Identification of Residues in Yeast Spo11p Critical for Meiotic DNA Double-Strand Break Formation

Abstract

Saccharomyces cerevisiae Spo11 protein (Spo11p) is thought to generate the DNA double-strand breaks (DSBs) that initiate homologous recombination during meiosis. Spo11p is related to a subunit of archaebacterial topoisomerase VI and appears to cleave DNA through a topoisomerase-like transesterase mechanism. In this work, we used the crystal structure of a fragment of topoisomerase VI to model the Spo11p structure and to identify amino acid residues in yeast Spo11p potentially involved in DSB catalysis and/or DNA binding. These residues were mutated to determine which are critical for Spo11p function in vivo. Mutation of Glu-233 or Asp-288, which lie in a conserved structural motif called the Toprim domain, abolished meiotic recombination. These Toprim domain residues have been implicated in binding a metal ion cofactor in topoisomerases and bacterial primases, supporting the idea that DNA cleavage by Spo11p is Mg 2؉ dependent. Mutations at an invariant arginine (Arg-131) within a second conserved structural motif known as the 5Y-CAP domain, as well as three other mutations (E235A, F260R, and D290A), caused marked changes in the DSB pattern at a recombination hotspot, suggesting that Spo11p contributes directly to the choice of DNA cleavage site. Finally, certain DSB-defective mutant alleles generated in this study conferred a semidominant negative phenotype but only when Spo11p activity was partially compromised by the presence of an epitope tag. These results are consistent with a multimeric structure for Spo11p in vivo but may also indicate that the amount of Spo11 protein is not a limiting factor for DSB formation in normal cells.Homologous recombination during meiosis in Saccharomyces cerevisiae proceeds via the formation and subsequent repair of DNA double-strand breaks (DSBs) (reviewed in references 24 and 38). Formation of these DSBs requires the products of at least 10 genes, including SPO11. The Spo11 protein (Spo11p) was found covalently linked to the 5Ј-strand termini of DSBs in certain mutants (e.g., rad50S) that are defective for the normal 5Ј-to-3Ј nucleolytic processing of DSB ends (23), and Spo11p shares sequence similarity with a subunit of an archaebacterial topoisomerase (6). Based on these observations, it is thought that Spo11p is the catalytic subunit of the meiotic DNA cleaving activity and that it cuts DNA by a topoisomerase-like transesterification reaction. The role of Spo11p in promoting meiotic recombination initiation is widely conserved, as functional homologs have been characterized in fungi, plants, and animals (3,10,11,16,18,26,30,31,35,43).It is likely that all of the archaebacterial Spo11p homologs function as type II topoisomerases, but topoisomerase activity has been directly demonstrated only for the enzyme from Sulfolobus shibatae (5,8). Because the amino acid sequence of this topoisomerase is unlike the previously known eukaryotic and prokaryotic type II enzymes, it was named topoisomerase VI to distinguish it from these proteins (6). Topoisomerase VI is an A 2 B 2 heterot...

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

“…Alternatively, glycine-202 may be required for a transition bend between the β-sheet and α-helix: Insertion of glutamate might destabilize the bend or alter local structure in such a way as to disrupt interactions with DNA. Site-directed mutagenesis of budding yeast Spo11 also suggests the presence of a DNA binding pocket required for function (Diaz et al, 2002). Substitutions at three residues reduced recombination more than 100-fold and one of these (Spo11-Y292=Rec12-H233) also maps to the face of the DNA pocket of Top6A, less than 3.4Å from the corresponding position of Rec12-G202.…”

“…A comparison of the amino acid sequence of Rec12 (Spo11) to the sequence and structure of Top6A (Nichols et al, 1999) allows one to identify specific residues of Rec12 (Spo11) with likely functions and to ascribe potential functions to specific residues altered by mutation (Diaz et al, 2002;Sharif et al, 2002). Glycine-202 of Rec12 is a nonpolar residue that maps to the base of a DNA binding pocket between a stretch of β-sheet and α-helix (Fig.…”

“…Substitutions at three residues reduced recombination more than 100-fold and one of these (Spo11-Y292=Rec12-H233) also maps to the face of the DNA pocket of Top6A, less than 3.4Å from the corresponding position of Rec12-G202. In addition, mutation of a phenylalanine adjacent to the position of Rec12-G202 (Spo11-F260=Rec12-F203) also reduced recombination as much as to 15-fold (Diaz et al, 2002). Thus in two widely diverged eukaryotes, budding yeast and fission yeast, a conserved region of Rec12 (Spo11) is implicated to interact with DNA in a fashion similar to that of the archaeal ortholog, topoisomerase subunit Top6A (Nichols et al, 1999).…”

Interactions between Mei4, Rec114, and other proteins required for meiotic DNA double-strand break formation in Saccharomyces cerevisiae

“…The cells were then washed and resuspended in the indicated sporulation medium (either 2% potassium acetate or SPM, which is 0.3% potassium acetate and 0.02% raffinose) and incubated at 30°C with vigorous aeration. Return-to-growth assays for recombination between arg4-N and arg4-B heteroalleles were conducted as described (Diaz et al 2002).…”

TopoVIL

“…Lorsque cela a pu être testé, l'absence de Spo11 se caractérise par un défaut de CDB méiotiques accompagnée d'une diminution drastique de la recombinaison homologue ainsi que par des erreurs de ségrégation des chromosomes en méiose I et finalement par une très forte diminution de la fertilité [11]. En dépit de son rôle clé dans la reproduction, la caractérisation biochimique de l'activité de Spo11 n'a pas été réalisée, principalement du fait de son insolubilité, et les données concernant son mode d'action restent parcellaires [12]. À l'instar de ce qui est observé pour les TopoVI, les données obtenues suggèrent que Spo11 est active sous la forme d'un dimère qui catalyse la génèse de CDB via la formation de deux brèches simple brin dans la molécule d'ADN.…”