A monomeric mutant of restriction endonuclease EcoRI nicks DNA without sequence specificity.
We have mutated the monomer-monomer interface of the restriction endonuclease EcoRI in order to destabilize the homodimer and to stabilize heterodimers. Mutations of Leu158 to charged amino acid residues result in strong destabilization of the dimer. The largest effect was detected for the L158D mutant which is monomeric even at higher concentrations. It unspecifically degrades DNA by cleaving both single strands independently every 15 nucleotides on the average. Although cleavage is reproducible, it is not determined by nucleotide sequence but by general properties like conformation or deformability as has been found for other unspecific nucleases. Mutations of Ile230, which is in direct contact with Leu158 of the other subunit, cause structural changes with the loss of about ten percent alpha-helix content, but interfere only marginally with homodimerization and double strand cleavage. Again the mutation to aspartate shows the strongest effects. Mixtures of single mutants, one containing aspartate at one of the two positions and the other lysine at the corresponding position, form heterodimers. These are mainly stabilized compared to the homodimers by re-establishment of the wild-type hydrophobic interaction at the not mutated residues while an interaction of aspartate and lysine seems energetically unfavorable in this structural context.
Fritsche
Petra
P
Institut für Biophysikalische Chemie, Medizinische Hochschule Hannover, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany.
Alves
Jürgen
J
D004252:DNA Mutational Analysis / Q000379:methods; D004262:DNA Restriction Enzymes / Q000737:chemistry / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics; D015246:Deoxyribonuclease EcoRI / Q000737:chemistry / Q000235:genetics*; D009154:Mutation; D011487:Protein Conformation; D017422:Sequence Analysis, DNA / Q000379:methods*
D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D004262:DNA Restriction Enzymes; D015246:Deoxyribonuclease EcoRI