Open AccessSite directed mutagenesis experiments suggest that Glu lll, Glu 144 and Arg 145 are essential for endonucleolytic activity of EcoRI
Author(s) -
Heiner Wolfes,
Jürgen Alves,
Anja Fließ,
Robert Geiger,
Alfred Pingoud
Publication year - 1986
Publication title -
nucleic acids research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 9.008
H-Index - 537
eISSN - 1362-4954
pISSN - 0305-1048
DOI - 10.1093/nar/14.22.9063
Subject(s) - ecori , biology , plasmid , mutant , restriction enzyme , site directed mutagenesis , mutagenesis , microbiology and biotechnology , genetics , gene , dna , transformation (genetics) , structural gene
We have constructed a plasmid (pRIF 309+) carrying the EcoRI restriction endonuclease gene and the f1 origin of replication. Upon transformation of this plasmid into E. coli and infection with bacteriophage f1 single stranded plasmids are produced which can be used for sequencing and site directed mutagenesis. Using this single stranded DNA and synthetic oligodeoxynucleotides we have introduced point mutations at defined positions of the EcoRI gene. Since in pRIF309+ the EcoRI gene is under the control of the pL-promoter, high level expression of the mutated EcoRI gene could be obtained upon induction. Mutant EcoRI enzymes were purified to homogeneity and characterized in structural and functional terms. Our results demonstrate that the Glu 111----Gln, Glu 144----Gln and Arg 145----Lys -mutants adopt a very similar conformation as the wild type enzyme, but have by two orders of magnitude smaller specific activities than the wild type enzyme, mainly due to a reduction of the Vmax-value.