restriction endonucleases

Recombinant Dna technology could not be constructed without the use of restriction endonucleases/ restriction enzymes.
Such enzymes are able to cleave the double stranded DNA molecules at a specific nucleotide pair sequence called restriction site / restriction enzyme recognition site sequence.

in bacteria it is seen that their natural function is to protect the organis against the invading virus.
the bacteria modifies its own restriction sites by methylation of its restriction sites thereby making it possible for bacteria restriction enzymes to cleave viral dna.

Over 400 restriction enzymes ahve been isolated.they are usually named from the nyme from which they ahve been isolated.
Conventionally three letter name is used followed by a roman numeral.
Additional letters are used sometimes to identify the strain from which they are used.
Bgl II
this is from the bacteria Bacillus globigii.
the genus name is si the first letter and capitalised and the second and third letter are in small letter which is the species name.
the roman letter indicates that it is the second enzyme isolated the above organism.

Restriction enzymes are divided into three types.
1) Type I
mode of action (MOD): These enzymes recognise unmodified recognition sequence in a duplex DNA molecule and then move 1000-5000 bp along the molecule.
Type I restriction enzymes possess three subunits called HsdR, HsdM, and HsdS; HsdR is required for restriction, HsdM is necessary for adding methyl groups to host DNA (methyltransferase activity) and HsdS is important for specificity of cut site recognition in addition to its methyltransferase activity.
 the above require Mg2+ ions, atp and S-adenosyl methionine.

2)Type II
(MOD): Type II restriction endonucleases are components of restriction modification systems that protect bacteria and archaea against invading foreign DNA. Most are homodimeric or tetrameric enzymes that cleave DNA at defined sites of 4-8 bp in length and require Mg2+ ions for catalysis. They differ in the details of the recognition process and the mode of cleavage, indicators that these enzymes are more diverse than originally thought.they are usually used th gene manipulation.
This category also has subgroups:
Type IIB restriction enzymes
 (e.g. BcgI and BplI) are multimers, containing more than one subunit. They cleave DNA on both sides of their recognition to cut out the recognition site. They require both AdoMet and Mg2+ cofactors.
 Type IIE restriction endonucleases
(e.g. NaeI) cleave DNA following interaction with two copies of their recognition sequence. One recognition site acts as the target for cleavage, while the other acts as an allosteric effector that speeds up or improves the efficiency of enzyme cleavage.
Similar to type IIE enzymes, type IIF restriction endonucleases
 (e.g. NgoMIV) interact with two copies of their recognition sequence but cleave both sequences at the same time.
 Type IIG restriction endonucleases
(Eco57I) do have a single subunit, like classical Type II restriction enzymes, but require the cofactor AdoMet to be active.
 Type IIM restriction endonucleases
 such as DpnI, are able to recognize and cut methylated DNA.
 Type IIS restriction endonucleases
 (e.g. FokI) cleave DNA at a defined distance from their non-palindromic asymmetric recognition sites. These enzymes may function as dimers. Similarly,
Type IIT restriction enzymes (e.g., Bpu10I and BslI) are composed of two different subunits. Some recognize palindromic sequences while others have asymmetric recognition sites.


3)Type III
(MOD): These enzymes have sequences that are usually palindromic and have axis of symmetry at the midpoint ,ie; having two point rotational symmetry.

All the above require Mg2+ ions, atp and S-adenosyl methionine.


the basic cuts made by restriction endonucleases is by cleaving dna backbone between the 3' carbon end of dna and the phosphate. this cleaving leaves a 5' phosphate group and a oh group at the 3' end.

more on endonucleases and staggered cuts, types of restrictions endonucleases(RE) , and important applications later on.
If u find any thing wrong or worth mentioning do leave ur comment.

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