Saturation mutagenesis of the DNA site bound by the small carboxy-terminal domain of gamma delta resolvase.
We have analyzed the sequence requirements for the binding of the carboxy-terminal (DNA binding) domain of gamma delta resolvase to its recognition site. Using an efficient procedure for saturation mutagenesis we have obtained 31 of the possible 36 base substitutions within the 12 bp minimal binding sequence (using a modified right half of resolvase binding site I as the model sequence). Binding assays in vitro with the 43 residue DNA binding domain show that certain substitutions at eight of the 12 positions strongly inhibit complex formation, increasing the dissociation constant by 100-fold or more. The critical positions fall into two groups: the outside 6 bp of the binding sequence (positions 1-6) and positions 9-10. These positions correspond to the regions where the DNA binding domain spans the major and minor grooves, respectively, of its binding site. Base substitutions at the intervening positions (7 and 8) have more modest (less than 20-fold) effects on binding while substitutions at the inner two positions (11 and 12) are virtually neutral. The hierarchies of base preferences within each critical segment suggest that resolvase makes base-specific contacts in both major and minor grooves.
Rimphanitchayakit
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V
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06510.
Grindley
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ND
D001482:Base Composition; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D009713:Nucleotidyltransferases / Q000378:metabolism*; D010957:Plasmids; D015183:Restriction Mapping; D013379:Substrate Specificity; D019895:Transposases
D004247:DNA; D009713:Nucleotidyltransferases; D019895:Transposases