TY - JOUR
AU1 - Kobayashi,, Wataru
AU2 - Liu,, Enwei
AU3 - Ishii,, Hajime
AU4 - Matsunaga,, Sachihiro
AU5 - Schlögelhofer,, Peter
AU6 - Kurumizaka,, Hitoshi
AB - Abstract In eukaryotes, homologous recombination plays a pivotal role in both genome maintenance and generation of genetic diversity. Eukaryotic RecA homologues, RAD51 and DMC1, are key proteins in homologous recombination that promote pairing between homologous DNA sequences. Arabidopsis thaliana is a prominent model plant for studying eukaryotic homologous recombination. However, A. thaliana RAD51 and DMC1 have not been biochemically characterized. In the present study, we purified A. thaliana RAD51 (AtRAD51) and DMC1 (AtDMC1). Biochemical analyses revealed that both AtRAD51 and AtDMC1 possess ATP hydrolyzing activity, filament formation activity and homologous pairing activity in vitro. We then compared the homologous pairing activities of AtRAD51 and AtDMC1 with those of the Oryza sativa and Homo sapiens RAD51 and DMC1 proteins. Arabidopsis thaliana, DMC1, homologous pairing, homologous recombination, RAD51 Homologous recombination is highly conserved among eukaryotes, and functions in both mitotic cells and meiotic cells. In mitotic cells, homologous recombination is important for repairing DNA double-strand breaks (DSBs), which may be induced by ionizing radiation or DNA replication errors (1–3). In meiotic cells, homologous recombination creates linkages between maternal and paternal chromosomes that ensure accurate homologous chromosome segregation during meiotic cell division I, and also exchange of genetic material (4, 5). Homologous recombination is initiated by DSBs, at which the DNA ends are resected to generate single-stranded DNA (ssDNA) regions. The ssDNA region then invades the homologous double-stranded DNA (dsDNA), and new base pairs are formed between the ssDNA and the complementary strand of dsDNA. This process is called ‘homologous pairing’, and is a central reaction in homologous recombination (1–3, 6, 7). In eukaryotes, two RecA homologues, RAD51 and DMC1, are key proteins in the homologous recombination process (8–13). Biochemical studies revealed that both RAD51 and DMC1 catalyze homologous pairing in an ATP-dependent manner (14–20). RAD51 and DMC1 reportedly have different expression profiles (10–13). RAD51 is expressed in both mitotic cells and meiotic cells, whereas DMC1 is only produced in meiotic cells. In previous studies, RAD51 was shown to be responsible for mitotic recombinational repair of DSBs, and also to play a role in meiotic recombination as an accessory factor to stimulate DMC1-mediated homologous pairing (8–11, 21–23). In contrast, DMC1 is required for inter-homolog recombination, which forms a physical connection between homologous chromosomes as chiasma (12, 13). These facts indicate that RAD51 and DMC1 have both overlapping and distinct roles in homologous recombination. Genetic analyses in budding yeast showed that rad51 mutant cells are viable, but hypersensitive to ionizing radiation (10). Yeast cells carrying defective DMC1 genes exhibit an abnormality in meiotic recombination, concomitant with the failure in synaptonemal complex formation (12). Similarly, yeast cells carrying defective RAD51 gene also exhibited defective meiotic recombination (10). In vertebrates, Rad51-/- mice exhibit embryonic lethality (24, 25). In contrast, Dmc1-/- mice grow normally, but are infertile due to the failure of meiotic chromosome segregation (26, 27). In the plant Arabidopsis thaliana, RAD51 null mutant plants grow normally, but are completely sterile (28). Furthermore, RAD51 mutants are hypersensitive to the DNA damage agent Mitomycin C, due to defective mitotic recombinational repair of DNA (22). These results suggest that RAD51 functions in both mitotic and meiotic cells in A. thaliana. In contrast, a DMC1 null mutant reduced fertility to 1.5%, without substantial defects in plant growth (29). However, the biochemical characteristics of A. thaliana RAD51 (AtRAD51) and DMC1 (AtDMC1) have not been analysed. In the present