Restriction Endonucleases Overview

Study Notes

Restriction endonucleases are enzymes that make cuts (cleavage) within DNA molecules.
A restriction endonuclease (restriction enzyme) is a bacterial enzyme that cuts double-stranded DNA (dsDNA) into fragments after recognizing a specific nucleotide sequence, known as a recognition or restriction site.
Restriction enzymes are believed to have evolved in bacteria to defend against viral attack.
They are also known as molecular scissors.

The term "restriction enzyme" originated from studies of bacteriophages.
In 1960, Werner Arber and Matthew Meselson made an early observation about type I restriction enzymes.
In 1970, Hamilton O. Smith, Thomas Kelly, and Kent Wilcox isolated and characterized the first type II restriction enzyme (HindII) from the bacterium Haemophilus influenzae.
For their work on restriction enzymes, Werner Arber, Daniel Nathans, and Hamilton O. Smith received the 1978 Nobel Prize in Physiology or Medicine.

Restriction enzymes are categorized into three major groups: Type I, Type II, and Type III.

Enzymes are categorized based on their composition, co-factor requirements, target sequence nature, and the position of their DNA cleavage site relative to the target sequence.

Blunt ends: Cut DNA at the same position on both strands, leaving no overhanging nucleotides. The blunt ends are less specific.
Sticky ends: Cut DNA at different positions on both strands, creating overhanging, complementary single-stranded ends. These are more specific.
Sticky ends are essential for recombinant DNA experiments because they allow fragments with complementary ends to bond together easily.

Type II enzymes are highly stable and make cuts within or outside their symmetrical recognition sequences.
They function with only one co-factor: magnesium.
The first type II enzyme isolated was HindII in 1970.
Type II restriction endonucleases are commonly used in restriction mapping and gene cloning due to their ability to precisely cleave DNA at specific locations.

The naming convention for restriction enzymes uses the first letter of the bacterial genus, the first letter of the bacterial species, and a strain designation, followed by a roman numeral. An example is EcoRI.

The recognition sequences of Type II restriction enzymes are palindromes.
They are often four to six nucleotides long and rich in guanine-cytosine base pairs.

Type I enzymes exhibit endonuclease and methylase activity, require multiple subunits, and cleave away from the recognition site.
Type II enzymes also display endonuclease and methylase activity but use only Magnesium, have single subunits and cut within their recognition site.
Type III enzymes are intermediate in complexity between type I and type II enzymes They need ATP and AdoMet cofactors for methylation and restriction functions, produce staggered cuts, and cleave 20-30 nucleotides from their recognition site.

Type I enzymes have three subunits: HsdR, HsdM, and HsdS
HsdR is for restriction
HsdM adds methyl groups to host DNA for DNA methylation.
HsdS contributes to cut site specificity and depends on methyltransferase activity

Used in gene cloning and protein expression experiments.
Used to create recombinant DNA to produce human insulin from E. Coli as well as hepatitis B & HPV vaccines.
Restriction fragment length polymorphism (RFLP) analysis to study fragment lengths and identify differences among individuals in biotechnology.
DNA fragments are separated by agarose gel electrophoresis.