Relaxed circular SV40 DNA as cleavage intermediate of two restriction endonucleases

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Superhelical SV40 DNA (Form I) is a common substrate for studying DNA-protein interactions.
• Restriction endonucleases are crucial tools for DNA manipulation and analysis.
• Understanding the precise mechanism of DNA cleavage is essential for molecular biology techniques.

Purpose of the Study:

• To elucidate the cleavage mechanism of superhelical SV40 DNA by EcoRI and HpaII restriction endonucleases.
• To determine the order of strand cleavage and identify intermediate DNA forms.
• To propose a kinetic reaction scheme for the enzymatic DNA cleavage process.

Main Methods:

• Agarose gel electrophoresis was employed to analyze DNA fragments.
• Dark field electron microscopy provided direct visualization of DNA structures.
• Kinetic analysis was performed to determine rate constants for enzymatic reactions.

Main Results:

• Both EcoRI and HpaII initially produced single-nicked circular DNA (Form II) from superhelical DNA (Form I).
• Linear SV40 DNA (Form III) was observed only after a lag period, indicating a two-step cleavage process.
• The first-order rate constant for the second strand cleavage by EcoRI was determined.

Conclusions:

• Restriction endonucleases EcoRI and HpaII exhibit a sequential, two-step cleavage mechanism on superhelical DNA.
• The initial step involves nicking one DNA strand, followed by a delayed cleavage of the complementary strand.
• A kinetic model is proposed to describe the cleavage pathway for these enzymes.