A novel switch-activating site (SAS1) and its cognate binding factor (SAP1) required for efficient mat1 switching in Schizosaccharomyces pombe.
The pattern of parental DNA strand inheritance at the mating type locus (mat1) determines the pattern of mat1 switching in a cell lineage by regulating the formation of the site-specific double-stranded break (DSB) required for mating type interconversion in Schizosaccharomyces pombe. To study the molecular basis of this programmable cell type change, we conducted structural and functional analyses of the DNA sequence flanking the DSB at mat1. We have identified and characterized a DNA-binding activity that interacts with a specific sequence located 140 bp from the DSB site. Deletion analysis of DNA sequences located distal to mat1 cassette revealed the presence of at least two switch-activating sites (SAS1 and SAS2), both of which are required for generating an efficient level of DSBs and consequently, for efficient switching. We found that SAS1 overlaps with the target site of the DNA-binding activity called SAP1 (for switch-activating protein). Point mutations generated in the SAS1 element that adversely affect binding of SAP1 protein in vitro were found to reduce the efficiency of switching in vivo, suggesting the requirement of SAP1 for switching. Pedigree analysis revealed that SAS1 is equally required for initial switching (one switch in four grand-daughters of a cell) and for consecutive switching (where the sister of a recently switched cell switches again), indicating that the two developmentally asymmetric cell divisions required to generate a particular pattern of switching share the same molecular control mechanism.
Arcangioli
B
B
NCI-Frederick Cancer Research and Development Center, ABL Basic Research Program, MD 21701.
Klar
A J
AJ
D001483:Base Sequence; D015139:Blotting, Southern; D002853:Chromatography, Liquid; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005800:Genes, Fungal; D049770:Genes, Mating Type, Fungal; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D012568:Schizosaccharomyces / Q000235:genetics*; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics*
D004271:DNA, Fungal; D004268:DNA-Binding Proteins; C516545:SAS1 protein, S pombe; D029702:Schizosaccharomyces pombe Proteins