Identification of the DNA-binding domains of the switch-activating-protein Sap1 from S.pombe by random point mutations screening in E.coli.
Mating type switching in fission yeast, Schizosaccharomyces pombe, is initiated by a site-specific double-strand break (DSB) at the mat1 locus. The DSB is controlled from a distance by cis- and trans-acting elements. The switch-activating protein, Sap1 binds to the SAS1 cis-acting element which controls the frequency of the DSB at the mat1 locus and, consequently the efficiency of mating type switching. We developed a general method for screening randomly mutagenized expression libraries of DNA-binding protein in E.coli. Sap1 gene was mutagenized by PCR under conditions of reduced Taq polymerase fidelity. The mutated DNA was expressed in E.coli and screened for SAS1-recognition. This method was used to isolated 16 point mutations that abolished SAS1 interaction together with 18 mutations that did not affect binding. The position of these point mutations allowed the identification of three protein domains located in the N-terminal part of Sap1 that are essential for DNA-binding. Deletions and biochemical analysis showed that Sap1 is a dimer both in solution and when bound to SAS1 sequence. The dimerization domain was localized C-terminally to the three domains described above and when used in exess it inhibited DNA binding.
Arcangioli
B
B
Oncogenic Viruses Unit, URA 1644 of the CNRS, Department of Biotechnology, Pasteur Institute, Paris, France.
Ghazvini
M
M
Ribes
V
V
D001483:Base Sequence; D001665:Binding Sites; D004271:DNA, Fungal / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D015245:Deoxyribonuclease BamHI; D015252:Deoxyribonucleases, Type II Site-Specific; D004926:Escherichia coli / Q000235:genetics*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016296:Mutagenesis; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D012568:Schizosaccharomyces / Q000737:chemistry* / Q000235:genetics; D029702:Schizosaccharomyces pombe Proteins; D013329:Structure-Activity Relationship
D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; C085981:Sap1 protein, S pombe; D029702:Schizosaccharomyces pombe Proteins; D015245:Deoxyribonuclease BamHI; C034510:CTCGAG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific