1. Restriction Endonucleases By Stephanie, Jennice, Jessica

2. Definition Restriction enzymes (restriction endonucleases) are enzymes that cut double stranded DNA into fragments at specific nucleotide sequences

3. The Recognition Sites Each type of restriction enzyme recognises a characteristic sequence of nucleotides known as its recognition site ex. restriction enzyme EcoRI binds to – 5 ’ -GAATTC- 3 ’ /3 ’ - CTTAAG -5 ’ – Recognition sites are usually palindromic and consist of four to eight nucleotides

4. Recognition Site Diagram

5. Sticky & Blunt Ends EcoRI produces "sticky" ends, SmaI restriction enzyme produces "blunt" ends

6. Sticky & Blunt Ends Sticky ends are more useful because they are easily joined to other sticky end fragments through complementary base pairing Recognition sites → usually four base pair to eight-base-pair sequences → low cut frequency – Important for excising a piece of DNA that includes the whole gene Restriction enzymes that cleave at six- base-pair recognition sites → frequency of cuts used for many applications

7. Joining of Sticky Ends

8. Isolating Restriction Enzymes Restriction enzymes are isolated and purified from bacteria – One role of restriction enzymes in bacterium → crude immune system – Bacteriophage attacks & injects DNA into bacterium (Figure A) – Bacterium detects foreign DNA, the bacteria’s restriction endonuclease searches for recognition sites (foreign DNA will inevitably have recognition sites)

9. Isolating Restriction Enzymes – Restriction endonuclease cleaves the bacteriophage DNA into fragments → bacteriophage DNA fragments can’t be transcribed/translated into anything useful (Figure B) – Bacteria cell’s genome is protected & continues to function

10. Isolating Restriction Enzymes P.g 280

11. Examples of Restriction Enzyme Naming BamHIHindII B – genus Bacillus am – species amyloliquefaciens H – strain I – first endonuclease isolated from the strain H – genus Haemophilus in – species influenza d – strain Rd II – second endonuclease isolated from the strain Naming of Restriction Endonucleases

12. Methylases In prokaryotes, methylases → prevent restriction endonucleases from cutting a bacterium's own DNA by adding a methyl group to its DNA Foreign DNA is not methylated → defenceless against the restriction endonucleases Important tools for molecular biologists that work with prokaryotic organisms → protect a gene fragment from being cleaved in an undesired location

13. DNA Ligase Once DNA is cut by restriction endonucleases,DNA ligases rejoin the fragments Hydrogen bonding between two complementary sticky ends is not strong enough → DNA ligases reform the phosphodiester linkage between the backbones of the double strand – Does this by driving out a water molecule. Joining stands with blunt ends using DNA ligase is very inefficient Molecular biologists use T4 DNA ligase.

14. Video time!!!!! http://www.youtube.com/watch?v=aA5fyWJh5S0&feature=related