Restriction EndonucleasesBy Stephanie, Jennice, Jessica

Definition

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

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

Recognition Site Diagram

Sticky & Blunt Ends

EcoRI produces "sticky" ends,

SmaI restriction enzyme produces "blunt" ends

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

Joining of Sticky Ends

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)

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

Isolating Restriction Enzymes

P.g 280

Examples of Restriction Enzyme Naming

BamHI HindII

B – genus Bacillusam – species amyloliquefaciensH – strainI – first endonuclease isolated from the strain

H – genus Haemophilusin – species influenzad – strain RdII – second endonuclease isolated from the strain

Naming of Restriction Endonucleases

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

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.

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