The essential DNA-binding protein sap1 of Schizosaccharomyces pombe contains two independent oligomerization interfaces that dictate the relative orientation of the DNA-binding domain.
The sap1 gene from Schizosaccharomyces pombe, which is essential for mating-type switching and for growth, encodes a sequence-specific DNA-binding protein with no homology to other known proteins. We have used a reiterative selection procedure to isolate binding sites for sap1, using a bacterially expressed protein and randomized double-strand oligonucleotides. The sap1 homodimer preferentially selects a pentameric motif, TA(A/G)CG, organized as a direct repeat and spaced by 5 nucleotides. Removal of a C-terminal dimerization domain abolishes recognition of the direct repeat and creates a new specificity for a DNA sequence containing the same pentameric motif but organized as an inverted repeat. We present evidence that the orientation of the DNA-binding domain is controlled by two independent oligomerization interfaces. The C-terminal dimerization domain allows a head-to-tail organization of the DNA-binding domains in solution, while an N-terminal domain is involved in a cooperative interaction on the DNA target between pairs of dimers.
Ghazvini
M
M
URA 1644, Centre National de la Recherche Scientifique, Department of Biotechnology, Pasteur Institute, Paris, France.
Ribes
V
V
Arcangioli
B
B
D001483:Base Sequence; D001665:Binding Sites; D016384:Consensus Sequence; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D012091:Repetitive Sequences, Nucleic Acid; D012568:Schizosaccharomyces; D029702:Schizosaccharomyces pombe Proteins; D012641:Selection, Genetic; D013329:Structure-Activity Relationship
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D009838:Oligodeoxyribonucleotides; C085981:Sap1 protein, S pombe; D029702:Schizosaccharomyces pombe Proteins; D004247:DNA