study, we purified AtRAD51 and AtDMC1 as recombinant proteins. We then performed biochemical analyses, and compared the AtRAD51 and AtDMC1 activities with those of the Oryza sativa and Homo sapiens RAD51 and DMC1 proteins in vitro. Materials and Methods Protein purification H. sapiens RAD51 (HsRAD51), H. sapiens DMC1 (HsDMC1), O. sativa RAD51A1 (OsRAD51A1), O. sativa RAD51A2 (OsRAD51A2), O. sativa DMC1A (OsDMC1A) and O. sativa DMC1A (OsDMC1B) were purified by the methods described previously (30–33). For the AtRAD51 protein production, a codon-optimized DNA fragment encoding AtRAD51 was ligated into the NdeI-BamHI sites of the pET-15b expression vector (Novagen, Darmstadt, Germany). The DNA fragment encoding AtDMC1 was ligated into the NdeI-XhoI sites of the pET-15b expression vector (Novagen). AtRAD51 and AtDMC1 were produced as N-terminally His6-tagged proteins, and were purified by the same methods used for OsRAD51 and OsDMC1, respectively (32, 33). In these methods, the N-terminal His6-tag peptide was removed from the purified AtRAD51 and AtDMC1 proteins. DNA substrates Single-stranded (ss) ϕX174 viral (+) strand DNA and double-stranded (ds) ϕX174 RF II DNA were purchased from New England Biolabs (Ipswich, USA). Supercoiled dsDNA containing tandem repeats of the 5S rDNA sequences (CP943) was used in the D-loop formation assay. The single-stranded oligonucleotide was purchased from Nihon Gene Research Laboratory (Sendai, Japan) with the following sequence: 5S 70-mer, 5´ CCGGT ATATT CAGCA TGGTA TGGTC GTAGG CTCTT GCTTG ATGAA AGTTA AGCTA TTTAA AGGGT CAGGG 3´. The supercoiled dsDNA was prepared by a method that avoided alkaline denaturation (34, 35). ATPase assay AtRAD51, OsRAD51A1, OsRAD51A2 or HsRAD51 (1.5 μM) was incubated at 37°C for 90 min in the presence or absence of ϕX174 circular ssDNA (20 μM in nucleotides), in reaction buffer containing 26 mM HEPES (pH 7.5), 95 mM NaCl, 0.03 mM EDTA, 3% glycerol, 1 mM DTT, 1 mM MgCl2, 0.1 mg/ml BSA, 0.6 mM 2-mercaptoethanol, 5 μM ATP and 5 nCi [γ-32P]ATP (NEG502A, PerkinElmer). For AtDMC1, OsDMC1A, OsDMC1B and HsDMC1 (1.5 μM), the reactions were conducted at 37°C for 90 min, in buffer containing 24 mM HEPES (pH 7.5), 100 mM KCl, 0.05 mM EDTA, 2% glycerol, 1 mM DTT, 1 mM MgCl2, 0.1 mg/ml BSA and 0.4 mM 2-mercaptoethanol. The amounts of [γ-32P]ATP remaining after the reactions were measured by thin layer chromatography as described previously (23). Electron microscopy AtRAD51 or AtDMC1 (1 μM) was incubated with ϕX174 dsDNA (1 μM in nucleotides) at 37°C for 10 min. For AtRAD51, the reaction was conducted in buffer, containing 28 mM HEPES (pH 7.5), 160 mM NaCl, 20 mM KCl, 0.04 mM EDTA, 4% glycerol, 1 mM DTT, 1 mM MgCl2, 1 mM ATP and 0.8 mM 2-mercaptoethanol. For AtDMC1, the reaction was conducted in buffer, containing 28 mM HEPES (pH 7.5), 200 mM KCl, 0.1 mM EDTA, 4% glycerol, 1 mM DTT, 1 mM MgCl2, 1 mM ATP and 0.8 mM 2-mercaptoethanol. The samples were adsorbed on a carbon grid and stained with 2% uranium acetate. The samples were visualized with a JEM-2010 electron microscope (JEOL, Akishima, Japan). D-loop formation assay The D-loop formation assay was performed as described previously (6, 23). The reactions were conducted in the presence or absence of 2 mM CaCl2. For the analyses of RAD51, the indicated amounts of AtRAD51, OsRAD51A1, OsRAD51A2 and HsRAD51 were incubated with the 32P-labelled 5S 70mer ssDNA (1 μM in nucleotides) in reaction buffer, containing 24 mM HEPES (pH 7.5), 55 mM NaCl, 0.02 mM EDTA, 2% glycerol, 1 mM DTT, 1 mM MgCl2, 1 mM ATP, 20 mM creatine phosphate, 75 μg/ml creatine kinase, 0.1 mg/ml BSA and 0.4 mM 2-mercaptoethanol. For the analyses of DMC1, the reactions were conducted in buffer, containing 22 mM HEPES (pH 7.5), 50 mM KCl, 0.025 mM EDTA, 1% glycerol, 1 mM DTT, 1 mM MgCl2, 1 mM ATP, 20 mM creatine phosphate, 75 μg/ml creatine kinase, 0.1 mg/ml BSA and 0.2 mM 2-mercaptoethanol. The reaction mixture was incubated at 37°C for 10 min. Supercoiled dsDNA (30 μM in nucleotides) was then added to the reaction mixture, and the samples were further incubated