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.

FIG. 1.

(A) Orthogonal views of the M. jannaschii Top6A crystal structure, with B-form DNA (yellow) modeled into the putative DNA binding channel. The Top6A monomers are colored to indicate the 5Y-CAP (green) and Toprim (red) domains; the bound Mg²⁺ ions are represented by magenta spheres. (B) Stereo view of the M. jannaschii Top6A crystal structure, showing the positions of residues equivalent to those mutated in yeast Spo11p in this study (yellow spheres). One monomer is shaded gray; the other is colored as in panel A. The residues and numbering are according to the yeast Spo11p sequence. (C) Alignment of eukaryotic Spo11p and archaeal Top6A amino acid sequences. Portions of the sequences around the residues mutated in this study are shown (see reference for a more detailed alignment). Conserved residues are highlighted in black; conservative substitutions are highlighted in gray. The positions of the mutated S. cerevisiae residues are indicated by arrows.

FIG. 2.

(A) Effects of selected spo11 mutations on DSB formation. DSBs that were formed at the his4::LEU2 locus during meiosis in a spo11Δ rad50S strain (SKY103) carrying each of the indicated spo11-HA3His6 alleles on an ARS/CEN vector were analyzed by Southern blotting. Results are shown for a representative subset of the mutations in this study. The positions of the parental (unbroken) DNA and of the fragments generated by cleavage at either of the two prominent DSB sites in this region are indicated. The band marked with the asterisk derives from variable cross-hybridization of the probe to the vector carrying the spo11 alleles. (B) Steady-state levels of Spo11-HA3His6p. Whole-cell extracts were prepared 4 h after transfer to sporulation conditions from cultures of SKY10 (spo11Δ) carrying the indicated spo11-HA3His6 alleles on ARS/CEN vectors. Levels of Spo11-HA3His6p were determined by Western blotting with an anti-HA antibody. Stripping and reprobing the same blot with antitubulin antibodies controlled for loading. A representative experiment is shown. The small apparent variations in steady-state levels were not reproducible (data not shown), so they are presumably due to small differences in the synchronization and/or sporulation efficiency of individual cultures.

FIG. 3.

Alteration of the distribution of cleavage events at a recombination hotspot. kbp, kilobase pairs. ARS/CEN vectors carrying the indicated SPO11 alleles (with or without the HA3His6 tag, as indicated) were introduced into SKY103 (spo11Δ rad50S). DNA was prepared from meiotic cultures 6 h after transfer to sporulation medium, and DSBs at the his4::LEU2 hotspot were analyzed by Southern blotting after restriction enzyme digestion and electrophoresis on 0.8% agarose (A) or 5% polyacrylamide (B). The restriction-enzyme-and-probe combination shown in panel B detects DSBs only at site I.

FIG. 4.

Spore viability patterns indicative of meiosis I nondisjunction in strains carrying semidominant configurations of SPO11 mutant alleles. Tetrads were dissected from strains with the indicated epitope-tagged alleles integrated at the SPO11 locus (configuration B in Table 2), and the frequencies of four-spore-viable, three-spore-viable, etc., tetrads were determined.

FIG. 5.

Overexpression partially rescues the recessivity of spo11-HA3His6. Freq., frequency of; hr, hours. High-copy-number 2μm (2μ) vectors pDA7 (spo11-HA3His6) or pRS426 (vector control) were introduced into strain SKY545 [heterozygous for untagged spo11(Y135F) and tagged spo11-HA3His6]. Whole-cell extracts were prepared at the indicated times after transfer to sporulation medium and were analyzed for Spo11-HA3His6p levels by Western blotting with an anti-HA antibody. Stripping and reprobing the same blot with antitubulin antibodies controlled for loading. Cells were also collected at 8 h and plated to determine the frequency of intragenic recombination between his4::LEU2 heteroalleles (mean ± standard deviation of three independent cultures).