Restriction Enzymes

Restriction enzymes cut DNA* at specific sites based on the sequence of bases along the strand at the cut site. These enzymes were first identified and studied in strains of the bacteria E. Coli in the 1950’s and 60’s. The term restriction was used to describe them because their activity restricted the growth of viruses that infect E. coli.

Restriction enzymes are nucleases - enzymes that cut nucleic acid polymers (i.e. DNA and RNA). There are two types of nuclease: endonuclease and exonuclease. Endonucleases make cuts within a DNA polymer. Exonucleases remove individual nucleotides* from the end of a strand. Restriction enzymes are a type of endonuclease - they cut at specific sites in the middle of DNA strands.

The ability of these enzymes to cut DNA at specific sites provide bacteria with a type of immune system that cuts up and, therefore, deactivates foreign DNA such as that introduced by viruses. To be effective, the patterns recognized by each bacteria’s restriction enzymes do not recognize any sequence patterns found in that bacteria’s genome. The specificity of the activity of restriction enzymes has made them useful tools in many molecular biology procedures and techniques.



An important aspect of restriction recognition sites is that they are palindromic. This means the short recognition sequence reads the same way on both strands resulting in the enzyme cutting both strands of a double-stranded DNA molecule.

There are several ways to classify restriction enzymes. The most obvious way is in terms of the base pattern each enzyme recognizes. Many of the recognition sequences used in molecular biology are six bases long, but recognition sequence pattern and lengths vary from enzyme to enzyme. The most widely used enzymes require a perfect match to cut, but others allow for some variation.

Another useful classification system for restriction enzymes is the position of the cut. Many restriction enzymes do not cut in the center of their recognition sequence resulting in overhanging or ‘sticky ends’. Others cut at the mid-point of the recognition sequence leaving no overhang. These are referred to as blunt end cuts.

An example of an enzyme that leaves blunt ends is SmaI. SmaI recognizes the six base sequence: CCCGGG. When cut the end of the two strands are:

SmaI

Contrast this with the enzyme XmaI which recognizes the same six base pattern: CCCGGG, but cuts in a way that leaves overhangs on either side of the cut. The result of this type of cut is two new, matching strand ends:

XmaI

This cutting pattern leaves two matching ends with the four base 3`-CCGG… overhang.

The predictable way in which restriction enzymes cut DNA at specific locations makes them extremely useful for molecular biology. Knowing the recognition site of a particular restriction enzyme and the sequence of a DNA strand makes it possible to predict the number of cuts and the position of the cuts that that enzyme will make on that length of DNA. Cutting a set of strands with a panel of restriction enzymes and running them out on a gel is the basis for the comparative technique known as restriction mapping.

Cutting two sequences with a restriction enzyme and then gluing them back together with a ligase is a common and relatively easy way to make hybrid DNA sequences in the lab.

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