Restriction Enzymes- Mode of action and its Types

 

Restriction Enzymes

Restriction endonucleases are DNases that operate on specific places or sequences of DNA. Restriction endonucleases and restriction enzymes (RE) recognise particular sequences, or restriction sites, and those sequences are called recognition sequences or restriction sites. The palindromic sequences here are: Restriction enzymes vary in their restriction sites, which might be recognised by distinct or the same restriction enzymes. However, the restriction site will be cleaved at two separate sites. Restriction enzymes that are defined as isoschizomers are known as isoschizomers. When used on their own, a restriction enzyme will cut at different restriction sites; however, the restriction enzymes in a single bacterium are known to work at the same location.

Mode of action

To check for methylation, the restriction enzyme attaches to the recognition site (presence of methyl group on the DNA at a specific nucleotide). It just falls off the DNA without cutting if there is methylation in the recognition sequence. The methylation at the recognition site (RE) only occurs when one strand of the DNA molecule is methylated while the other strand is not. The methyl group is provided by the restriction enzymes (the RE) via the modification site found in S-adenosyl methionine. Type II restriction enzymes, however, employ the assistance of another enzyme called methylase, which converts the unmethylated DNA into its more permanent form, known as DNA methylation. DNA is then cleared by RE. RE cleaves the DNA only if there is no methylation on both the strands of DNA. Since methylation is required for the removal of RE, which is present in bacteria, but doesn't cleave the bacterial DNA, it follows that RE does not digest bacterial DNA but only foreign DNA. These restriction enzymes behave in the exact opposite manner, as the name implies. When they find a methylate in the DNA, they'll cut it.

Star activity

In severe conditions, such as low ionic strength, restriction enzymes can detect and cleave the DNA strand at the recognition site in any of the following sequence combinations: GA TCC, NGA TCC, GPOA TCC, GGNTCC. Activities done by the RE, like stars, are called star activities.

 


Nomenclature of Restriction

Enzymes As a large number of restriction enzymes have been discovered, a uniform nomenclature system is adopted to avoid confusion. Smith and Nattens proposed this terminology for the first time in 1973. Every limiting enzyme would have an individual name that uniquely identifies it. The first three letters are entitled to the biological source of the enzymes, the first to the genus' beginning and the third to the first two letters of its name. The first two letters are in italics. Therefore, Escherichia coli restriction enzymes are named Eco, Haemophilus influenza becomes Hin, Diplococcus pneumoniae Dpn, etc. A letter also comes with a reference to bacterial strain: Eco R for strain R. In the case where there is more than one in the strain in question there is finally a roman number, for the first enzyme E. coli R and Eco RII for the second. Eco RI for the second.

Types

Endonucleases restriction can be divided up in the types I, II and III in three categories. ATP-based restrain activity and modified activity in the same multimeric protéin are associated with Type I and Type III. The unmethylated DNA recognition sequences are recognised by these two types. Enzymes of Type I randomly split DNA, while enzymes of Type III split at a certain location. The Type II restrictions system has different enzymes, the most commonly utilised for genetic manipulation, for endonuclease and methylase activity.

Type I Restriction Enzymes

These restriction enzymes acknowledge the region of recognition but split the DNA from 400 base pairs (bp) to 10 000 bp or to 10 kbp right or left. It is not a dedicated site for cleavage. These enzymes consist of three multi-function peptides. For cleavaging and/or enzyme hydrolysis of DNA, these enzymes require Mg++, ATP and S adenosyl methionine. These enzymes are explored not as practical tools for genetic engineering but for general interest.

 Type II Restriction Enzymes

This type of restriction enzyme recognises the restriction site and cleaves the DNA within the recognition site or sequence. These enzymes need Mg++ as a cofactor to cleave and can produce 5 -PO4 or 3 -OH. Because of their selectivity, enzymes of this class are extremely significant. Type II restriction enzymes are further classified into two categories based on how they cut.

Type II Restriction Enzymes - Blend End Cutters

End cutters with a rounded edge This class of type II restriction enzymes cuts the DNA strands at identical locations on both strands of DNA inside the recognition sequence. The resulting DNA strands are entirely base-matched. These kinds of fragments are known as blunt-ended or flush terminated fragments.

Type II Restriction Enzymes - Cohesive End Cutters

 This class of type II restriction enzymes cuts the DNA stands at different positions on both strands of DNA inside the recognition sequence. At the end, they generate a short single-stranded sequence. This brief single-strand pattern is referred to as a sticky or cohesive end. Depending on the terminal molecule, this cohesive end may include 5 -PO 4 or 3 -OH (5 -PO4 or 3 -OH). These enzymes are further subdivided into 5 -end cutters (if 5 -PO 4 is present) and 3 -end cutters (if 3' -OH is present). Restriction Enzymes of Type III This type of restriction enzyme recognizes the recognition site but cuts the DNA 1 kbp distant from the restriction site. These enzymes consist of two peptides or subunits. A TP, Mg++, and S-adenosyl methionine are required for these enzymes to function.

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