Restriction Endonuclease

03/09/2013 17:27

Restriction enzymes are commonly classified into three types, which differ in their structure and whether they cut their DNA substrate at their recognition site, or if the recognition and cleavage sites are separate from one another.

To cut DNA, all restriction enzymes make two incisions, once through each sugar-phosphate backbone (i.e. each strand) of the DNA double helix.

These enzymes are found in bacteria and archaea and provide a defense mechanism against invadingviruses.

 Inside a prokaryote, the restriction enzymes selectively cut up foreign DNA in a process called restriction; while host DNA is protected by a modification enzyme (a methylase) that modifies the prokaryotic DNA and blocks cleavage. Together, these two processes form the restriction modification system.

Over 3000 restriction enzymes have been studied in detail, and more than 600 of these are available commercially.These enzymes are routinely used for DNA modification and manipulation in laboratories, and are a vital tool in molecular cloning.

Restriction-Modification Systems

A large majority of restriction enzymes have been isolated from bacteria, where they appear to serve a host-defense role. 

The idea is that foreign DNA, for example from an infecting virus, will be chopped up and inactivated ("restricted") within the bacterium by the restriction enzyme.

The presence of restriction enzymes immediately begs the question of why they do not chew up the genomic DNA of their host. In almost all cases, a bacterium that makes a particular restriction endonuclease also synthesizes a companion DNA methyltransferase, which methylates the DNA target sequence for that restriction enzyme, thereby protecting it from cleavage. This combination of restriction endonuclease and methylase is referred to as a restriction-modification system.

By convention, restriction enzymes are named after their host of origin. For example, EcoRI was isolated from Escherichia coli(strain RY13), Hind II and Hind III from Haemophilus influenzae, and XhoI from Xanthomonas holcicola.

Recognition site

Restriction enzymes recognize a specific sequence of nucleotides and produce a double-stranded cut in the DNA. The recognition sequences usually vary between 4 and 8 nucleotides, and many of them are palindromic, meaning the base sequence reads the same backwards and forwards.

 In theory, there are two types of palindromic sequences that can be possible in DNA.

The mirror-like palindrome is similar to those found in ordinary text, in which a sequence reads the same forward and backwards on a single strand of DNA strand, as in GTAATG.

The inverted repeat palindrome is also a sequence that reads the same forward and backwards, but the forward and backward sequences are found in complementary DNA strands (i.e., of double-stranded DNA), as in GTATAC (GTATAC being complementary to CATATG). 

Inverted repeat palindromes are more common and have greater biological importance than mirror-like palindromes.

Patterns of DNA Cutting by Restriction Enzymes

Restriction enzymes hydrolyze the backbone of DNA between deoxyribose and phosphate groups. This leaves a phosphate group on the 5' ends and a hydroxyl on the 3' ends of both strands. A few restriction enzymes will cleave single stranded DNA, although usually at low efficiency.

The restriction enzymes generate one of three different types of ends.

 In the diagrams below, the recognition site is boxed in yellow and the cut sites indicated by red triangles.

• 5' overhangs: The enzyme cuts asymmetrically within the recognition site such that a short single-stranded segment extends from the 5' ends. BamHI cuts in this manner.
  
• 3' overhangs: Again, we see asymmetrical cutting within the recognition site, but the result is a single-stranded overhang from the two 3' ends. KpnI cuts in this manner.
  
• Blunts: Enzymes that cut at precisely opposite sites in the two strands of DNA generate blunt ends without overhangs. SmaI is an example of an enzyme that generates blunt ends.
  

The 5' or 3' overhangs generated by enzymes that cut asymmetrically are called sticky ends or cohesive ends, because they will readily stick or anneal with their partner by base pairing.

Recognition sequences in DNA differ for each restriction enzyme, producing differences in the length, sequence and strand orientation (5' end or the 3' end) of a sticky-end "overhang" of an enzyme restriction.

Different restriction enzymes that recognize the same sequence are known as neoschizomers. Eg. MaeII and TaiI. These often cleave in different locales of the sequence. Different enzymes that recognize and cleave in the same location are known as isoschizomers. Eg. SacI and SstI 

Types

Naturally occurring restriction endonucleases are categorized into four groups (Types I, II III, and IV) based on their composition and enzyme cofactor requirements, the nature of their target sequence, and the position of their DNA cleavage site relative to the target sequence.

All types of enzymes recognize specific short DNA sequences and carry out the endonucleolytic cleavage of DNA to give specific fragments with terminal 5'-phosphates. They differ in their recognition sequence, subunit composition, cleavage position, and cofactor requirements, as summarised below:

·         Type I enzymes- cleave at sites remote from recognition site; require both ATP and S-adenosyl-L-methionine to function; multifunctional                                                 protein with both restriction and methylase  activities. Eg.K-12 and B of E.coli.

·         Type II enzymes- cleave within or at short specific distances from recognition site; most require magnesium; single function (restriction)                                               enzymes independent of methylase. E.g. BcgI and BplI

·         Type III enzymes-cleave at sites a short distance from recognition site; require ATP (but do not hydrolyse it); S-adenosyl-L-methionine                                                    stimulates reaction but is not required; exist as part of a complex with a modification methylase. E.g. EcoP15

·         Type IV enzymes target modified DNA, e.g. methylated, hydroxymethylated and glucosyl-hydroxymethylated DNA

Nomenclature

Derivation of the EcoRI name
Abbreviation	Meaning	Description
E	Escherichia	genus
co	coli	species
R	RY13	strain
I	First identified	order of identification in the bacterium

Since their discovery in the 1970s, more than 100 different restriction enzymes have been identified in different bacteria.

Each enzyme is named after the bacterium from which it was isolated using a naming system based on bacterial genus, species and strain.

For example, the name of the EcoRI restriction enzyme was derived as shown in the box.

 

 

 

 

Isolated restriction enzymes are used to manipulate DNA for different scientific applications.

They are used to assist insertion of genes into plasmid vectors during gene cloning and protein expression experiments.

Restriction enzymes can also be used to distinguish gene alleles by specifically recognizing single base changes in DNA known as single nucleotide polymorphisms (SNPs). This is only possible if a SNP alters the restriction site present in the allele. In this method, the restriction enzyme can be used to genotype a DNA sample without the need for expensive gene sequencing. The sample is first digested with the restriction enzyme to generate DNA fragments, and then the different sized fragments separated by gel electrophoresis. In general, alleles with correct restriction sites will generate two visible bands of DNA on the gel, and those with altered restriction sites will not be cut and will generate only a single band. The number of bands reveals the sample subject's genotype, an example of restriction mapping.

In a similar manner, restriction enzymes are used to digest genomic DNA for gene analysis by Souther blot. This technique allows researchers to identify how many copies (or paralogues) of a gene are present in the genome of one individual, or how many gene mutations (polymorphisms) have occurred within a population. The latter example is called restriction fragment length polymorphism (RFLP).

 

Sources:

Content: 

https://nsdl.niscair.res.in/bitstream/123456789/301/2

https://en.wikipedia.org/wiki/Restriction_enzyme

https://www.vivo.colostate.edu/hbooks/genetics/biotech/enzymes/renzymes.html

 

Images:

https://www.vivo.colostate.edu/hbooks/genetics/biotech/enzymes/renzymes.html