at 37°C for 10 min. The homologous pairing products, D-loops, were separated by agarose gel electrophoresis, and were detected as described previously (23). Results AtRAD51 and AtDMC1 purification In A. thaliana, single protein species of AtRAD51 and AtDMC1 exist, as in H. sapiens RAD51 (HsRAD51) and DMC1 (HsDMC1) (11, 13, 36). In contrast, in O. sativa, two protein species of RAD51 (OsRAD51A1 and OsRAD51A2) and DMC1 (OsDMC1A and OsDMC1B) have been reported (33, 37–39). AtRAD51 shares 88%, 85% and 69% amino acid identities with OsRAD51A1, OsRAD51A2 and HsRAD51, respectively. AtDMC1 shares 81%, 82% and 60% amino acid identities with OsDMC1A, OsDMC1B and HsDMC1, respectively. (Fig. 1A and B). Therefore, RAD51 and DMC1 are closely conserved among plants. Fig. 1 View largeDownload slide Purification of AtRAD51 and AtDMC1. (A, B) The amino acid sequence alignments of A. thaliana, O. sativa and H. sapiens RAD51 and DMC1 are shown. Non-conserved amino acids sequences are shown in white backgrounds. (C) Purified AtRAD51, OsRAD51A1, OsRAD51A2 and HsRAD51 (lanes 2–5, each 0.75 μg). Lane 1 indicates the molecular markers. (D) Purified AtDMC1, OsDMC1A, OsDMC1B and HsDMC1 (lanes 2–5, each 0.75 μg). Lane 1 indicates the molecular markers. Fig. 1 View largeDownload slide Purification of AtRAD51 and AtDMC1. (A, B) The amino acid sequence alignments of A. thaliana, O. sativa and H. sapiens RAD51 and DMC1 are shown. Non-conserved amino acids sequences are shown in white backgrounds. (C) Purified AtRAD51, OsRAD51A1, OsRAD51A2 and HsRAD51 (lanes 2–5, each 0.75 μg). Lane 1 indicates the molecular markers. (D) Purified AtDMC1, OsDMC1A, OsDMC1B and HsDMC1 (lanes 2–5, each 0.75 μg). Lane 1 indicates the molecular markers. We produced AtRAD51 and AtDMC1 as hexahistidine (His6)-tagged recombinant proteins in Escherichia coli cells. His6-tagged AtRAD51 and AtDMC1 were purified by Ni-NTA agarose chromatography, followed by His6-tag peptide removal by thrombin proteinase treatment. The resulting AtRAD51 and AtDMC1 proteins without the His6-tag peptide were further purified by gel filtration chromatography and Heparin Sepharose chromatography, respectively (Fig. 1C and D, lane 2). For comparison, we also purified HsRAD51, HsDMC1, OsRAD51A1, OsRAD51A2, OsDMC1A and OsDMC1B (Fig. 1C and D, lanes 3–5). ATPase activities of AtRAD51 and AtDMC1 Both RAD51 and DMC1 hydrolyze ATP in the presence of ssDNA (14, 15, 18, 19). Therefore, we tested whether the ATP hydrolyzing activity is conserved in AtRAD51 and AtDMC1 (Fig. 2A). As shown in Fig. 2B, AtRAD51 exhibited ssDNA-dependent ATP hydrolyzing activity, which was quite similar to those of OsRAD51A1 and OsRAD51A2. In contrast, the ATP hydrolyzing activity of AtDMC1 was detected in the ssDNA-dependent manner, but it was quite low, as compared with those of the O. sativa and H. sapiens RAD51 and DMC1 proteins (Fig. 2C). Fig. 2 View largeDownload slide The ATPase activities of AtRAD51 and AtDMC1. (A) Schematic representation of the ATPase assay. (B, C) The reactions were conducted in the presence or absence of ϕx174 circular ssDNA (20 μl in nucleotides) and protein (1.5 μM). White bars represent the reactions in the absence of ssDNA. Black bars represent the reactions in the presence of ssDNA. The average values of three independent experiments are presented with the SD values. Fig. 2 View largeDownload slide The ATPase activities of AtRAD51 and AtDMC1. (A) Schematic representation of the ATPase assay. (B, C) The reactions were conducted in the presence or absence of ϕx174 circular ssDNA (20 μl in nucleotides) and protein (1.5 μM). White bars represent the reactions in the absence of ssDNA. Black bars represent the reactions in the presence of ssDNA. The average values of three independent experiments are presented with the SD values. AtRAD51 and AtDMC1 form helical filaments on DNA During the promotion of homologous pairing, RAD51 and DMC1 form helical filaments on DNA (20, 40, 41). Therefore, we tested the filament formation activities of AtRAD51 and AtDMC1. Purified AtRAD51 or AtDMC1 was mixed with circular