Clustering of null mutations in the EcoRI endonuclease

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Abstract
EcoRI endonuclease mutants were isolated in a methylase-deficient background following in vitro hydroxylamine mutagenesis of plasmid pKG2 (Kuhn et al.: Gene 44:253–263, 1986). Mutants which survived high-level endonuclease expression (IPTG induction) were termed null mutants. Sixtytwo of 121 null mutants tested by Western blot contained normal levels of endonuclease cross-reacting protein. The complete endonuclease gene was scquenced for 27 null mutants. This group was found to consist of 20 signle base-change missense mutations, 6 double mutations, and 1 triple mutation. Ten of the 20 signle mutations were clustered between residues 139 and 144. When examined with respect to the structure of the EcoRI-DNA complex (McClarin et al.: Science 234:1526–1541, 1986), these alterations werre found to fall predominantly into two classes: substitutions at the protein-DNA interface or substitutions at the protein-protein (dimer) interface. Protein from several of the mutants was purified and sized by using HPLC. Wild-type EcoRI endonuclease and protein from three of the DNA interface mutations (A1a139→Thr, Gly140→Ser, Arg203→Gln) appeared to be dimeric, while protein from subunit interface mutations (Glu144→Lys, Glu152→Lys, Gly210→Arg) migrated as monomers.