Restriction enzyme : class xii

Restriction enzyme 

What is a resriction enzyme? 
Restriction enzyme, also called restriction endonuclease, a protein produced by bacteria that cleaves DNA at specific sites

What do restriction enzyme do in bacterial cell?
In the bacterial cell, restriction enzymes cleave foreign DNA, thus eliminating infecting organisms. 
A bacterium uses a restriction enzyme to defend against bacterial viruses called bacteriophages, or phages. When a phage infects a bacterium, it inserts its DNA into the bacterial cell so that it might be replicated. The restriction enzyme prevents replication of the phage DNA by cutting it into many pieces. Restriction enzymes were named for their ability to restrict, or limit, the number of strains of bacteriophage that can infect a bacterium.

Why restriction enzymes are important for us?
Restriction enzymes can be isolated from bacterial cells and used in the laboratory to manipulate fragments of DNA, such as those that contain genes; for this reason they are indispensible tools of recombinant DNA technology (genetic engineering).
Restriction enzymes are used in biotechnology to cut DNA into smaller strands in order to study fragment length differences among individuals. This is referred to as restriction fragment length polymorphism (RFLP). They’re also used for gene cloning.

What are recognition sequences?
Each restriction enzyme recognizes a short, specific sequence of nucleotide bases (the four basic chemical subunits of the linear double-stranded DNA molecule—adenine, cytosine, thymine, and guanine). These regions are called recognition sequences and are randomly distributed throughout the DNA. Different bacterial species make restriction enzymes that recognize different nucleotide sequences.

How do restriction enzymes cleave the DNA sequence? 
When a restriction endonuclease recognizes a sequence, it snips through the DNA molecule by catalyzing the hydrolysis (splitting of a chemical bond by addition of a water molecule) of the bond between adjacent nucleotides.

How does Bacteria prevent their own DNA from being degraded by RE?
Bacteria prevent their own DNA from being degraded in this manner by disguising their recognition sequences. Enzymes called methylases add methyl groups (—CH3) to adenine or cytosine bases within the recognition sequence, which is thus modified and protected from the endonuclease. The restriction enzyme and its corresponding methylase constitute the restriction-modification system of a bacterial species.

What are different types of restriction enzymes? How are they different from each other? 
Traditionally, four types of restriction enzymes are recognized, designated I, II, III, and IV, which differ primarily in structure, cleavage site, specificity, and cofactors. 
Types I and III enzymes are similar in that both restriction and methylase activities are carried out by one large enzyme complex, in contrast to the type II system, in which the restriction enzyme is independent of its methylase. 
Type II restriction enzymes also differ from types I and III in that they cleave DNA at specific sites within the recognition site; the others cleave DNA randomly, sometimes hundreds of bases from the recognition sequence. Several thousand type II restriction enzymes have been identified from a variety of bacterial species. These enzymes recognize a few hundred distinct sequences, generally four to eight bases in length. 
Type IV restriction enzymes cleave only methylated DNA and show weak sequence specificity.

When and how were the restriction enzymes discovered? 
Restriction enzymes were discovered and characterized in the late 1960s and early 1970s by molecular biologists Werner Arber, Hamilton O. Smith, and Daniel Nathans. The ability of the enzymes to cut DNA at precise locations enabled researchers to isolate gene-containing fragments and recombine them with other molecules of DNA—i.e., to clone genes.

Explain about nomenclature of RE?
The names of restriction enzymes are derived from the genus, species, and strain designations of the bacteria that produce them; for example, the enzyme EcoRI is produced by Escherichia coli strain RY13.
First restriction enzyme to be discovered was endonuclease-HindII. It was isolated from Haemophilus influenza bacterium and this it’s so named.
The convention for naming these enzymes proceeds in a way that the first letter of the name comes from the genes and the second two letters come from the species of prokaryotic cell, from which they were isolated, e.g., Eco RI comes from E. coli RY13. The letter ‘R’ is derived from the name of strain. Roman numbers following the names, indicate the order in which the enzymes were isolated from that strain of bacteria.
EcoRI →
‘E’ represents Escherichia bacteria,
‘co’ represents coli the species,
‘R’ represents the RY13 strain of the bacteria and
‘I’ it is the 1st to be discovered from that strain.
HindII →
‘H’ represents Haemophilus bacteria,
‘in’ represents influenzae species,
‘d’ represents Rd strain and
‘II’ it is 2nd to be discovered from that strain.

Restricted sites of EcoRI:
5′ GAATTC 3′
3′ CTTAAG 5′

Restricted sites of Hind II:
5′ GTCGAC 3′
3′ CAGCTG 5′

Where do restriction enzymes come from?
Restriction enzymes are found in bacteria. Bacteria use restriction enzymes to kill viruses – the enzymes attack the viral DNA and break it into useless fragments.

When are restriction enzymes used?
Restriction enzymes are a basic tool for biotechnology research. They are used for DNA cloning and DNA fingerprinting.

DNA fragments: Blunt or sticky ends?
DNA consists of two complementary strands of nucleotides that spiral around each other in a double helix. Restriction enzymes cut through both nucleotide strands, breaking the DNA into fragments, but they don’t always do this in the same way.
SmaI is an example of a restriction enzyme that cuts straight through the DNA strands, creating DNA fragments with a flat or blunt end.
Other restriction enzymes, like EcoRI, cut through the DNA strands at nucleotides that are not exactly opposite each other. This creates DNA fragments with one nucleotide strand that overhangs at the end. This overhanging nucleotide strand is called a sticky end because it can easily bond with complementary DNA fragments.

What is the differences between Exonucleases and Endonucleases?
Exonucleases:These nucleases cleave base pairs of DNA at their terminal ends

They act on single – strand of DNA or gaps in double –stranded DNA. They do not cut RNA

Endonucleases:

They cleave DNA at any Pont except the terminal ends

They cleave one strand (figure below) or both strands (figure below) of double – stranded DNA. They may cut RNA

What are molecular scissors? Why are they called so?
Restriction endonucleases are known as “molecular scissors” in genetic engineering.
They are capable of breaking DNA at specific sites called the restricted sites.
What are Palindromic sequences?
Palindromic sequences are sequences that have the same meaning when read from both the sides just like the word ‘MADAM’ & ‘MALAYALAM’.
5‘ → 3‘
GTAC
CATG
3‘ → 5‘

https://youtu.be/rhd_fBPyzSM