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Home      Recombinant DNA Technology      1. Restriction endonucleases
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Restriction Endonucleases (RE) are known as scissors of recombinant DNA Technology. These are the enzymes that cut single and double staranded DNA at specific site recognized by these enzymes. It is believed that these enzymes have evolved to enhance the defend mechanism of the bacteria where there one prime function is to degrade foreign DNA. These enzymes recognize specific nucleotide sequence, which differ for each RE.

Bacteria protect its own DNA by methylation. The recognition sequences in DNA differ for each restriction enzyme, so different RE produces different length of fragments of DNA. Restriction endonucleases which recognize the same nucleotide sequence are called as neoschizomers, which cleave at different locates of the sequence. If enzymes recognize & cuts at the same location then it is called as isoschizomers.

Restriction Enzyme on digestion produce two kind of ends in DNA molecules on cutting

Sticky Ends                                                             Blunt Ends

example: as in case of EcoR1                                                      example: as in case of Sma1

                                           

Sometime the ends produced are sticky and sometime the digestion results in blunt ends,this completely depends on the restriction enzyme which is used for digestion.

Type of Restriction Enzymes

Depending on the sequence specificity, enzyme co-factor & their cleavage site relative to the site they recognize. They have been classified into four types. At molecular level there are extraordinary varieties so the classification in future may evolve to more than four types.

Type I: These are restriction-modification enzymes & these restriction enzymes cleave DNA at 1000-5000bp away from the site they recognize. These have recognition sequence of around 15bp. Type I restriction enzymes are not employed in recombinant DNA technology as they do not produce discrete DNA fragment

They require Mg+², ATP and S-adenosyl methionine co factors for function, example Eco K

Type II: These are most explored type of restriction enzymes in the molecular biology laboratories because of their specificity to cut at the recognized site. This group of enzyme have separate enzyme for modification and restriction. They recognize the site of usually 4-8bp nucleotide and cut at very close to the or in the recognized site. They produce well defined small DNA fragmentsin the presence of Mg+² as co factors. Majority of them cleave at unmethylated target but some of them are capable of cleaving at methylated as well unmethylated sites. Cleaving leaves a 3´ OH on one side of each cut & 5´ phosphate on the other side. There are more than 3500 type II restriction enzyme discovered. Hind II in 1970 was the first type II enzyme to be isolated. These enzymes are essential tools for recombinant DNA technology and only class that helps in DNA analysis, gene clonning and restriction mapping formation of chimeric DNA and in many other techniques.

NOTE:

TypeII enzymes which cleave in the recognition site are the most common with small in size, subunit between 200-350 amino acid range

Type IIs enzymes are those type II enzymes which cleave outside the recognition site and have separate distinct domain for DNA binding and DNA cleavage. Their size varies between 400-650 amino acids in length.

Type IIG restriction enzymes are those type II enzymes which cleave outside the recognized site but in these kind of RE the restriction and modification activities reside in the same protein chain. Their size varies between 850-1250 amino acids in length

Type III: These are considered intermediate between type I & type II. These recognize the short two non-palindromic inversely oriented sequences within the same DNA molecule for the cleavage. They require Mg+² & ATP.

Type IV: These have combined feature of modification and restriction in one polypeptide chain. Atleast six restriction enzymes that belong to this type IV restriction-modification system are i) R.Eco571 ii) R.Bce831 iii) R.HaeIV iv)R.Mmel v)R.BspLU11III vi) Bse MII

These recognize the asymmetric DNA sequence. All of them cleave at definite distance from the recognition site except R.HaeIV, which cleave at both the side of recognition site. Some scientists say that type IV enzymes are intermediate between type IIs & type III enzymes. There activity is positively affected by S-adenonsine methionine but ATP plays no role in their activity.