title: A DNA-binding surface of SPO11-1, an Arabidopsis SPO11 orthologue required for normal meiosis.
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.
authors.affiliation: Cellular & Molecular Biology Laboratory, RIKEN Advanced Science Institute, Wako-shi, Saitama, Japan.
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Shingu
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Mikawa
authors.forename: Mariko
authors.initials: M
authors.lastname: Onuma
authors.forename: Takashi
authors.initials: T
authors.lastname: Hirayama
authors.forename: Takehiko
authors.initials: T
authors.lastname: Shibata
mesh_terms: D000595:Amino Acid Sequence / Q000235:genetics; D019943:Amino Acid Substitution / Q000502:physiology; D000596:Amino Acids / Q000235:genetics; D017360:Arabidopsis / Q000033:anatomy & histology / Q000235:genetics / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D004247:DNA / Q000378:metabolism*; D026942:DNA Topoisomerases / Q000096:biosynthesis / Q000235:genetics; D024202:Electrophoretic Mobility Shift Assay; D029968:Escherichia coli Proteins / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D005816:Genetic Complementation Test; D008540:Meiosis; D008958:Models, Molecular; D010446:Peptide Fragments / Q000096:biosynthesis / Q000235:genetics; D019696:Peptidylprolyl Isomerase / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs / Q000502:physiology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012639:Seeds / Q000235:genetics / Q000254:growth & development; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D000099257:Meiotic Recombination Protein SPO11
chemical_list: D000596:Amino Acids; D029681:Arabidopsis Proteins; D004247:DNA; D026942:DNA Topoisomerases; D029968:Escherichia coli Proteins; D010446:Peptide Fragments; D019696:Peptidylprolyl Isomerase; D011994:Recombinant Proteins; C524069:PRD1 protein, Arabidopsis; C477460:Spo11-1 protein, Arabidopsis; D000099257:Meiotic Recombination Protein SPO11; C403324:trigger factor, E coli; C515046:SPO11-2 protein, Arabidopsis