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Preferential cleavage by restriction endonuclease HinfIII.

Acta Biochimica Polonica

|January 1, 1984

Summary

Restriction endonuclease HinfIII shows variable cleavage efficiency across recognition sites. Its activity and methylation patterns depend on the number and sequence of these sites in DNA substrates.

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Area of Science:

Molecular Biology
Enzymology
Genetics

Background:

Restriction enzymes like HinfIII are crucial tools in molecular biology for DNA manipulation.
Understanding the specificity and efficiency of these enzymes is vital for various genetic applications.

Purpose of the Study:

To investigate the variable efficiency of endonucleolytic cleavage by restriction endonuclease HinfIII at different recognition sites.
To determine the influence of the number of recognition sites and S-adenosylmethionine on HinfIII activity.
To analyze the nucleotide sequences associated with high-frequency cleavage and methylation by HinfIII.

Main Methods:

Analysis of specific unit length linear molecules to determine relative cleavage frequencies.
Comparative analysis of DNA substrates with varying numbers of HinfIII recognition sites.
Investigation of HinfIII activity in the presence and absence of S-adenosylmethionine.
Sequencing of high-frequency cleavage and methylation sites.

Main Results:

Cleavage efficiency of HinfIII is highly dependent on the specific recognition site sequence.
HinfIII-mediated cleavage requires a minimum of three recognition sites on the DNA substrate, irrespective of S-adenosylmethionine presence.
HinfIII exhibits preferential methylation at specific sites, even with a single recognition site present.
Nucleotide sequence comparison revealed distinct patterns at frequently cleaved and methylated sites.

Conclusions:

The efficiency and specificity of HinfIII are complex, influenced by both DNA sequence and site multiplicity.
HinfIII's activity is regulated by the number of recognition sites, with a threshold effect observed for cleavage.
Preferential methylation by HinfIII suggests sequence-specific recognition beyond simple cleavage.

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