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Determinants of EcoRI endonuclease sequence discrimination
M C Needels1, S R Fried, R Love
1Department of Biochemistry and Biophysics, University of California, San Francisco 94143.
Summary
Mutations in EcoRI endonuclease at position 200 reveal insights into DNA sequence recognition. Lysine substitution at Arg-200 shows the highest in vivo activity, though in vitro activity is significantly reduced.
Area of Science:
- Molecular Biology
- Enzymology
- Protein Engineering
Background:
- EcoRI endonuclease recognizes the GAATTC DNA sequence.
- Arginine at position 200 (Arg-200) is crucial for sequence discrimination via hydrogen bonding to guanine.
Purpose of the Study:
- To investigate the role of Arg-200 in EcoRI endonuclease activity and sequence specificity.
- To characterize mutant EcoRI endonucleases with substitutions at position 200.
Main Methods:
- Site-directed mutagenesis of Arg-200 in EcoRI endonuclease.
- Assessment of mutant enzyme activities in vivo and in vitro.
- Analysis of DNA binding specificity.
Main Results:
- Lysine substitution at position 200 yielded the highest in vivo activity among mutants, but with <1% of wild-type in vitro activity.
- Cysteine, Valine, and Serine mutants retained some wild-type site specificity in vivo despite losing hydrogen bonding potential.
- Proline and Tryptophan mutants showed methylase-independent in vivo activity.
Conclusions:
- Arg-200's hydrogen bonding is important but not solely responsible for EcoRI's sequence specificity.
- Mutant analysis provides insights into the complex mechanisms of DNA-protein recognition.