DNA supercoiling during ATP-dependent DNA translocation by the type I restriction enzyme EcoAI

Area of Science:

• Molecular Biology
• Enzymology
• Biochemistry

Background:

• Type I restriction enzymes translocate DNA via ATP hydrolysis, moving the DNA towards themselves from both directions.
• This translocation process generates DNA loops that appear supercoiled under electron microscopy.

Purpose of the Study:

• To investigate the mechanism of DNA translocation by type I restriction enzymes.
• To determine if DNA supercoiling is the trigger for DNA cleavage.

Main Methods:

• Utilized a DNA cleavage-deficient mutant of EcoAI (a type I restriction enzyme) in DNA supercoiling assays.
• Employed eubacterial and eukaryotic DNA topoisomerase I to probe reaction intermediates and assess supercoiling.
• Observed DNA supercoiling and cleavage activity with both mutant and wild-type EcoAI.

Main Results:

• EcoAI mutant introduced positive supercoils into relaxed plasmid DNA, dependent on ATP hydrolysis.
• Wild-type EcoAI exhibited similar supercoiling activity followed by DNA cleavage.
• Eukaryotic DNA topoisomerase I, which relaxes both positive and negative supercoils, prevented supercoil accumulation and relaxed the products.

Conclusions:

• EcoAI translocation generates positive supercoils ahead and negative supercoils behind the enzyme complex.
• The topological changes induced by translocation are not the primary trigger for DNA cleavage by EcoAI.
• Highly supercoiled DNA is cleaved more slowly by wild-type EcoAI than relaxed DNA.