Clustering of null mutations in the EcoRI endonuclease.
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. Sixty-two of 121 null mutants tested by Western blot contained normal levels of endonuclease cross-reacting protein. The complete endonuclease gene was sequenced for 27 null mutants. This group was found to consist of 20 single base-change missense mutations, 6 double mutations, and 1 triple mutation. Ten of the 20 single 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 were 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 (Ala139----Thr, Gly140----Ser, Arg203----Gln) appeared to be dimeric, while protein from subunit interface mutations (Glu144----Lys, Glu152----Lys, Gly210----Arg) migrated as monomers.
Yanofsky
S D
SD
Department of Biochemistry and Biophysics, University of California at San Francisco 94143.
Love
R
R
McClarin
J A
JA
Rosenberg
J M
JM
Boyer
H W
HW
Greene
P J
PJ
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics*; D001665:Binding Sites; D004262:DNA Restriction Enzymes / Q000235:genetics*; D004269:DNA, Bacterial / Q000378:metabolism; D015246:Deoxyribonuclease EcoRI; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics*; D005796:Genes; D005798:Genes, Bacterial; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000494:pharmacology; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000235:genetics
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D006898:Hydroxylamines; D011994:Recombinant Proteins; D019811:Hydroxylamine; D004262:DNA Restriction Enzymes; D015246:Deoxyribonuclease EcoRI