dsDNA (ϕX174: replication form II) in the presence of ATP and Mg2+, and the complexes, stained with uranyl acetate, were visualized by electron microscopy. As shown in Fig. 3, both AtRAD51 and AtDMC1 formed helical nucleoprotein filaments on dsDNA. These images are quite similar to those of the RAD51 and DMC1 proteins from O. sativa and H. sapiens (20, 32, 33, 40). Fig. 3 View largeDownload slide Helical filament formation by AtRAD51 and AtDMC1. (A, B) Electron microscopy visualizations of the AtRAD51-dsDNA complex (panel A) and the AtDMC1-dsDNA complex (panel B). AtRAD51 or AtDMC1 was incubated with circular dsDNA (ϕx174 RF II). The bar represents 100 nm. Fig. 3 View largeDownload slide Helical filament formation by AtRAD51 and AtDMC1. (A, B) Electron microscopy visualizations of the AtRAD51-dsDNA complex (panel A) and the AtDMC1-dsDNA complex (panel B). AtRAD51 or AtDMC1 was incubated with circular dsDNA (ϕx174 RF II). The bar represents 100 nm. Homologous pairing activity of AtRAD51 We tested the homologous pairing activity by the D-loop formation assay (Fig. 4A). 32P-labelled ssDNA was incubated with RAD51 or DMC1, and the presynaptic filament containing ssDNA and RAD51 or DMC1 was assembled (Fig. 4A). The homologous pairing reaction was initiated by the addition of superhelical dsDNA. D-loops, in which a 32P-labelled ssDNA pairs with a complementary strand of superhelical dsDNA, were detected as the products of homologous pairing (Fig. 4A). Fig. 4 View largeDownload slide Homologous pairing activities of RAD51. (A) Schematic representation of the D-loop formation assay. (B) Homologous pairing activities of RAD51 in the absence of Ca2+. The protein concentrations were 0.2 μM (lanes 2, 6, 10 and 14), 0.4 μM (lanes 3, 7, 11 and 15), 0.8 μM (lanes 4, 8, 12 and 16) and 1.2 μM (lanes 5, 9, 13 and 17). Lane 1 indicates a negative control experiment without proteins. (C) Graphical representation of the experiments shown in panel (B). The average values of three independent experiments are presented with the SD values. (D) Homologous pairing activities of RAD51 in the presence of 2 mM CaCl2. Protein concentrations as in panel (B). (E) Graphical representation of the experiments shown in panel (D). The average values of three independent experiments are presented with the SD values. Fig. 4 View largeDownload slide Homologous pairing activities of RAD51. (A) Schematic representation of the D-loop formation assay. (B) Homologous pairing activities of RAD51 in the absence of Ca2+. The protein concentrations were 0.2 μM (lanes 2, 6, 10 and 14), 0.4 μM (lanes 3, 7, 11 and 15), 0.8 μM (lanes 4, 8, 12 and 16) and 1.2 μM (lanes 5, 9, 13 and 17). Lane 1 indicates a negative control experiment without proteins. (C) Graphical representation of the experiments shown in panel (B). The average values of three independent experiments are presented with the SD values. (D) Homologous pairing activities of RAD51 in the presence of 2 mM CaCl2. Protein concentrations as in panel (B). (E) Graphical representation of the experiments shown in panel (D). The average values of three independent experiments are presented with the SD values. It has been reported that the homologous pairing reactions by HsRAD51 and HsDMC1 are robustly stimulated in the presence of Ca2+ (42, 43). In contrast, Ca2+ ion does not significantly stimulate the homologous pairing reactions by OsRAD51A1 and OsRAD51A2 (33). We therefore performed D-loop formation assays in the presence or absence of Ca2+. Consistent with the previous reports, the homologous pairing reaction promoted by HsRAD51 was stimulated by Ca2+ (42), but those promoted by OsRAD51A1 and OsRAD51A2 were not affected by Ca2+ (33) (Fig. 4). AtRAD51 substantially promoted homologous pairing in the absence of Ca2+ (Fig. 4B and C). In contrast to HsRAD51, the homologous pairing activity of AtRAD51 was not significantly enhanced by Ca2+ (Fig. 4D and E). Therefore, the characteristics of AtRAD51 are quite similar to those of OsRAD51A1 and OsRAD51A2 (Fig. 4D and E). Homologous pairing activity of AtDMC1 We next