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BMC Mol Biol. 2012; 13: 1.
Published online Jan 16, 2012. doi:  10.1186/1471-2199-13-1
PMCID: PMC3273433
The double-stranded break-forming activity of plant SPO11s and a novel rice SPO11 revealed by a Drosophila bioassay
Yoshinori Shingu,#1,2 Takeshi Tokai,#3 Yasuo Agawa,#4 Kentaro Toyota,3 Selina Ahamed,3 Makiko Kawagishi-Kobayashi,5 Akira Komatsu,5 Tsutomu Mikawa,1,2 Masa-Toshi Yamamoto,4 Kyo Wakasa,corresponding author3 Takehiko Shibata,corresponding author1,2 and Kohji Kusanocorresponding author4
1Cellular & Molecular Biology Laboratory, RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
2Department of Supramolecular Biology, Graduate School of Nanobioscience, Yokohama City University, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan
3Department of Agriculture, Tokyo University of Agriculture, Atsugi, Kanagawa 243-0034, Japan
4Center for Genetic Resource Education & Development, Kyoto Institute of Technology, Saga-Ippongi-cho, Ukyo-ku, Kyoto 616-8354, Japan
5National Institute of Crop Science, 2-1-8 Kannondai, Tsukuba, Ibaraki 305-8518, Japan
corresponding authorCorresponding author.
#Contributed equally.
Yoshinori Shingu: shingu/at/riken.jp; Takeshi Tokai: ta.tokai/at/shoko-sc.co.jp; Yasuo Agawa: agawa/at/kindaisuiken.jp; Kentaro Toyota: Toyota.Kentaro/at/insent.co.jp; Selina Ahamed: selinapapri/at/yahoo.com; Makiko Kawagishi-Kobayashi: makikokk/at/affrc.go.jp; Akira Komatsu: akomatsu/at/affrc.go.jp; Tsutomu Mikawa: mikawa/at/riken.jp; Masa-Toshi Yamamoto: yamamoto/at/kit.jp; Kyo Wakasa: k3wakasa/at/nodai.ac.jp; Takehiko Shibata: tshibata/at/riken.jp; Kohji Kusano: kusano/at/dgrc.kit.ac.jp
Received May 26, 2011; Accepted January 16, 2012.
Abstract
Background
SPO11 is a key protein for promoting meiotic recombination, by generating chromatin locus- and timing-specific DNA double-strand breaks (DSBs). The DSB activity of SPO11 was shown by genetic analyses, but whether SPO11 exerts DSB-forming activity by itself is still an unanswered question. DSB formation by SPO11 has not been detected by biochemical means, probably because of a lack of proper protein-folding, posttranslational modifications, and/or specific SPO11-interacting proteins required for this activity. In addition, plants have multiple SPO11-homologues.
Results
To determine whether SPO11 can cleave DNA by itself, and to identify which plant SPO11 homologue cleaves DNA, we developed a Drosophila bioassay system that detects the DSB signals generated by a plant SPO11 homologue expressed ectopically. We cytologically and genetically demonstrated the DSB activities of Arabidopsis AtSPO11-1 and AtSPO11-2, which are required for meiosis, in the absence of other plant proteins. Using this bioassay, we further found that a novel SPO11-homologue, OsSPO11D, which has no counterpart in Arabidopsis, displays prominent DSB-forming activity. Quantitative analyses of the rice SPO11 transcripts revealed the specific increase in OsSPO11D mRNA in the anthers containing meiotic pollen mother cells.
Conclusions
The Drosophila bioassay system successfully demonstrated that some plant SPO11 orthologues have intrinsic DSB activities. Furthermore, we identified a novel SPO11 homologue, OsSPO11D, with robust DSB activity and a possible meiotic function.
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