TAILIEUCHUNG - Báo cáo khoa học: A DNA-binding surface of SPO11-1, an Arabidopsis SPO11 orthologue required for normal meiosis

Meiotic recombination is initiated by DNA double-stranded breaks intro-duced by the SPO11 protein. Despite a decade of research, the biochemical functions of SPO11 remain largely unknown, perhaps because of difficulties in studying the functionally active SPO11. | ễFEBS Journal A DNA-binding surface of SPO11-1 an Arabidopsis SPO11 orthologue required for normal meiosis Yoshinori Shingu1 Tsutomu Mikawa2 Mariko Onuma1 Takashi Hirayama3 and Takehiko Shibata1 1 Cellular Molecular Biology Laboratory RIKEN Advanced Science Institute 2-1 Hirosawa Wako-shi Saitama Japan 2 BiometalScience Laboratory RIKEN Spring-8 Center Mikazuki cho Hyogo Japan 3 Molecular Membrane Biology Laboratory RIKEN Advanced Science Institute 2-1 Hirosawa Wako-shi Saitama Japan Keywords AtSPO11-1 homology modelling meiotic recombination topoisomerase VI transgenic Arabidopsis Correspondence T. Shibata Cellular Molecular Biology Laboratory RIKEN Advanced Science Institute 2-1 Hirosawa Wako-shi Saitama 351-0198 Japan Fax 81 48 462 1227 Tel 81 48 467 9528 E-mail tshibata@ Received 28 November 2009 revised 9 March 2010 accepted 15 March 2010 doi Meiotic recombination is initiated by DNA double-stranded breaks introduced by the SPO11 protein. Despite a decade of research the biochemical functions of SPO11 remain largely unknown perhaps because of difficulties in studying the functionally active SPO11. Arabidopsis thaliana encodes three SPO11-related proteins two of which SPO11-1 and SPO11-2 are required for and cooperate in meiosis. We isolated soluble SPO11-1 fused with or free of a trigger factor-tag at its N terminus. The tag-free SPO11-1 needed to interact physically with soluble SPO11-1 to maintain its solubility suggesting a multimeric active form including a solubilizing protein cofactor. An N-terminal fragment of PRD1 a SPO11-1-interacting protein required for normal meiosis but not SPO11-2 forms a soluble complex with trigger factor-tagged SPO11-1 but the trigger factor-tag was required for the solubility. Formation of the complex is not sufficient to express endonuclease activity. Trigger factor-tagged SPO11-1 exhibited DNA-bind-ing activities Glu substitutions of the invariant Gly215 and Arg222 and of

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