1. Restriction Enzymes Gabriela Perales 1

2. Restriction Enzymes  Restriction enzymes, also called restriction endonucleases, are molecules that cut double stranded DNA at specific sites  Cut by hydrolysis (digestion) of the phosphodiester backbone 2

3. Restriction Enzymes  Were discovered about 40 years ago during investigations of the phenomenon of host-specfic restriction and modification of bacterial viruses.  Bacteria resist infections by new viruses and this restriction of viral growth stems from endonucleases within the cells that destroy foreign DNA molecules.  Restriction enzymes protect bacteria from infections by viruses. They function as microbial immune systems. 3

4. Restriction Enzymes  Some of the first restriction enzymes to be purified were EcoRI and EcoRII from Escherichia coli and Hind II and HindIII from Haemophilus influenzae.  In 1978, the Nobel Prize was awarded jointly to Werner Arber, Daniel Nathans, and Hamilton O. Smith for the discovery of restriction enzymes and their application to molecular genetics.  In 1980’s, restriction enzymes began to be isolated and purified commercially by the New England BioLabs 4

5. Naming Restriction Enzymes  The enzyme name is comprised of the first letter of the genus name and the first two letters of the species name. Escherichia coli = Eco  The strain or identification follows. RY13=EcoR  when a strain has more then one restriction enzyme, they are numbered in the order they were discovered. EcoR 1 5

6. Restriction Enzymes  Restriction enzymes are found only in bacteria and archaea  They range in size from Pvu II(157 amino acids) to Cje I (1250 amino acids).  Restriction enzymes usually occur in combination with other modification enzymes that protect the cell’s own DNA from cleavage by the restriction enzyme.  These modification enzymes recognize the same DNA as the restriction enzyme they accompany, but instead of cleaving the sequence, they methylate one of the bases in each of the DNA strands.  The methyl group extends into the major grove of DNA at the binding site and prevent the enzyme from acting upon it. 6

7. Restriction Enzymes  Restriction enzymes are traditionally classified into three categories, (Type I, II, and III) on the basis of subunit composition, cleavage position… 7

8. Type I Enzymes  Complex multi-subunit that cut DNA at random far from their recognition sequences.  They do not produce discrete restriction fragments or distinct gel banding patterns so they are not a good biotech interest.  found in E. coli and its relatives, are the most complex known. They are comprise of three polypeptides, R (restriction), M (modification), and S (specificity).  The resulting complex is both an endonuclease and a methyltransferase that work together. 8

9. Type II Enzymes  Cut DNA at defined positions that are close or within the recognition sequences.  Produce discrete restriction fragments and distinct gel patterns.  Are composed of a collection of different proteins that greatly differ in their amino acid sequence.  These are that ones that are used in a laboratory for DNA analysis and gene cloning.  Endonucleases and methyltransferases act independently 9

10. Type II Enzymes  Most common type of enzymes cleave within the recognition sequences and are symmetric 5’…AˇAGCTT…3’ 3’…TTCGAˆA…5’  Other types cleave outside recognition sequence and are symmetric and interrupted. 5’…CCA(N) 9 ˇTGG…3’ 3’…GGT(N) 9 ˆACC…5’ 10

11. Type III Enzymes  Composed of large combination of R-M enzymes.  They cleave outside their recognition sequences and need two sequences in opposite orientation in order to cleave.  Not used in laboratories. 11

12. Recognition Sites  Restriction enzymes bind to DNA at a specific sequence  This sequence is usually 4 to 8 bases pairs long.  These sequences form an inverted repeat called a palindrome 5’…GˇAATTC…3’ 3’…CTTAAˆG…5’ 12

13. Sticky Ends vs Blunt Ends  Depending on where and how the enzyme cuts it can leave a blunt end or sticky end. 13

14. Applications  Recombining DNA  Methylation-Specific Restriction Digestion  Restriction Fragment Length Polymorphism Analysis (RFLP)  Fingerprinting  Cloning  In class: cloned copies of bacteria that contained our PCR product in their plasmids; restriction enzymes were most likely used to cleave the plasmid and insert PCR product in the recognition sites of a specific restriction enzyme. 14