A DNA binding motif of meiotic recombinase Rec12 (Spo11) defined by essential glycine-202, and persistence of Rec12 protein after completion of recombination

Abstract

The Rec12 (Spo11) protein of the fission yeast Schizosaccharomyces pombe is a meiosis-specific ortholog of the catalytic subunit of type VI topoisomerases and is thought to catalyze doublestrand DNA breaks that initiate recombination. We tested the hypothesis that the rec12-117 allele affects the choice of pathways by which recombination is resolved. DNA sequence analysis revealed a single missense mutation in the coding region (rec12-G202E). The corresponding glycine-202 residue of Rec12 protein is strictly conserved in proteins of the Rec12/Spo11/Top6A family. It maps to the base of the DNA binding pocket in the crystal structure of the archaeal ortholog, Top6A. The rec12-G202E mutants lacked crossover and non-crossover recombination, demonstrating that rec12-G202E does not affect choice of resolution pathway. Like rec12-D15 null mutants, the rec12-G202E mutants suffered chromosome segregation errors in meiosis I. The Rec12-G202E protein was as stable as wild-type Rec12, demonstrating that glycine-202 is essential for a biochemical activity of Rec12 protein, rather than for its stability. These findings suggest that Rec12 facilitates binding of the meiotic recombinase to its substrate, DNA. Interestingly, the bulk of Rec12 protein persisted until the time of anaphase I, and a portion of Rec12 protein persisted until the time of anaphase II, after which it was undetectable. This suggests that Rec12 protein has additional meiotic functions after completion of recombination in prophase, as inferred previously from genetic studies.

The C-terminus of S. pombe DDK subunit Dfp1 is required for meiosis-specific transcription and cohesin cleavage

“…Interestingly, Rec12 protein persists and has been suggested to play a role in MII (DeWall et al, 2005), so there may be some additional effects of proper pairing of homologous chromosomes in the context of recombination that is independent of DDK. These results provide a caveat in the use of haploid pat1 strains to model meiotic progression.…”

DNA Repair Pathways in Trypanosomatids: from DNA Repair to Drug Resistance

“…Our bioinformatic analyses revealed that the Spo11 gene is conserved in trypanosomes and Leishmania major but not in Leishmania infantum. It has been reported (228,229) that fission yeast Spo11 (Rec12) contains a distinct catalytic motif composed of amino acids R94, D95, Y97, and Y98 and a DNA-binding motif which is defined by glycine 202. Interestingly, these residues are conserved in L. major, T. brucei, and T. cruzi (Fig.…”

Spo11 and the Formation of DNA Double-Strand Breaks in Meiosis

“…More recently, mutational analyses of S. cerevisiae Spo11 and the S. pombe ortholog Rec12 have confirmed the biochemical and structural similarity of Spo11 to the TopoVIA subunit (Diaz et al 2002;Sharif et al 2002;DeWall et al 2005;Nag et al 2006). In particular, residues have been identified in Spo11 and Rec12 that are equivalent to the catalytic tyrosine and acidic residues that coordinate a divalent metal ion in archaeal TopoVI.…”