Localization of a protein–DNA interface by random mutagenesis

Abstract

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 wildtype activity and six from mutants lacking activity. There is a strong correlation between phenotype and DNA-binding affinity, as determined by fluorescence anisotropy.

Type I Restriction Systems: Sophisticated Molecular Machines (a Legacy of Bertani and Weigle)

“…It seems significant that the amino acid sequences and predicted secondary structures of type I enzymes have led to model structures similar to those deduced experimentally for type II enzymes, in particular, the active site of type I methyltransferases (44) and the regions of the TRDs located close to the DNA target (131,166). Irrespective of family, or even type, the evidence from the protein domains is consistent with the concept that R-M systems have common building blocks.…”

“…The experimental approach of O'Neill et al (131) aimed to localize the protein-DNA interface by random mutagenesis. It was anticipated that amino acids that could be changed without loss of R-M activity were unlikely to be involved in target recognition, while substitutions that resulted in an r Ϫ m Ϫ phenotype would include amino acids involved in a specific interaction with DNA.…”

“…Mutations conferring an r Ϫ m ϩ phenotype have been identified in the hsdS genes of type IA and IC systems. In EcoR124I (189) these mutations are at the junction between a conserved region and a TRD, while in EcoKI they can be close to the junction (197) or at various positions within the TRD (131). A cautious interpretation of r Ϫ phenotypes for EcoKI, however, is required by the recent discovery that mutations in hsdM that result in inadequate modification induce host-mediated alleviation of restriction (111) (see section on modulation of restriction activity).…”

Crystal structure of DNA sequence specificity subunit of a type I restriction-modification enzyme and its functional implications

“…Among these, two loops (between ␤6 and ␤7 and between ␤8 and ␤9) could be aligned to the DNA binding regions that were experimentally confirmed by random point mutagenesis studies in the type IA R-M enzyme EcoKI (29). Transformation of the bound DNA on the TaqI-MTase into the DNA binding clefts of TRDs provides each TRD with a DNA complex model, where the double helical DNA model is bound by using its major groove.…”

Genetic analysis of nonstructural genes (NS1 and NS2) of H9N2 and H5N1 viruses recently isolated in Israel

“…The NS2, or nuclear export protein (NEP), consists of 121 amino acid residues [38]; it mediates the export of newly synthesized RNPs from the nucleus to the cytoplasm [39,40]. The NS2 protein interacts with the viral matrix protein (M1) and with cellular exportin, a family of cellular proteins that mediate nuclear export [39].…”