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Mutational analysis of the function of Met137 and Ile197, two amino acids implicated in sequence-specific DNA
T Ivanenko1, J Heitman, A Kiss
1Institute of Biochemistry, Biological Research Center of the Hungarian Academy of Sciences, Szeged, Hungary.
Abstract:
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.
Insights
Site-directed mutagenesis of EcoRI endonuclease revealed that M137 is crucial for DNA recognition, while I197 plays a lesser role. Substitutions at M137 significantly impaired enzyme function and phage restriction.
Area of Science:
- Molecular Biology
- Enzymology
- Protein Engineering
Background:
- EcoRI endonuclease recognizes the 5'-GAATTC-3' DNA sequence.
- Specific amino acids, M137 and I197, were hypothesized to be involved in target DNA recognition.
Purpose of the Study:
- To investigate the roles of M137 and I197 in EcoRI endonuclease DNA binding and function.
- To engineer EcoRI variants with altered DNA recognition properties.
Main Methods:
- Site-directed mutagenesis was used to introduce substitutions at M137 and I197.
- Mutant enzyme activity was assessed by DNA damage, phage restriction, and viability assays in the absence of EcoRI methyltransferase.
Main Results:
- Most M137 substitutions abolished phage restriction, indicating a critical role in DNA recognition.
- I197 substitutions had varied effects, with conservative changes retaining activity and non-conservative changes impairing it.
- Substitutions at M137 were generally more detrimental than those at I197.
- Double mutants affecting both M137 and I197 showed phenotypes similar to M137 single mutants.
- No mutations resulted in altered DNA recognition specificity.
Conclusions:
- M137 is essential for EcoRI endonuclease substrate recognition.
- I197 appears to play a supporting or less critical role in DNA binding.
- The study provides insights into the structure-function relationship of EcoRI endonuclease.