3. RESTRICTION ENDONUCLEASES
• Also known as enzyme for cutting.
• Present day dna technology is totally dependent on the ability to cut dna
molecules at specific sites with restriction endonucleases.
• Restriction means the identification of incoming dna to the cell and it’s
destruction by cleaving pieces if it is recognised as foreign dna.
• The phenomenon of restriction and modification has been explained by the
behaviour of lambda phage on two ecoli host strain c and k.
4. • Modification is process of protection of the cells own dna by methylation by
certain bases so that host dna is not cleaved.
• For eg if stock preparation of phage lambda is grown on ecoli strain c and is then
titred on c and k strains of ecoli.
• At last it was obsereved that when restricted phages adsorb on the surface of
restrictive host and inject their dna it is degraded soon,restriction endonuclease is
responsible for this degradation.
6. TYPES OF RESTRICTION ENDONUCLEASE
• Type 1- these are complex nucleases example ecok and ecoB.
• They function simulataneously as an endonucleases and a methylase and requires
ATP,mg2+ and s-adenosyl methionine as cofactors.
• The recognition site of class 1 enzyme is 15 b.p. In length which can be
methylated at the adenine position in the recognition site,and cleavage site is
approx 1000 b.p away from the 5’ end of the sequence TCA located in the 15 b.p
recognition site.
7.
8. • Type 2- They are simple enzymes that consist of polypeptide.
• They have separate endonuclease and methylase activities.
• They recognize a specific nucleotide sequence and cut a dna molecule at this
sequence and nowhere else.
• They are very stable and only require mg2+ as cofactors.
• The majority of the known recognition sequence have an axis of rotational
symmetry I.eplaindromic sequences.
9. • Many restriction endonuclease make a simple double stranded cut in the middle
of the recognition sequences,reultimg a blunt or flush end e.g. Pvu2
• Other restriction endonucleases donot cut the two dna strands at exactly the
same position. The cleavage is staggered ususally by 2,4 nucleotides.
• So that resulting dna fragments have short single stranded overhungs at each
end.they are called sticky or cohesive ends. E.g Ecor1.
11. • Type 3-they have two different subunits.one unit is responsible for site
recognition and modification while the other is responsible for nuclease action.
• They require mg2+ and ATP as cofactors.
• The recognition sites are asymmetric.these nuclease cleaves at non palindromic
sequences cleavage takes place by nicking one strand in the immediate vicinity of
the recognition site,two sites in opposite orientation are necessary to break the
dna duplex.
• In contrast to staggered end generated by type 2 nuclease the single strand ends
generated by type 3 always differ and cannot recombined.
