Restriction Endonucleases (45 mins) | Lecture 48/90 | ASRB NET (ICAR-NET) Veterinary Microbiology

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Techniques in Microbiology and VaccinologyPrinciple of Molecular Techniques

Restriction endonucleases

Restriction endonucleases are part of the natural defence mechanisms of bacteria against incoming DNA, which may be from viruses or plasmids from a foreign population of cells.

These enzymes were first recognized by their ability to restrict the growth of certain viruses in particular strains of Escherichia coli, and were named accordingly.

The restriction enzymes are associated with modifying enzymes, which methylate the DNA. Methylation protects the DNA from cleavage by endonucleases, and this stops the cell from degrading its own DNA.

Invading DNA that has not been correctly methylated will be degraded unless it can be modified by the cell՚s methylating enzymes quickly enough, which happens only rarely.

DNA, once modified, remains protected even after replication. This is because semiconservative replication of a molecule methylated on both strands results in two daughter molecules that are hemi methylated (i.e.. methylated on one strand), and hemi methylation is sufficient to confer protection against cleavage by an endonuclease. The non-methylated strand can then be modified before replication takes place again.

Three types of restriction/modification system are recognized. These are called Types (or Classes) I, II and III.

All the enzymes recognize particular DNA sequences, but only the Type II endonucleases cut within those recognition sequences.

If all nucleotides occurred with equal frequencies (both in the DNA to be cut and in the enzyme, recognition sites) and at random, a particular four-nucleotide motif would be expected to occur on average once every 4⁴ (i.e.. 256) nucleotides.

So, the average length of fragments generated by enzymes with such sites would be 256 base-pairs.

Similarly, enzymes with a six-nucleotide recognition sequence would generate fragments with an average size of 46(i.e.. 4096) base-pairs.