Mutational analysis of the function of Met137 and Ile197, two amino acids implicated in sequence-specific DNA recognition by the EcoRI endonuclease.
The gene encoding the EcoRI endonuclease was altered by site-directed mutagenesis to introduce multiple substitutions of M137 and 1197, two amino acids which were suggested by the revised crystal structure to mediate recognition of the cytosines in the 5'-GAATTC-3' target sequence. Eight substitutions of M137 and ten substitutions of 1197 were isolated. With the exception of M137W, M137P and M137K, all mutant enzymes retained enough activity to damage cellular DNA in the absence of the EcoRI methyltransferase. All M137 replacements abolished the ability of the enzyme to restrict phage growth. Conservative replacements at 1197 (L, V) did not impair phage restriction, whereas non-conservative changes reduced (G, W) or abolished (D, P) restriction. In general, substitutions at M137 were more deleterious than substitutions at I197. Double mutants with combinations of M137G/A and I197G/A mutations exhibited a phenotype characteristic for the respective single M137 mutant. Double mutants carrying combinations of the M137G/A replacements and substitutions at R200 were viable even in the absence of the methyltransferase, suggesting that disrupting contacts to both bases of the GC base pair inactivates the enzyme. None of the replacements resulted in relaxed recognition specificity. In summary, our findings are consistent with a role for M137 but do not support such a role for I197 in substrate recognition by the EcoRI endonuclease.
Ivanenko
T
T
Institute of Biochemistry, Biological Research Center of the Hungarian Academy of Sciences, Szeged, Hungary.
Heitman
J
J
Kiss
A
A
D010582:Bacteriophage lambda / Q000235:genetics; D004279:DNA, Viral / Q000378:metabolism; D015246:Deoxyribonuclease EcoRI / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D007532:Isoleucine / Q000235:genetics / Q000378:metabolism*; D008715:Methionine / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D004279:DNA, Viral; D007532:Isoleucine; D008715:Methionine; D015246:Deoxyribonuclease EcoRI