protein. Rrp1 and Rrp2 form co- Nucleofilament formation by Rad51 recombinase at Sussex Language Institute on September 10, 2014 localizing methyl methanesulphonate–induced foci (formerly Rhp51 in Schizosaccharomyces pombe) (2) is in nuclei, further suggesting they function as a assisted by a group of proteins called recombination me- complex. To place the Rrp1/2 proteins more accur- diators (3–5). The main mediator, Rad52 (formerly Rad22 ately within HR sub-pathways, we carried out exten- in S. pombe), localizes to the 30 ends of ssDNA and is sive epistasis analysis between mutants defining required for the exchange of RPA for Rad51 (6–8). Rrp1/2, Rad51 (recombinase), Swi5 and Rad57 (HR- Human and mice homologs of Rad52 exist but are mediators) plus the anti-recombinogenic helicases thought to play a minor role in vertebrate HR; for Srs2 and Rqh1. We confirm that Rrp1 and Rrp2 act example, knockout mice exhibit almost no phenotype in DNA recombination or repair (9). In its place, the human together with Srs2 and Swi5 and independently of tumor suppressor protein BRCA2 plays a key role in HR, Rad57 and show that Rqh1 also acts independently recruiting vertebrate RAD51 on RPA-coated ssDNA and of Rrp1/2. Mutants devoid of Srs2 are characterized stabilizing presynaptic filaments (10,11). Recently, verte- by elevated recombination frequency with a con- brate RAD52 has been shown, especially in the absence of comitant increase in the percentage of conversion- BRCA2, to anneal RPA-coated ssDNA (12,13). type recombinants. Strains devoid of Rrp1 or Rrp2 Additional proteins or complexes are required to facili- did not show a change in HR frequency, but tate the formation and/or stabilization of the Rad51 the number of conversion-type recombinants was nucleoprotein filament. The Rad55/Rad57 complex increased, suggesting a possible function for (formerly Rhp55/Rhp57 in S. pombe) was shown to Rrp1/2 with Srs2 in counteracting Rad51 activity. stabilize the Rad51 nucleofilament and enhance Rad51- Our data allow us to propose a model placing Rrp1 mediated strand exchange (14–16). In Saccharomyces cerevisiae, the Rad55/Rad57 heterodimer counteracts the and Rrp2 functioning together with Swi5 and Srs2 in disruption of nucleoprotein filament by the Srs2 a synthesis-dependent strand annealing HR repair antirecombinase in a manner requiring direct interaction pathway. between Rad55/Rad57 and Srs2 (17). In S. pombe,a *To whom correspondence should be addressed. Tel: +4871 375 6238; Fax: +4871 375 6234; Email: [email protected] Present address: Karol Kramarz, Faculty of Biological Sciences, University of Wroclaw, 51-148 Wroclaw, Poland. ß The Author(s) 2013. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/3.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] Nucleic Acids Research, 2013, Vol. 41, No. 17 8197 second mediator complex, Sfr1/Swi5 (18) acts in parallel Media and general methods to Rad55/Rad57 (19,20) to stabilize and activate Rad51- Media used for S. pombe growth were as described (43). ssDNA filaments in an ATP-dependent manner (21). Sfr1/ Yeast cells were cultured at 30C in complete yeast extract Swi5 is conserved in mice and humans, and its depletion plus supplements (YES) medium or Edinburgh minimal results in sensitivity to ionizing radiation, suggesting a medium (EMM). Thiamine was added, where required, conserved role in HR (22,23). A corresponding complex at 5 mg/ml and geneticin (ICN Biomedicals) at 100 mg/ in S. cerevisiae, Sae3/Mei5 functions exclusively in meiosis ml. For YES low Ade plates, the concentration of together with Dmc1, the meiotic Rad51 homolog (24,25). adenine was reduced 10-fold. HR plays a critical role in both DSB DNA repair and in the recovery of arrested replication forks (26–28). In the Spot assays absence of HR, spontaneously occurring barriers to repli- cation fork progression contribute to a slow growth Cells were grown to mid log phase, serially 10-fold diluted phenotype and to constitutive checkpoint activation. and 2 ml aliquots were spotted onto YES plates, which ssDNA is a common intermediate in many aspects of were either UV irradiated using Stratalinker (Stratagene) DNA metabolism including replication arrest and can or contained one of the