A monomeric mutant of restriction endonuclease EcoRI nicks DNA without sequence specificity

Abstract

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.

Homodimerisation-independent cleavage of dsRNA by a pestiviral nicking endoribonuclease

“…Monomeric restriction endonuclease such as BcnI 65 or dimeric BfiI that possesses only a single active site 66 , cut dsDNA by sequentially nicking the strands individually. Analogously, a monomeric mutant of EcoRI cleaves both strands of DNA independently 67 , a mechanism reminiscent of nicking endonucleases 68 . Thus, it may well be envisaged that E rns independently cleaves both strands of dsRNA – with or without unwinding the dsRNA as discussed above – and the double strand is entirely cleaved into smaller fragments when both nicks in the opposite strands are sufficiently close to each other.…”

Catalytic Activity Control of Restriction Endonuclease—Triplex Forming Oligonucleotide Conjugates

“…Otherwise, a free enzyme left in the reaction mixture upon decaging will become engaged in the nonaddressed cleavage reactions directed solely by the REase specificity. This limitation can be overcome by the controlled oligomerization approach which makes use of the observation that subunit interactions of some orthodox REases can be attenuated by mutations. , For example, the R226A mutation at the dimerization/tetramerization interface of restriction endonuclease Bse634I converts the native tetramer into inactive monomers which are still able to form a functional dimer at increased protein concentrations. Fusion of the two REase monomers to different TFOs should bring inactive monomers into close proximity at the Bse634I recognition site and promote the assembly of the catalytically active dimer.…”