A monomeric mutant of restriction endonuclease EcoRI nicks DNA without sequence specificity
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Petra Fritsche
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 α-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.
References
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Articles in the same Issue
- Wolfgang Baumeister – Felix Hoppe-Seyler Lecturer 2004
- Mapping molecular landscapes inside cells
- Paper of the Year 2003: Award to Dieter Hoffmann
- Structural basis of denitrification
- Roles of nectins in cell adhesion, migration and polarization
- Designing novel spectral classes of proteins with a tryptophan-expanded genetic code
- Imprinted small RNA genes
- Inter- and intra-molecular distances determined by EPR spectroscopy and site-directed spin labeling reveal protein-protein and protein-oligonucleotide interaction
- Combined transport of water and ions through membrane channels
- How do ABC transporters drive transport?
- X-ray structure of fumarylacetoacetate hydrolase family member Homo sapiens FLJ36880
- Generation of glycogen- and albumin-producing hepatocyte-like cells from embryonic stem cells
- Imaging morphological details and pathological differences of red blood cells using tapping-mode AFM
- A database search for double-strand containing RNAs in Dictyostelium discoideum
- Segregation of partly melted molecules: isolation of CpG islands by polyacrylamide gel electrophoresis
- A monomeric mutant of restriction endonuclease EcoRI nicks DNA without sequence specificity