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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

Robert L. Diaz,1 Alston D. Alcid,1,2 James M. Berger,3 and Scott Keeney1,2*

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.

* Corresponding author. Mailing address: Memorial Sloan-Kettering Cancer Center, 1275 York Ave., Box 97, New York, NY 10021. Phone: (212) 639-5182. Fax: (212) 717-3627. E-mail: s-keeney{at}ski.mskcc.org.

Molecular and Cellular Biology, February 2002, p. 1106-1115, Vol. 22, No. 4
0022-538X/01/$04.00+0     DOI: 10.1128/MCB.22.4.1106-1115.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

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