Localization of a protein-DNA interface by random mutagenesis

The type I restriction and modification enzymes do not possess obvious DNA-binding motifs within their target recognition domains (TRDs) of 150-180 amino acids. To identify residues involved in DNA recognition, changes were made in the amino-TRD of EcoKI by random mutagenesis. Most of the 101 substitutions affecting 79 residues had no effect on the phenotype. Changes at only seven positions caused the loss of restriction and modification activities. The seven residues identified by mutation are not randomly distributed throughout the primary sequence of the TRD: five are within the interval between residues 80 and 110. Sequence analyses have led to the suggestion that the TRDs of type I restriction enzymes include a tertiary structure similar to the TRD of the HhaI methyltransferase, and to a model for a DNA-protein interface in EcoKI. In this model, the residues within the interval identified by the five mutations are close to the protein-DNA interface. Three additional residues close to the DNA in the model were changed; each substitution impaired both activities. Proteins from twelve mutants were purified: six from mutants with partial or wild-type activity and six from mutants lacking activity. There is a strong correlation between phenotype and DNA-binding affinity, as determined by fluorescence anisotropy.     

The deformation behavior of the matrix near a planar interface in a two-phase medium

The behavior of three pile-ups of screw dislocations situated one above the other at a planar interface is analyzed by the method of continuously distributed dislocations. The behavior of three shear cracks situated one above the other at the planar interface is obtained by treating them as double pile-ups of screw dislocations. The analysis is used to discuss the deformation behavior near a planar interface and the stress required to extend the shear cracks. Max Planck Institut fur Eisenforschung, Dusseldorf. Part of this work was done when K. S. RAGHAVAN was on a Fellowship granted by the Humboldt Foundation.