Optimizing the nucleotide sequence of a meiotic recombination hotspot in Schizosaccharomyces pombe.
The ade6-M26 mutation of Schizosaccharomyces pombe created a meiotic recombination hotspot. Previous analyses indicated that the heptamer 5'-ATGACGT-3' was necessary and sufficient for hotspot activity; the Atf1-Pcr1 transcription factor binds to this sequence and activates M26. After finding cases in which the M26 heptamer in ade6 was, surprisingly, not active as a hotspot, we used an in vitro selection method (SELEX) that revealed an 18-bp consensus sequence for Atf1-Pcr1 binding, 5'-GNVTATGACGTCATNBNC-3', containing the M26 heptamer at its core. Using this consensus sequence as a guide, we made mutations on each side of the heptamer at two separate sites in ade6. These mutations increased the intracellular hotspot activity of the heptamer, in some cases by >15-fold. These results show that M26, the eukaryotic recombination hotspot with the most precisely defined nucleotide sequence, is larger than previously thought, and they provide valuable information for clarifying the role of M26, and perhaps other hotspots, in meiotic recombination.
Steiner
Walter W
WW
Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109-1024, USA.
Smith
Gerald R
GR
D000483:Alleles; D001483:Base Sequence; D001665:Binding Sites; D015825:Chromosomes, Fungal; D003433:Crosses, Genetic; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D005821:Genetic Techniques; D008540:Meiosis; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic; D012568:Schizosaccharomyces / Q000235:genetics*; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics / Q000502:physiology; D012689:Sequence Homology, Nucleic Acid
D029702:Schizosaccharomyces pombe Proteins
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