Research The Arabidopsis RAD51 paralogs RAD51B, RAD51D and XRCC2 play partially redundant roles in somatic DNA repair and gene regulation Yingxiang Wang1, Rong Xiao2*, Haifeng Wang1,3*, Zhihao Cheng1, Wuxing Li2, Genfeng Zhu1, Ying Wang1 and Hong Ma1,2,3 1State Key Laboratory of Genetic Engineering and Institute of Genetics, Institute of Plant Biology, Center for Evolutionary Biology, School of Life Sciences, Fudan University, Shanghai 200433, China; 2Department of Biology, Huck Institutes of the Life Sciences, Pennsylvania State University, University Park, PA 16802, USA; 3Institutes of Biomedical Sciences, Fudan University, Shanghai 200032, China Summary Author for correspondence: The eukaryotic RAD51 gene family has seven ancient paralogs conserved between plants Hong Ma and animals. Among these, RAD51, DMC1, RAD51C and XRCC3 are important for homolo- Tel: +86 21 55665569 gous recombination and/or DNA repair, whereas single mutants in RAD51B, RAD51D or Email: [email protected] XRCC2 show normal meiosis, and the lineages they represent diverged from each other Received: 12 May 2013 evolutionarily later than the other four paralogs, suggesting possible functional redundancy. Accepted: 14 August 2013 The function of Arabidopsis RAD51B, RAD51D and XRCC2 genes in mitotic DNA repair and meiosis was analyzed using molecular genetic, cytological and transcriptomic approaches. New Phytologist (2014) 201: 292–304 The relevant double and triple mutants displayed normal vegetative and reproductive doi: 10.1111/nph.12498 growth. However, the triple mutant showed greater sensitivity than single or double mutants to DNA damage by bleomycin. RNA-Seq transcriptome analysis supported the idea that the Key words: Arabidopsis, DNA repair, gene triple mutant showed DNA damage similar to that caused by bleomycin. On bleomycin treat- regulation, homologous recombination, ment, many genes were altered in the wild-type but not in the triple mutant, suggesting that molecular evolution, RAD51 paralogs. the RAD51 paralogs have roles in the regulation of gene transcription, providing an explana- tion for the hypersensitive phenotype of the triple mutant to bleomycin. Our results provide strong evidence that Arabidopsis XRCC2, RAD51B and RAD51D have complex functions in somatic DNA repair and gene regulation, arguing for further studies of these ancient genes that have been maintained in both plants and animals during their long evolutionary history. correct genetic information in the repair process (West , Introduction et al. 2004; Bleuyard et al., 2006). In addition to its function in somatic Genome stability is important for cellular homeostasis and an DNA repair, HR is also required for normal meiosis to maintain organism must be able to repair DNA damage. Among a variety the association of homologous chromosomes and contributes to of DNA damage, double-strand DNA breaks (DSBs) are caused the redistribution of genetic diversity among progeny. by ionizing radiation, genotoxic chemicals or errors in DNA repli- The genes involved in HR were first identified in budding cation (Kuzminov, 2001; Tonami et al., 2005). Failure to correctly yeast and mainly belong to the RAD52 epistasis group, including repair DSBs can cause genome instability, mutations, cell cycle RAD50, RAD51, RAD52, RAD54, RDH54/TID1, RAD55, arrest and even cell death (Glazer & Glazunov, 1999; Mills et al., RAD57, RAD59, MRE11 and XRS2 (Paques & Haber, 1999; 2003; Dudasova et al., 2004; Sasaki et al., 2004). DSBs are known Krogh & Symington, 2004). Further identification of their to be repaired by two major pathways: homologous recombination homologs in animals and plants suggests that the HR repair path- (HR) and non-homologous end-joining (NHEJ). The NHEJ way is highly conserved (Krogh & Symington, 2004; Bleuyard pathway involves the rejoining of two broken DNA ends without et al., 2006). Among them, members of the RAD51 family, a template of similar sequence, often resulting in deletions or inser- including DMC1, RAD51 and five RAD51 paralogs (RAD51B, tions. By contrast, HR is a relatively accurate pathway that RAD51C, RAD51D, XRCC2 and XRCC3) have crucial roles in depends on the homologous DNA sequence, thereby retaining the HR or DNA repair in mammals. Mutations in several of these genes lead not only to elevated sensitivity to DNA damaging *These authors contributed equally to this work. agents, but also to embryonic lethality (Tsuzuki et al., 1996; Shu 292 New Phytologist (2014) 201: 292–304 Ó 2013 The Authors www.newphytologist.com New Phytologist Ó 2013 New Phytologist Trust New Phytologist Research 293 et al., 1999; Deans et al., 2000; Pittman & Schimenti, 2000), Characterization of the double and triple mutants suggesting that they are important for DNA repair during the mitotic cell cycle. F1 double heterozygous plants were generated by crosses between Homologs of DMC1 and RAD51 have been studied in many xrcc2, rad51b and rad51d homozygous single mutant plants. The eukaryotes, including fungi, invertebrate animals and plants triple heterozygous F1 plants were generated by crossing the (Bishop et al., 1992; Habu et al., 1996; Klimyuk & Jones, 1997; rad51b xrcc2 double homozygous mutant with rad51d. The F2 Couteau et al., 1999). In Arabidopsis thaliana, they function in or F3 progeny plants were genotyped with each of the gene- DNA repair via HR in somatic or meiotic cells (Couteau et al., specific primers for RAD51B and XRCC2, combining with the 1999; Bleuyard & White, 2004; Li et al., 2004, 2005; Abe et al., T-DNA left board primer (Supporting Information Table S1). 