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Substrate recognition and selectivity in the type IC DNA modification methylase M.EcoR124I
1Biophysics Laboratories, School of Biological Sciences, University of Portsmouth, UK.
Nucleic Acids Research
|October 25, 1993
Summary
The DNA methylase M.EcoR124I targets specific adenines for methylation. Methylating one site significantly speeds up methylation at the other, but reduces enzyme binding.
Area of Science:
- Molecular Biology
- Enzymology
- DNA Methylation
Background:
- Type I DNA modification methylase M.EcoR124I exhibits sequence-specific DNA binding.
- The enzyme protects a 25bp DNA fragment containing its recognition sequence from exonuclease III digestion.
Purpose of the Study:
- To investigate the catalytic properties of M.EcoR124I methylase.
- To identify the specific sites of methylation on the DNA substrate.
- To understand the effect of methylation on enzyme-DNA interactions.
Main Methods:
- Utilized modified synthetic oligonucleotide duplexes for experiments.
- Employed competitive gel retardation assays to assess DNA binding affinity.
- Analyzed catalytic properties and methylation site specificity.
Main Results:
- Identified specific adenine bases on each DNA strand as the methylation sites.
- Demonstrated a >100-fold increase in methylation rate when one target adenine is pre-methylated.
- Observed a significant decrease in methylase affinity for DNA with both adenines methylated.
- Showed that methylation of a non-target adenine causes minimal changes in binding and methylation rate.
Conclusions:
- The methylation of M.EcoR124I recognition sites exhibits cooperative kinetics.
- Sequential methylation of target adenines enhances catalytic rate but reduces enzyme binding affinity.
- Methylation of non-target sites has a negligible impact on enzyme activity and binding.