1. Topics to cover Biological origin and function of restriction enzymes
Type of restriction enzymes
Isoschizomers
Blunt / sticky ends
Compatible ends
Sensitivity to methylation
Star activity

2. Restriction Enzymes
All cells contain enzymes that can chemically modify DNA. One major class of such enzymes is the restriction endonucleases, or restriction enzymes
Restriction enzymes recognize specific base sequences (recognition sequences) within DNA and cut the DNA
In vivo restriction enzymes protect prokaryotes from aggressive foreign DNA such as virus genomes.
Restriction enzymes are basic tools for in vitro DNA manipulation, and the field of genetic engineering

3. Restriction enzyme and methylase
DNA sequence specific restriction enzymes and their corresponding methylase protect host organism by degrading foreign DNA, which is not methylated
Restriction site typically four, six, eight, ten, or twelve nucleotides long, palindromic sequence

4. The biological background of restriction endonucleases

5. Types of Restriction Enzymes
Restriction endonucleases are divided into three major classes
The type I and III restriction enzymes bind to the DNA at their recognition sequences but cut the DNA a considerable distance away
In contrast, the type II restriction enzymes cleave the DNA within their recognition sequences, making this class of enzymes much more useful for the specific manipulation of DNA

6. Different types of restriction endonucleases

7. Restriction enzymes are homodimeric proteins
Most restriction enzymes are homodimeric proteins; that means that they are composed of two identical polypeptide subunits and each subunit recognizes and cuts the DNA on one of its two strands.
This results in a double-stranded break
EcoRI crystal structure. Dimer bound to DNA.
Homodimer of a 31 kilodalton subunit consisting of one globular domain of the α/β architecture. Each subunit contains a loop which sticks out from the globular domain and wraps around the DNA when bound

8. Mechanism of Restriction Enzymes
6-base-pair (bp) sequence that is recognized and cleaved by the restriction enzyme from Escherichia coli called EcoRI
The cleavage sites are indicated by arrows and the axis of symmetry by a dashed line
Note that the two strands of the recognition sequence have the same sequence if one is read from the left and the other from the right (or if both are read from 5’ to 3’)

9. “Sticky” versus “blunt” ends
Many type II restriction enzymes make staggered cuts, leaving short, single-stranded overhangs known as “sticky ends” at the ends of the two fragments
Sticky ends are generally used in molecular cloning experiments

10. Blunt ends
Other restriction enzymes cut both strands of the DNA directly opposite each other, yielding blunt ends

11. Restriction enzymes as tools
A restriction digest is a procedure used in molecular biology to prepare DNA for analysis or processing
Site specific cleavage of DNA
Type of cleaving product

12. Isoschizomeric restriction endonucleases

13. Recognition and cleavage site of selected type-II restriction enzymes

14. Compatible restriction enzymes
Enzymes that generate DNA fragments with complementary
overhanging ends that can be ligated.

15. Ligation of fragments with non-compatible ends

16. „Star activity“ of restriction enzymes
= relaxed specificity that is exhibited by some restriction enzymes under suboptimal reaction conditions.

17. Interference of restriction and methylation

18. Use of EcoRI methylase in cDNA cloning

19. See also: https://www.neb.com/