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Nucleic Acids Research, 1986, Vol. 14, No. 22 9063-9080
© 1986

Articles

Site directed mutagenesis experiments suggest that Glu lll, Glu 144 and Arg 145 are essential for endonucleolytic activity of EcoRI

Heiner Wolfes, Jürgen Alves, Anja Fliess, Robert Geiger and Alfred Pingoud

Abteilung Biophysikalische Chemie, Zentrum Biochemie, Medizinische Hochschule Hannover, Konstanty-Gutschow-Str.8, D-3000 Hannover, FRG

Received September 30, 1986. Revised October 22, 1986. Accepted October 22, 1986.

We have constructed a plasmid (pRIF 309+) carrying the EcoRI restriction endonuclease gene and the fl origin of replication. Upon transformation of this plasmid into E.coli and infection with bacteriophage fl single stranded plasmids are produced which can be used for sequencing and site directed mutagenesis. Using this single stranded DNA and synthetic oligodeoxynucleo-tides 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.


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