Identification of residues in yeast Spo11p critical for meiotic DNA double-strand break formation Journal Article
| Authors: | Diaz, R. L.; Alcid, A. D.; Berger, J. M.; Keeney, S. |
| Article Title: | Identification of residues in yeast Spo11p critical for meiotic DNA double-strand break formation |
| 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. |
| Keywords: | controlled study; unclassified drug; mutation; dna-binding proteins; nonhuman; protein conformation; protein domain; protein motif; phenotype; meiosis; homologous recombination; alleles; bacteria (microorganisms); dna strand breakage; double stranded dna; amino acid sequence; molecular sequence data; saccharomyces cerevisiae; sequence alignment; recombination, genetic; epitope; amino acid; models, molecular; protein structure, tertiary; binding sites; saccharomyces cerevisiae proteins; catalysis; protein structure; sequence homology; dna binding; cell level; enzyme subunit; site directed mutagenesis; glutamic acid; aspartic acid; archaea; fungal protein; molecular model; arginine; dna cleavage; dna topoisomerase iv; dna topoisomerases, type ii, eukaryotic; dna topoisomerase; esterases; spores, fungal; dna, fungal; magnesium; metal ion; saccharomyces; dna primase; priority journal; article; protein spo11p |
| Journal Title: | Molecular and Cellular Biology |
| Volume: | 22 |
| Issue: | 4 |
| ISSN: | 0270-7306 |
| Publisher: | American Society for Microbiology |
| Date Published: | 2002-02-01 |
| Start Page: | 1106 |
| End Page: | 1115 |
| Language: | English |
| DOI: | 10.1128/mcb.22.4.1106-1115.2002 |
| PUBMED: | 11809802 |
| PROVIDER: | scopus |
| PMCID: | PMC134631 |
| DOI/URL: | |
| Notes: | Export Date: 14 November 2014 -- Source: Scopus |
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