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In vivo specificity of EcoRI DNA methyltransferase
D W Smith1, S W Crowder, N O Reich
1Department of Chemistry, University of California, Santa Barbara 93106.
Nucleic Acids Research
|November 25, 1992
Summary
EcoRI adenine DNA methyltransferase shows high specificity for its canonical GAATTC site in vivo. Noncanonical sites are methylated only upon overexpression, suggesting DNA repair mechanisms ensure specificity.
Area of Science:
- Molecular Biology
- Bacterial Genetics
- Enzymology
Background:
- EcoRI restriction/modification systems involve DNA methyltransferases and endonucleases.
- Methyltransferases protect specific DNA sequences from endonuclease cleavage.
- The canonical EcoRI site is GAATTC.
Purpose of the Study:
- To investigate the in vivo methylation specificity of EcoRI adenine DNA methyltransferase.
- To determine if the methyltransferase acts on noncanonical EcoRI sites.
- To elucidate the mechanisms maintaining high specificity in vivo.
Main Methods:
- In vivo methylation assays under normal and overexpression conditions.
- In vitro analysis of methyltransferase activity and sequence discrimination.
- Analysis of noncanonical EcoRI sites (TAATTC, CAATTC, GTATTC, GGATTC, GAGTTC).
Main Results:
- EcoRI methyltransferase efficiently methylates the canonical GAATTC site in vivo.
- Noncanonical sites show minimal to no methylation in vivo under standard conditions.
- Partial methylation of noncanonical sites is observed only during methyltransferase overexpression.
- In vitro studies reveal low sequence discrimination by the methyltransferase.
Conclusions:
- The methyltransferase exhibits high specificity for the canonical EcoRI site in vivo.
- Noncanonical sites are not protected by this methyltransferase under normal physiological conditions.
- High in vivo specificity is likely maintained by DNA repair systems actively removing methylated noncanonical sequences.