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Clustering of null mutations in the Eco RI endonuclease
Author(s) -
Yanofsky Stephen D.,
Love Robert,
McClarin Judith A.,
Rosenberg John M.,
Boyer Herbert W.,
Greene Patricia J.
Publication year - 1987
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/prot.340020403
Subject(s) - microbiology and biotechnology , mutant , endonuclease , biology , mutagenesis , restriction enzyme , mutant protein , gene , dna , mutation , ecori , genetics
Eco RI 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 Eco RI‐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 Eco RI 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.