Identification of residues in yeast Spollp critical for meiotic DNA double-strand break formation

Robert L. Diaz, Alston D. Alcid, James M. Berger, Scott Keeney

Research output: Contribution to journalArticle

Abstract

Saccharomyces cerevisiae Spoll protein (Spollp) is thought to generate the DNA double-strand breaks (DSBs) that initiate homologous recombination during meiosis. Spollp 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 Spollp structure and to identify amino acid residues in yeast Spollp potentially involved in DSB catalysis and/or DNA binding. These residues were mutated to determine which are critical for Spollp 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 Spollp 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 Spollp 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 Spollp activity was partially compromised by the presence of an epitope tag. These results are consistent with a multimetic structure for Spollp in vivo but may also indicate that the amount of Spoll protein is not a limiting factor for DSB formation in normal cells.

Original languageEnglish (US)
Pages (from-to)1106-1115
Number of pages10
JournalMolecular and cellular biology
Volume22
Issue number4
DOIs
StatePublished - Feb 5 2002
Externally publishedYes

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

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