RESTRICTION ENZYMES

BRIEFLY EXPLAINED PPT ABOUT RESTRICTION ENZYMES, THEIR WORKING SITES, TYPES, ARTIFICIALLY GENERATED RESTRICTION ENZYMES, THEIR MECHANISM OF ACTION, TYPES OF CUTS THEY MAKE, THEIR NOMENCLATURE ETC.

1. Restriction Enzymes By : Shabana and Alisha

2. Introduction A restriction enzyme or restriction endonuclease is an enzyme that cuts DNA at or near specific recognition nucleotide sequences known as restriction sites . Restriction enzymes are molecular scissors. Natural function of it is to protect the cells from foreign DNA ( they restrict the function of infecting DNA. Optimum conditions are necessary for the expected result. Under extreme conditions such s elevated pH or low ionic strength, RE are capable of cleaving sequences which are similar but not identical to their recognition sequence.

3. Restriction site Restriction enzymes recognize a specific sequence of nucleotides and produce a double-stranded cut in the DNA. Many of them are palindromic. 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 backward on a single strand of DNA, as in GTAATG. The inverted repeat palindrome is also a sequence that reads the same forward and backward, 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. Palindromes

4. Types of RE These types are categorized based on: Their composition Enzymes co-factor requirement The nature of their target sequence Position of their DNA cleavage site relative to the target sequence Type 1 : Enzymes recognize DNA sequences but cut the DNA in random sites that can be as far as 1,000 base pairs away from the recognized site. Type 2 : Enzymes recognize and cut within the recognized site. Type 3 : Enzymes recognize sequences but cut at a different location within 25 base pairs of the recognized site. Type 4 : Recognition sequences have not been well defined.

5. Type 1 : Capable of both restriction and modification activities. Contains : 2 R (restriction) subunits, 2 M(methylation) subunits, 1S(specificity) subunits . Type 2: Most commonly available and used restriction enzymes. Composed of only 1 subunit Their recognition sites are usually undivided and palindromic and 4-8 nucleotides in length. Do Not use ATP for their activity Usually require only Mg2+ as a cofactor. Subtypes : 1. Type Ⅱ P- Palindromic specificity (one domain) 2. Type Ⅱ S - Shifted cleavage (two domains) 3. Type Ⅱ C - Combined ‘restriction and modification’ (three domains) 4. Type Ⅱ T - two different catalytic sites; heterodimers

6. Type 3 : Recognize 2 separate non-palindromic sequences that are inversely oriented. These contain more than 1 subunit. Require ATP for restriction. Type 4 : Cleave only normal and modified DNA ( methylated, hydroxymethylated and glucosyl-hydroxymethylated bases). Cleavage takes places ∼30 bp away ron one of the sites.

7. Artificial RE Generated by fusing a natural or engineered DNA binding domain to a nuclease domain. Can target large DNA sites (up to 36 bp) Can be engineered to bind to desired DNA sequences. Zinc finger nucleases are the most commonly used artificial restriction enzymes. Generally used in genetic engineering applications.

8. Mechanism of action Restriction Endonuclease scan the length of the DNA, binds to the DNA molecule when it recognizes a specific sequence and makes one cut in each of the sugar phosphate backbones of the double helix- by hydrolyzing the phosphodiester bond.

9. Types of cuts done by restriction enzymes Blunt ends Sticky ends Blunt ends

10. These blunt ended fragments can be joined to any other DNA with blunt ends. e.g. HaeIII Sticky ends E.g. EcoR1

11. Isoschizomers and Neoschizomers Isoschizomers : Restriction Enzymes that have the same recognition sequence as well as the same cleavage site. Neoschizomers : Restriction Enzymes that have the same recognition sequence but cleave the DNA at a different site within that sequence.

12. The restriction enzymes are named on basis of their bacterial Genus, Species, Strain and Type E.g. EcoR1 Genus: Escherichia Species: coli Strain: R Order discovered: 1 Nomenclature

13. Why don’t bacteria destroy their own DNA with their restriction enzymes? Methylation

14. Applications They are used in gene cloning and protein expression experiments. Restriction enzymes are used in biotechnology to cut DNA into smaller strands in order to study fragment length differences among individuals ( Restriction Fragment Length Polymorphism- RFLP). Each of these methods depends on the use of agarose gel electrophoresis for separation of the DNA fragments.