tested the homologous pairing activity of AtDMC1. As shown in Fig. 5A and B, the homologous pairing activity of AtDMC1 was very low in the absence of Ca2+. OsDMC1B and HsDMC1 also exhibited low homologous pairing activities, although OsDMC1A robustly promoted the homologous pairing activity in the absence of Ca2+ (Fig. 5A and B). Interestingly, the addition of Ca2+ drastically enhanced the homologous pairing of AtDMC1, as well as those of OsDMC1B and HsDMC1 (Fig. 5C and D). In contrast, the homologous pairing activity of OsDMC1A was somewhat suppressed in the presence of Ca2+ (Fig. 5C and D). These differences may be caused by the Ca2+-induced conformational change of the active DMC1 proteins. Fig. 5 View largeDownload slide Homologous pairing activities of DMC1. (A) Homologous pairing activities of DMC1 in the absence of Ca2+. The protein concentrations were 0.2 μM (lanes 2, 6, 10 and 14), 0.4 μM (lanes 3, 7, 11 and 15), 0.8 μM (lanes 4, 8, 12 and 16) and 1.2 μM (lanes 5, 9, 13 and 17). Lane 1 indicates a negative control experiment without proteins. (B) Graphical representation of the experiments shown in panel (A). The average values of three independent experiments are presented with the SD values. (C) Homologous pairing activities of DMC1 in the presence of 2 mM CaCl2. Protein concentrations as in panel (A). (D) Graphical representation of the experiments shown in panel (C). The average values of three independent experiments are presented with the SD values. Fig. 5 View largeDownload slide Homologous pairing activities of DMC1. (A) Homologous pairing activities of DMC1 in the absence of Ca2+. The protein concentrations were 0.2 μM (lanes 2, 6, 10 and 14), 0.4 μM (lanes 3, 7, 11 and 15), 0.8 μM (lanes 4, 8, 12 and 16) and 1.2 μM (lanes 5, 9, 13 and 17). Lane 1 indicates a negative control experiment without proteins. (B) Graphical representation of the experiments shown in panel (A). The average values of three independent experiments are presented with the SD values. (C) Homologous pairing activities of DMC1 in the presence of 2 mM CaCl2. Protein concentrations as in panel (A). (D) Graphical representation of the experiments shown in panel (C). The average values of three independent experiments are presented with the SD values. Discussion Since plants are immobile, unlike animals, they must tolerate various environmental stresses, such as ultraviolet light, ionizing radiation, chemicals and heavy metal ions (44–46). Furthermore, in plants, the genomic mutations in somatic cells can be transmitted to the next generation (44, 46). Therefore, plants have evolved their own genome-maintenance system to survive under these circumstances. A. thaliana is one of the prominent model plants to study the eukaryotic homologous recombination system (47). In the present study, we found that AtRAD51 and AtDMC1 catalyze homologous pairing (Figs 4 and 5), indicating that AtRAD51 and AtDMC1 are bona fide recombinases in A. thaliana. We found that AtRAD51 possesses robust homologous pairing activity, which is comparable to those of OsRAD51A1 and OsRAD51A2. These plant RAD51 proteins possess higher homologous pairing activities than HsRAD51. Since RAD51 functions in DNA DSB repair in mitotic cells (8–11), the enhanced homologous pairing activities of the plant RAD51 proteins may be required for survival in situations with various environmental stresses (44–46). It has been reported that RAD51 null mutant plants grow normally in situations without environmental stresses in A. thaliana (28). Therefore, RAD51 may function to antagonize acute environmental stress response. DMC1 functions in meiotic homologous recombination, but not in mitotic DNA repair (12, 13). We found that AtDMC1 promotes homologous pairing, but its activity is quite low in the absence of Ca2+ (Fig. 5A and B). Interestingly, the AtDMC1-mediated homologous pairing was drastically stimulated in the presence of Ca2+ (Fig. 5C and D). Previously, Bugreev et al. reported that the HsDMC1-mediated homologous pairing is stimulated as a result of the conformational changes induced