A DNA-binding surface of SPO11-1, an Arabidopsis SPO11 orthologue required for normal meiosis

Yoshinori Shingu, Tsutomu Mikawa, Mariko Onuma, Takashi Hirayama, Takehiko Shibata

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

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-binding activities: Glu substitutions of the invariant Gly215 and Arg222 and of the nonconserved Arg223 and Arg226 in a conserved motif (G215E, R222E, R223E, R226E) reduced the DNA-binding ability in vitro, but substitutions of the conserved Arg130 and invariant Tyr103 (a residue in the putative endonuclease-active center) and of Arg residues outside conserved motifs by Glu or Phe (R130E, Y103F, R207E and R254E), did not. Tests for the ability of mutant spo11-1 proteins to complement the silique-defective phenotype of a spo11-1-homozygous mutant in vivo revealed that R222E and G215E induced serious deficiencies, while R130E caused a partial defect in silique formation. Thus, the Gly215, Arg222 and Arg223 residues of SPO11-1 form a DNA-binding surface that is functional in meiosis.

Original languageEnglish
Pages (from-to)2360-2374
Number of pages15
JournalFEBS Journal
Volume277
Issue number10
DOIs
Publication statusPublished - May 2010
Externally publishedYes

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Keywords

  • AtSPO11-1
  • Homology modelling
  • Meiotic recombination
  • Topoisomerase VI
  • Transgenic Arabidopsis

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

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