caa a22 4500 378941011 CHVBK 20180305123652.0 cr unu---uuuuu 161128e20041001xx s 000 0 eng 10.1515/BC.2004.127 doi (NATIONALLICENCE)gruyter-10.1515/BC.2004.127 A monomeric mutant of restriction endonuclease EcoRI nicks DNA without sequence specificity [Elektronische Daten] [Petra Fritsche, Jürgen Alves] 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. © Walter de Gruyter Biochemistry nationallicence Molecular biology nationallicence Cellular biology nationallicence heterodimer nationallicence nuclease nationallicence protein engineering nationallicence protein nucleic acid interaction nationallicence restriction enzyme nationallicence sequence specificity nationallicence Fritsche Petra Institut für Biophysikalische Chemie, Medizinische Hochschule Hannover, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany aut Alves Jürgen Institut für Biophysikalische Chemie, Medizinische Hochschule Hannover, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany aut Biological Chemistry Walter de Gruyter 385/10(2004-10-01), 975-985 1431-6730 385:10<975 2004 385 bchm https://doi.org/10.1515/BC.2004.127 text/html Onlinezugriff via DOI 1 research article jats NATIONALLICENCE 856 40 https://doi.org/10.1515/BC.2004.127 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Fritsche Petra Institut für Biophysikalische Chemie, Medizinische Hochschule Hannover, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany aut NATIONALLICENCE 700 1- Alves Jürgen Institut für Biophysikalische Chemie, Medizinische Hochschule Hannover, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany aut NATIONALLICENCE 773 0- Biological Chemistry Walter de Gruyter 385/10(2004-10-01), 975-985 1431-6730 385:10<975 2004 385 bchm CC0 http://creativecommons.org/publicdomain/zero/1.0 nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-gruyter