by Ca2+ (43). Intriguingly, we found that the homologous pairing reaction by OsDMC1A was not stimulated in the presence of Ca2+, in contrast to AtDMC1, OsDMC1B and HsDMC1 (Fig. 5). These DMC1 proteins may provide important biochemical tools to study the Ca2+-mediated activation mechanism for homologous pairing. In the present study, we performed the biochemical characterizations of AtRAD51 and AtDMC1. These new data provide important information for future studies of homologous recombination in A. thaliana. Funding This work was supported in part by JSPS KAKENHI (grant number JP17H01408) [to H.K.] and by the Kobayashi International Scholarship Foundation [to H.K. and E.L]. Conflict of Interest None declared. References 1 Symington L.S. ( 2002 ) Role of RAD52 epistasis group genes in homologous recombination and double-strand break repair . Microbiol. Mol. Biol. Rev . 66 , 630 – 670 Google Scholar Crossref Search ADS PubMed 2 West S.C. ( 2003 ) Molecular views of recombination proteins and their control . Nat. Rev. Mol. Cell Biol. 4 , 435 – 445 Google Scholar Crossref Search ADS PubMed 3 San Filippo J. , Sung P. , Klein H. ( 2008 ) Mechanism of eukaryotic homologous recombination . Annu. Rev. Biochem. 77 , 229 – 257 Google Scholar Crossref Search ADS PubMed 4 Petronczki M. , Siomos M.F. , Nasmyth K. ( 2003 ) Un ménage à quatre: the molecular biology of chromosome segregation in meiosis . Cell 112 , 423 – 440 Google Scholar Crossref Search ADS PubMed 5 Neale M.J. , Keeney S. ( 2006 ) Clarifying the mechanics of DNA strand exchange in meiotic recombination . Nature 442 , 153 – 158 Google Scholar Crossref Search ADS PubMed 6 Shibata T. , DasGupta C. , Cunningham R.P. , Radding C.M. ( 1979 ) Purified Escherichia coli recA protein catalyzes homologous pairing of superhelical DNA and single-stranded fragments . Proc. Natl. Acad. Sci. U.S.A . 76 , 1638 – 1642 Google Scholar Crossref Search ADS PubMed 7 Renkawitz J. , Lademann C.A. , Jentsch S. ( 2014 ) Mechanisms and principles of homology search during recombination . Nat. Rev. Mol. Cell Biol. 15 , 369 – 383 Google Scholar Crossref Search ADS PubMed 8 Aboussekhra A. , Chanet R. , Adjiri A. , Fabre F. ( 1992 ) Semidominant suppressors of Srs2 helicase mutations of Saccharomyces cerevisiae map in the RAD51 gene, whose sequence predicts a protein with similarities to procaryotic RecA proteins . Mol. Cell. Biol. 12 , 3224 – 3234 Google Scholar Crossref Search ADS PubMed 9 Basile G. , Aker M. , Mortimer R.K. ( 1992 ) Nucleotide sequence and transcriptional regulation of the yeast recombinational repair gene RAD51 . Mol. Cell. Biol. 12 , 3235 – 3246 Google Scholar Crossref Search ADS PubMed 10 Shinohara A. , Ogawa H. , Ogawa T. ( 1992 ) Rad51 protein involved in repair and recombination in S. cerevisiae is a RecA-like protein . Cell 69 , 457 – 470 Google Scholar Crossref Search ADS PubMed 11 Shinohara A. , Ogawa H. , Matsuda Y. , Ushio N. , Ikeo K. , Ogawa T. ( 1993 ) Cloning of human, mouse and fission yeast recombination genes homologous to RAD51 and recA . Nat. Genet. 4 , 239 – 243 Google Scholar Crossref Search ADS PubMed 12 Bishop D.K. , Park D. , Xu L. , Kleckner N. ( 1992 ) DMC1: a meiosis-specific yeast homolog of E. coli recA required for recombination, synaptonemal complex formation, and cell cycle progression . Cell 69 , 439 – 456 Google Scholar Crossref Search ADS PubMed 13 Habu T. , Taki T. , West A. , Nishimune Y. , Morita T. ( 1996 ) The mouse and human homologs of DMC1, the yeast meiosis-specific homologous recombination gene, have a common unique form of exon-skipped transcript in meiosis . Nucleic Acids Res . 