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Molecular and Cellular Biology, February 2002, p. 1106-1115, Vol. 22, No. 4
0270-7306/01/$04.00+0     DOI: 10.1128/MCB.22.4.1106-1115.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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

Molecular Biology Program, Memorial Sloan-Kettering Cancer Center,¹ Weill Graduate School of Medical Sciences of Cornell University, New York, New York 10021,² Department of Molecular and Cell Biology, University of California, Berkeley, California 94720³

Received 29 June 2001/ Returned for modification 31 July 2001/ Accepted 15 November 2001

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 Mg²⁺ 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.

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