following compounds: methyl Downloaded from promote inappropriate recombination that, in turn, leads methanesulphonate (MMS), camptothecin (CPT) or to genomic instability (29). Thus, the action of Rad51 and hydroxyurea (HU) at the stated concentrations. Plates its mediators must be tightly controlled. There is substan- were incubated at 30C for 3–5 days and photographed. tial evidence that the choice of a mediator complex may All assays were repeated a minimum of three times. determine the final outcomes of HR: for example, Rad55/ Rad57, but not Swi5/Sfr1, is essential for crossover (CO) Complementation of rad57Drrp2" mutant phenotype by http://nar.oxfordjournals.org/ production in S. pombe (30,31). Selection of the mechan- overexpression of Rrp2 ism for recombination intermediate resolution is also im- The rrp2+ gene was polymerase chain reaction amplified portant: for example, the Mus81-Eme1 nuclease complex using genomic DNA as a template and cloned into the is required for CO formation in mitotic cells (32). NdeI-SmaI sites of pREP41/42-EGFPN (44), or into On the other hand, the role of numerous noncanonical SalI-SmaI sites of pREP41-Red Fluorescent Protein helicases in recombination is to restrict the formation of (RFP) plasmid (42). Both inserts were verified by CO. Anti-recombinogenic helicases Srs2 and Fml1 sequencing. For complementation experiments, rad57Á (FANCM ortholog) are essential for CO avoidance, rrp2Á mutant cells transformed with either pREP41- at Sussex Language Institute on September 10, 2014 acting to remove Rad51 from ssDNA and channel RFP-Rrp2 or the empty vector control were grown for repair into synthesis-dependent strand annealing (SDSA) 20 h in EMM-Leu medium without thiamine. Both (33–37). The RecQ homologs act as anti-recombinases by cultures were diluted and 2 ml aliquots were spotted on promoting Holiday junction (HJ) dissolution thus limiting YES plates either without drug or containing 1.5 mM CO formation (33,38). In S. pombe, an F-box helicase, CPT or 4 mM HU. Untransformed rad57Á and rad57Á Fbh1, has also been shown to limit CO formation, par- rrp2Á mutant cultures were plated for comparison. ticularly at stalled replication forks (39–41). Inappropriate activation of HR can lead to erroneous recombination, Survival assays promoting genetic instability, loss of heterozygosity, chromosome rearrangements and potentially cell death. For CPT survival, cells were grown to mid-log phase in 5 ml Thus, identifying all the players and understanding of YES medium, CPT was added to a final concentration of the interactions between Rad51 recombinase, its medi- 20 mM and cultures incubated at 30 C. At the stated ators plus the enzymes that can remodel and/or resolve timepoints, 500 ml aliquots were removed to Eppendorf joint molecules is crucial for building a model of HR regu- tubes, serially diluted and plated on YES plates. Plates lation in mitotic cells. Schizosaccharomyces pombe has were incubated at 30 C for 3–5 days. Colonies formed proven a good model system for this purpose: two new were counted and percent survival calculated against mediator complexes, Swi5/Sfr1 (18) and Rrp1/Rrp2 (42), samples taken before addition of the drug (time 0). were initially identified in this organism. Here we present For HU survival, cells were grown to mid-log phase in further characterization of the Rrp1 and Rrp2 proteins. 5 ml YES medium, serially diluted and plated on YES We show that they can form a complex with each other plates containing the indicated concentrations of HU. and co-localize at sites of DNA damage. We present Colonies formed after 4–6 days of incubation were genetic evidence that Rrp1 and Rrp2 act together in a counted and percent survival calculated against the no Srs2- and Swi5/Sfr1-dependent SDSA sub-pathway of drug control. All assays were repeated a minimum of HR for DSB repair and replication-dependent DNA three times for each strain. damage tolerance. For survival following acute HU treatment, cells were grown to mid-log phase in 5 ml YES medium and samples were taken, serially diluted and plated on YES medium to MATERIALS AND METHODS determine the initial number of cells in each culture.
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