L9. restriction endonucleases

Biotechnology Engineering

1. RESTRICTION ENDONUCLEASES 1 Lecture- 9

2. RESTRICTION ENDONUCLEASES Prokaryotic nucleases that recognize specific DNA sequences and cleave sugar-phosphate backbone, either within or close to recognition site. 2

3. 3 These enzymes are produced by bacteria and archaea for defending themselves against the foreign DNA molecules. Restriction enzymes recognize specific sequences and make cuts in the DNA molecules. Stuart Linn and Werner Arber in 1968 showed in vitro restriction of fd phage DNA by an E. coli cell extract. Soon after this discovery, Hamilton Smith and Kent Wilcox in the year 1970 isolated the first restriction enzyme, endonuclease R (later renamed as Hin dII ), from Hemophilus influenzae . In the subsequent year Kathleen Dana and Daniel Nathans pioneered the applications of restriction enzymes by showing specific cleavage of SV40 DNA by Hin dII . Werner Arber, Hamilton Smith and Daniel Nathans were awarded Nobel Prize in Physiology or Medicine in 1978 for the discovery and applications of restriction enzymes.

4. 4 After the isolation of Hin dII in 1970, many other restriction enzymes have been isolated from several bacterial and archaeal sources. Comprehensive information about all the characterized restriction enzymes is available in the database known as REBASE given by R. J. Roberts and his colleagues in 2007 . As per this database, 3805 restriction enzymes have been biochemically or genetically characterized till now.

5. 5 Restriction enzymes have been classified into four major types known as Type I, Type II, Type III and Type IV, depending on their composition, cofactor requirement, target sequence, position of cleavage site, etc. Among these, Type II enzymes have separate enzymes for endonuclease and methylase activities. A Type II enzyme cuts at or near its recognition sequence. Hence, Type II enzymes have been found to be most useful in recombinant DNA technology . Type IV recognize modified, typically methylated DNA As per the REBASE database, 611 Type II restriction enzymes are commercially available.

6. Werner Arber (Jun 3, 1929 - ) Nobel Prize in Physiology or Medicine 1978 showed in vitro restriction of fd phage DNA by E. coli extract 1968 6

7. 1970 Isolated and characterized the first restriction enzyme Endonuclease R ( later renamed HindII ) Hamilton O. Smith (Aug 23, 1931-) Nobel Prize in Physiology or Medicine 1978 7

8. 1971 Daniel Nathans (Oct 30, 1928 – Nov 16, 1999) Pioneered the application of restriction enzymes : Specific cleavage of SV 40 DNA Nobel Prize in Physiology or Medicine 1978 Kathleen Janet Danna 8

9. TYPES OF RESTRICTION ENDONUCLEASES Type I : EcoK , EcoB Type II: EcoRI , EcoRV Type III: EcoPI , EcoP15 Type IV: McrBC , Mrr 9

10. 10 On cutting DNA at specific sequences, some of the Type II enzymes produce blunt ends. The cut sites for Type II enzyme Sma I have been shown below: 5′ C C C ↓ G G G 3′ 3′ G G G↑C C C 5′ Some of the Type II enzymes produce cohesive ends. The cohesive ends may have 5′ overhangs as has been shown below in case of Bam HI enzyme. 5′ G ↓ G A T C C 3′ 3′ C C T A G↑G 5′ The cohesive ends produced by some of the Type II restriction enzymes have 3′ overhangs as given below in case of Pst I enzyme: 5′ C T G C A ↓ G 3′ 3′ G↑A C G T C 5′

11. PROPERTY TYPEI TYPEII TYPEIII Enzyme activities and structure Single multisubunit , having both endonuclease and methylase functions, Large size Separate endonuclease and methylase enzymes, Small size Separate subunits for restriction and methylation Recognition Sequence Assymetric Bipartite Palindromic with dyad symmetry Assymetric Unipartite Recognition site length Long, 8-16bp Short, 4-9bp Short, 5-7bp Methylation site Recognition Site Recognition Site Recognition Site Requirements for Restriction ATP , Mg 2+ , SAM Mg 2+ ATP , Mg 2+ , SAM (optional) Cleavage Site Random 1000bp away from Recognition Site At or Near Recognition Site ≈25bp downstream of Recognition Site Examples EcoK , EcoB EcoRI , EcoRV EcoPI , EcoP15 11

12. TYPES OF RESTRICTION SITES PALINDROMIC RECOGNITION SEQUENCES Sequences having two-fold axis of dyad symmetry. i.e. PHRASES THAT READ THE SAME BACKWARDS AS FORWARD , Madam I’m Adam Continuous : two half-sites of recognition sequence are adjacent. e.g. 5’ G AATTC 3’ Discontinuous : two half-sites are seperated or interrupted. e.g. 5’ GCCNNNN NGGC 3’ 12

13. Uni / Bipartite Recognition Sequence An interrupted, non- palindromic recognition sequence 5’ ATGCCNNNNNNNNNTAGCG 3’ 13

14. CLASSIFICATION ON THE BASIS OF LENGTH OF RECOGNITION SEQUENCE or FRAGMENT SIZE Frequency of occurrence of any recognition sequence in the random sequence of DNA is given by – 1/4 n where, n= length (in bp ) of recognition sequence. for e.g. a 4 base cutter that recognizes a tetranucleotide recognition sequence, would cleave DNA every 4 4 =256bp . 14

15. CLASSIFICATION ON THE BASIS OF TYPES OF ENDS GENERATED BLUNT-END CUTTERS - Endonucleases cut straight across the axis of symmetry of restriction site. e.g. Sma I 5’ 3’ 3’ 5’ 15

16. STICKY-END CUTTERS - Endonucleases cut DNA off the center of restriction site, but between two same bases on opposite strands. 5’ Overhang – e.g. Eco RI 3’ Overhang – e.g. Pst I 16