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Linear diffusion of restriction endonucleases on DNA.

H J Ehbrecht, A Pingoud, C Urbanke

The Journal of Biological Chemistry

|May 25, 1985

Summary

Restriction enzymes like EcoRI may use linear diffusion to find DNA sites, especially on longer DNA strands. This process is influenced by buffer conditions and blocked by proteins, suggesting limited in vivo importance.

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Area of Science:

Molecular Biology
Biochemistry
Genetics

Background:

Restriction enzymes are crucial tools in molecular biology for DNA manipulation.
Understanding how restriction enzymes locate specific recognition sites on DNA is fundamental.
Previous studies have explored enzyme-DNA interactions, but the role of substrate length and diffusion is less clear.

Purpose of the Study:

To investigate the impact of DNA substrate chain length on the cleavage rates of restriction enzymes EcoRI, HindIII, and BamHI.
To determine if linear diffusion of enzymes on DNA plays a role in site recognition.
To assess the influence of buffer conditions and DNA-binding proteins on enzyme activity and diffusion.

Main Methods:

Experiments were conducted using plasmid DNA digested to various lengths to serve as substrates.
Cleavage rates of EcoRI, HindIII, and BamHI were measured under different buffer conditions (e.g., MgCl2 concentrations).
The effect of the prokaryotic histone-like protein Hu (NS 2) on cleavage rates was evaluated.

Main Results:

Cleavage rates were dependent on substrate chain length, with longer DNA substrates sometimes cleaved faster than shorter ones.
Linear diffusion of EcoRI on DNA was observed at 1 mM MgCl2, with a mean diffusion length of approximately 1000 base pairs.
At 10 mM MgCl2, linear diffusion of EcoRI was negligible.
The presence of histone-like protein Hu blocked linear diffusion, leading to equal cleavage rates for short and long substrates.

Conclusions:

Linear diffusion of restriction enzymes on DNA can contribute to site localization under specific in vitro conditions.
Enzyme diffusion is influenced by ionic strength (MgCl2 concentration) and can be hindered by other DNA-bound proteins.
While linear diffusion is detectable in vitro, its significance for DNA site localization in vivo is likely limited.

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