Saturation mutagenesis of the DNA site bound by the small carboxy-terminal domain of gamma delta resolvase.

AUTOR(ES)

Rimphanitchayakit, V

RESUMO

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.

Documentos Relacionados

• Functional Analysis of the Carboxy-Terminal Domain of Bacillus anthracis Protective Antigen
• Site-directed mutagenesis of the conserved Asp-443 and Asp-498 carboxy-terminal residues of HIV-1 reverse transcriptase.
• The carboxy-terminal transactivation domain of Oct-4 acquires cell specificity through the POU domain.
• Phosphorylation of the RNA Polymerase II Carboxy-Terminal Domain by the Bur1 Cyclin-Dependent Kinase
• Assembly of Spikes into Coronavirus Particles Is Mediated by the Carboxy-Terminal Domain of the Spike Protein