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DNA recognition by the EcoRV restriction endonuclease probed using base analogues
Damian Parry1, Sarah A Moon, Hsaio-Hui Liu
1School of Cell and Molecular Biosciences, The University of Newcastle, NE2 4HH, Newcastle-upon-Tyne, UK.
Journal of Molecular Biology
|August 21, 2003
Summary
EcoRV restriction endonuclease uses specific DNA base-pair contacts for recognition and catalysis. Modifications to the GATATC sequence reveal how protein-DNA interactions influence binding and hydrolysis, highlighting the role of magnesium ions.
Area of Science:
- Molecular Biology
- Enzymology
- Biochemistry
Background:
- EcoRV restriction endonuclease recognizes the palindromic GATATC sequence.
- DNA recognition and cleavage by restriction enzymes require divalent metal ions, typically Mg(2+).
Purpose of the Study:
- To investigate the role of specific base-pairs in the GATATC sequence for EcoRV DNA recognition.
- To understand how protein-DNA contacts affect both binding affinity and catalytic activity.
Main Methods:
- Utilized base analogues to disrupt hydrogen bonding and hydrophobic interactions within the GATATC recognition site.
- Assessed DNA binding using dissociation constants with Ca(2+).
- Evaluated catalytic activity via single-turnover hydrolysis assays with Mg(2+).
Main Results:
- Loss of individual contacts at A:T base-pairs reduced binding as expected, while G:C base-pair modifications caused more significant effects.
- Modified bases generally decreased hydrolysis rates, and modifications on one strand impacted cleavage on the other.
- Magnesium ion (Mg(2+)) is crucial for mediating interactions between the DNA binding site and the active center, with Mn(2+) sometimes rescuing hydrolysis.
Conclusions:
- Specific contacts within the GATATC sequence are essential for tight EcoRV binding and proper catalytic site assembly.
- These interactions facilitate Mg(2+)-dependent acid/base catalysis and transition state stabilization.
- Communication exists between the DNA binding site and the enzyme's active site.