24 , 470 – 477 Google Scholar Crossref Search ADS PubMed 14 Sung P. ( 1994 ) Catalysis of ATP-dependent homologous DNA pairing and strand exchange by yeast RAD51 protein . Science 265 , 1241 – 1243 Google Scholar Crossref Search ADS PubMed 15 Baumann P. , Benson F.E. , West S.C. ( 1996 ) Human Rad51 protein promotes ATP-dependent homologous pairing and strand transfer reactions in vitro . Cell 87 , 757 – 766 Google Scholar Crossref Search ADS PubMed 16 Maeshima K. , Morimatsu K. , Horii T. ( 1996 ) Purification and characterization of XRad51.1 protein, Xenopus RAD51 homologue: recombinant XRad51.1 promotes strand exchange reaction . Genes Cells 1 , 1057 – 1068 Google Scholar Crossref Search ADS PubMed 17 Gupta R.C. , Bazemore L.R. , Golub E.I. , Radding C.M. ( 1997 ) Activities of human recombination protein Rad51 . Proc. Natl. Acad. Sci. U.S.A. 94 , 463 – 468 Google Scholar Crossref Search ADS PubMed 18 Li Z. , Golub E.I. , Gupta R. , Radding C.M. ( 1997 ) Recombination activities of HsDmc1 protein, the meiotic human homolog of RecA protein . Proc. Natl. Acad. Sci. U.S.A. 94 , 11221 – 11226 Google Scholar Crossref Search ADS PubMed 19 Hong E.L. , Shinohara A. , Bishop D.K. ( 2001 ) Saccharomyces cerevisiae Dmc1 protein promotes renaturation of single-strand DNA (ssDNA) and assimilation of ssDNA into homologous super-coiled duplex DNA . J. Biol. Chem. 276 , 41906 – 41912 Google Scholar Crossref Search ADS PubMed 20 Sehorn M.G. , Sigurdsson S. , Bussen W. , Unger V.M. , Sung P. ( 2004 ) Human meiotic recombinase Dmc1 promotes ATP-dependent homologous DNA strand exchange . Nature 429 , 433 – 437 Google Scholar Crossref Search ADS PubMed 21 Cloud V. , Chan Y.L. , Grubb J. , Budke B. , Bishop D.K. ( 2012 ) Rad51 is an accessory factor for Dmc1-mediated joint molecule formation during meiosis . Science 6099 , 1222 – 1225 Google Scholar Crossref Search ADS 22 Da Ines O. , Degroote F. , Goubely C. , Amiard S. , Gallego M.E. , White C.I. ( 2013 ) Meiotic recombination in Arabidopsis is catalysed by DMC1, with RAD51 playing a supporting role . PLoS Genet. 9 , e1003787 Google Scholar Crossref Search ADS PubMed 23 Kobayashi W. , Sekine S. , Machida S. , Kurumizaka H. ( 2014 ) Green fluorescent protein fused to the C terminus of RAD51 specifically interferes with secondary DNA binding by the RAD51-ssDNA complex . Genes Genet. Syst. 89 , 169 – 179 Google Scholar Crossref Search ADS PubMed 24 Lim D.S. , Hasty P. ( 1996 ) A mutation in mouse rad51 results in an early embryonic lethal that is suppressed by a mutation in p53 . Mol. Cell. Biol. 16 , 7133 – 7143 Google Scholar Crossref Search ADS PubMed 25 Tsuzuki T. , Fujii Y. , Sakumi K. , Tominaga Y. , Nakao K. , Sekiguchi M. , Matsushiro A. , Yoshimura Y. , Morita T. ( 1996 ) Targeted disruption of the Rad51 gene leads to lethality in embryonic mice . Proc. Natl. Acad. Sci. U.S.A . 93 , 6236 – 6240 Google Scholar Crossref Search ADS PubMed 26 Pittman D.L. , Cobb J. , Schimenti K.J. , Wilson L.A. , Cooper D.M. , Brignull E. , Handel M.A. , Schimenti J.C. ( 1998 ) Meiotic prophase arrest with failure of chromosome synapsis in mice deficient for Dmc1, a germline-specific RecA homolog . Mol. Cell 1 , 697 – 705 Google Scholar Crossref Search ADS PubMed 27 Yoshida K. , Kondoh G. , Matsuda Y. , Habu T. , Nishimune Y. , Morita T. ( 1998 ) The mouse RecA-like gene Dmc1 is required for homologous chromosome synapsis during meiosis . Mol. Cell 1 , 707 – 718 Google Scholar Crossref Search ADS PubMed 28 Li W. , Chen C. , Markmann-Mulisch U. , Timofejeva L. , Schmelzer E. , Ma H. , Reiss B. ( 2004 ) The Arabidopsis AtRAD51 gene is dispensable for vegetative development but required for meiosis . Proc. Natl. Acad. Sci. U.S.A . 101 , 10596 – 10601 Google Scholar Crossref Search ADS PubMed 29 Couteau F. , Belzile F. , Horlow C. , Grandjean O. , Vezon D. , Doutriaux M.P. ( 1999 ) Random chromosome segregation without meiotic arrest in both male and female meiocytes of a dmc1 mutant of Arabidopsis . Plant Cell 11 , 1623 – 1634 Google Scholar Crossref Search ADS PubMed 30 Ishida T. , Takizawa Y. , Sakane I. , Kurumizaka H. ( 2007 ) The Lys313 residue of the human Rad51 protein negatively regulates the strand-exchange activity . Genes Cells 13 , 91 – 103 Google Scholar Crossref Search ADS 31 Hikiba J. , Hirota K. , Kagawa W. , Ikawa S. , Kinebuchi T. , Sakane I. , Takizawa Y. , Yokoyama S. , Mandon-Pépin B. , Nicolas A. , Shibata T. , Ohta K. , Kurumizaka H. ( 2008 ) Structural and functional analyses of the DMC1-M200V polymorphism found in the human population . Nucleic Acids Res . 