2005; Bleuyard et al., 2005; Osakabe et al., 2005). For simplicity, To genotype rad51d, PCR products were digested with SphI to unless otherwise noted, the RAD51 paralogous genes and produce two fragments of 233 and 86 bp for the wild-type and mutants refer to those of Arabidopsis. Both rad51c and xrcc3 one fragment of 319 bp for the mutant allele. knockout mutants are hypersensitive to DNA damaging agents and sterile with striking meiotic chromosome fragmentation, sug- Light microscopy gesting that RAD51C and XRCC3 are involved in mitotic DNA repair by somatic and meiotic recombination (Bleuyard & Photographs of plants were taken with a Sony digital camera White, 2004; Abe et al., 2005; Bleuyard et al., 2005; Li et al., DSC-707 (Tokyo, Japan). The viability of mature pollen grains 2005). By contrast, the rad51b, rad51d and xrcc2 mutants show was examined after staining with Alexander’s solution (Alexander, normal fertility without detectable meiotic defects, but are sensi- 1969). Mitosis was examined using root tips of 1-wk-old seed- tive to various DNA damaging agents (Bleuyard et al., 2005), lings, as described previously (Li et al., 2004). Male meiosis was and RAD51B and XRCC2 seem to have a role in the suppression examined using chromosome spreading with 4′,6-diamidino- of meiotic recombination (Ines et al., 2013), suggesting that 2-phenylindole (DAPI) staining, as described previously (Ross RAD51B, RAD51D and XRCC2 are involved in somatic and mei- et al., 1996). Both pollen and meiotic cells were photographed otic HR. Furthermore, the Arabidopsis RAD51B, RAD51C and using a Nikon dissecting microscope (Tokyo, Japan) with an RAD51 proteins also interact in yeast two-hybrid systems, similar Optronics digital camera (Goleta, CA, USA). to their mammalian counterparts, suggesting that they have conserved functions (Osakabe et al., 2005). Treatment with DNA damaging agents In mammals, the embryonic lethality of mutations in RAD51 and its paralogs makes it difficult to analyze their function The eight genotypes of Col (wild-type), rad51b, rad51d (ssn1), in vivo. By contrast, none of the Arabidopsis RAD51 paralogs is xrcc2, rad51b rad51d, rad51d xrcc2, rad51b xrcc2 and rad51b required for survival in individual single mutants. It has been rad51d xrcc2 were treated with either of two types of DNA dam- reported that the RAD51B, RAD51D and XRCC2 homologs aging agent: the cross-linking agents cisplatin (Sigma P4394), form the three groups that occupy the last three branches in the methyl methanesulfonate (MMS; Sigma M4016) and mitomy- RAD51 family tree (Lin et al., 2006). Therefore, these genes cin-C (MMC; Sigma M4287); the DSB-inducing agent bleomy- might have overlapping/redundant functions in plant mitotic cell cin (Sigma B5507). Seeds were surface sterilized with 10% cycle or meiotic cells. It is also possible that RAD51B, RAD51D NaClO for 5 min and 75% ethanol for 5 min, and then sown on and XRCC2 are not required for normal meiosis, even when their Murashige and Skoog (MS) plates containing different concen- functions are lost simultaneously. Nevertheless, the Arabidopsis trations of MMC, bleomycin, cisplatin or MMS, as indicated in RAD51B, RAD51D and XRCC2 proteins might form a protein the text. The plates were placed at 4°C for 3 d, and then trans- complex that interacts with RAD51C in the process of DNA ferred to a growth chamber. The resistance or sensitivity was esti- repair by HR. To date, the genetic relationship among RAD51B, mated by the average fresh weight of four plants after growth for RAD51D and XRCC2 in Arabidopsis has not been studied. The 3 wk. study of their genetic interactions should provide clues to the understanding of the function and relationship of these genes The comet assay during their long evolutionary history. Fourteen-day-old plants grown on half-strength MS plates under 1 normal conditions were incubated in 2 glÀ of bleomycin for Materials and Methods l 6 h and then harvested in liquid nitrogen. Comet assay for DNA damage was performed according to a previously described proto- Plant materials and growth conditions col (Menke et al., 2001; Zhu et al., 2006) with minor modifica- The xrcc2 (SALK_029106) and rad51b (SALK_024755) T-DNA tions. Comet slides were prepared and subjected to 1 insertional lines have been characterized previously by Bleuyard electrophoresis on ice for 2 min (2 V cmÀ , 11 mA). Images of et al. (2005). rad51d (also named ssn1) was obtained from Profes- comets were captured under a Zeiss Axio Imager A2 fluorescence sor Xinnian Dong’s laboratory (Durrant et al., 2007).