Identification of residues in yeast Spo11p critical for meiotic DNA double-strand break formation.
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.
Diaz
Robert L
RL
Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.
Alcid
Alston D
AD
Berger
James M
JM
Keeney
Scott
S
D000483:Alleles; D000595:Amino Acid Sequence; D001665:Binding Sites; D004250:DNA Topoisomerases, Type II / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004271:DNA, Fungal / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004706:Endodeoxyribonucleases; D004950:Esterases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008540:Meiosis; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000502:physiology; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013172:Spores, Fungal / Q000235:genetics / Q000378:metabolism; D000099257:Meiotic Recombination Protein SPO11
D004250:DNA Topoisomerases, Type II; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D004706:Endodeoxyribonucleases; D004950:Esterases; D029701:Saccharomyces cerevisiae Proteins; D000099257:Meiotic Recombination Protein SPO11