36 , 4181 – 4190 Google Scholar Crossref Search ADS PubMed 32 Sakane I. , Kamataki C. , Takizawa Y. , Nakashima M. , Toki S. , Ichikawa H. , Ikawa S. , Shibata T. , Kurumizaka H. ( 2008 ) Filament formation and robust strand exchange activities of the rice DMC1A and DMC1B proteins . Nucleic Acids Res . 36 , 4266 – 4276 Google Scholar Crossref Search ADS PubMed 33 Morozumi Y. , Ino R. , Ikawa S. , Mimida N. , Shimizu T. , Toki S. , Ichikawa H. , Shibata T. , Kurumizaka H. ( 2013 ) Homologous pairing activities of two rice RAD51 proteins, RAD51A1 and RAD51A2 . PLoS One 10 , e75451 Google Scholar Crossref Search ADS 34 Cunningham P.R. , DasGupta C. , Shibata T. , Radding M.C. ( 1980 ) Homologous pairing in genetic recombination: recA protein makes joint molecule of gapped circular DNA and closed circular DNA . Cell 20 , 223 – 235 Google Scholar Crossref Search ADS PubMed 35 Kagawa W. , Kurumizaka H. , Ikawa S. , Yokoyama S. , Shibata T. ( 2001 ) Homologous pairing promoted by the human Rad52 protein . J. Biol. Chem . 37 , 35201 – 35208 Google Scholar Crossref Search ADS 36 Doutriaux M.-P. , Couteau F. , Bergounioux C. , White C. ( 1998 ) Isolation and characterisation of the RAD51 and DMC1 homologs from Arabidopsis thaliana . Mol. Gen. Genet. 257 , 283 – 291 Google Scholar Crossref Search ADS PubMed 37 Ding Z. , Wang T. , Chong K. , Bai S. ( 2001 ) Isolation and characterization of OsDMC1, the rice homologue of the yeast DMC1 gene essential for meiosis . Sex. Plant Reprod . 13 , 285 – 288 Google Scholar Crossref Search ADS 38 Shimazu J. , Matsukura C. , Senda M. , Ishikawa R. , Akada S. , Harada T. , Tabata S. , Niizeki M. ( 2001 ) Characterization of a DMC1 homologue, RiLIM15, in meiotic panicles, mitotic cultured cells and mature leaves of rice (Oryza sativa L.) . Theor. Appl. Genet . 102 , 1159 – 1163 Google Scholar Crossref Search ADS 39 Kathiresan A. , Khush G.S. , Bennett J. ( 2002 ) Two rice DMC1 genes are differentially expressed during meiosis and during haploid and diploid mitosis . Sex. Plant Reprod . 14 , 257 – 267 Google Scholar Crossref Search ADS 40 Benson F.E. , Stasiak A. , West S.C. ( 1994 ) Purification and characterization of the human Rad51 protein, an analogue of E. coli RecA . Embo J. 13 , 5764 – 5771 Google Scholar Crossref Search ADS PubMed 41 Ogawa T. , Yu X. , Shinohara A. , Egelman E.H. ( 1993 ) Similarity of the yeast RAD51 filament to the bacterial RecA filament . Science 259 , 1896 – 1899 Google Scholar Crossref Search ADS PubMed 42 Bugreev D.V. , Mazin A.V. ( 2004 ) Ca2+ activates human homologous recombination protein Rad51 by modulating its ATPase activity . Proc. Natl. Acad. Sci. U.S.A . 101 , 9988 – 9993 Google Scholar Crossref Search ADS PubMed 43 Bugreev D.V. , Golub E.I. , Stasiak A.Z. , Stasiak A. , Mazin A.V. ( 2005 ) Activation of human meiosis-specific recombinase Dmc1 by Ca2+ . J. Biol. Chem. 280 , 26886 – 26895 Google Scholar Crossref Search ADS PubMed 44 Schuermann D. , Molinier J. , Fritsch O. , Hohn B. ( 2005 ) The dual nature of homologous recombination in plants . Trends Genet. 21 , 172 – 181 Google Scholar Crossref Search ADS PubMed 45 Lieberman-Lazarovich M. , Levy A.A. ( 2011 ) Homologous recombination in plants: an antireview . Methods Mol. Biol. 701 , 51 – 65 Google Scholar Crossref Search ADS PubMed 46 Roy S. ( 2014 ) Maintenance of genome stability in plants: repairing DNA double strand breaks and chromatin structure stability . Front. Plant Sci . 5 , 487 Google Scholar Crossref Search ADS PubMed 47 Hays J.B. ( 2002 ) Arabidopsis thaliana, a versatile model system for study of eukaryotic genome-maintenance functions . DNA Repair 1 , 579 – 600 Google Scholar Crossref Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - Homologous pairing activities of Arabidopsis thaliana RAD51 and DMC1
JF - The Journal of Biochemistry
DO - 10.1093/jb/mvy105
DA - 2019-03-01
UR - https://www.deepdyve.com/lp/oxford-university-press/homologous-pairing-activities-of-arabidopsis-thaliana-rad51-and-dmc1-ml6n40mB0o
SP - 289
VL - 165
IS - 3
DP - DeepDyve
ER -