RESTRICTION ENDONUCLEASES PPT SANSKRUTI SHAHANE.pptx

This presentation is made to understand the overview of restriction endonuclease which is also called as Molecular scissors . This presentation includes everything from its defination to its type and mode of action .

1. Restriction endonucleases MADE BY SANSKRUTI YOGESH SHAHANE

2. CONTENTS OF THIS TEMPLATE 1.WHAT ARE RESTRICTION ENZYMES? 2.NOMENCLATURE OF RESTRICTION ENZYME 3 .WHAT ARE RESTRICTION ENDONUCLEASES? 4.TYPES OF RESTRICTION ENDONUCLEASES

3. WHAT ARE RESTRICTION ENZYMES ? (MOLECULAR SCISSORS)

4. “Restriction enzymes are the enzymes produced by certain bacteria that have the property of cleaving DNA molecule at or near specific base sequences.”

5. The restriction enzyme is a protein produced by bacteria that cleaves the DNA at specific sites. This site is known as the restriction site. The restriction enzymes protect the live bacteria from bacteriophages . They recognize and cleave at the restriction sites of the bacteriophage and destroy its DNA. Restriction enzymes are important tools for genetic engineering . They can be isolated from the bacteria and used in the laboratories. The restriction enzymes recognize short and specific nucleotide sequences in the DNA known as the recognition sequences. When the restriction enzyme recognizes a DNA sequence, it hydrolyzes the bond between adjacent nucleotide and cuts through the DNA molecule. The  bacteria  prevents its own DNA sequences from degradation by the addition of the methyl group at the adenine or cytosine bases within the recognition sequence with the help of enzyme methylases .

6. NOMENCLATURE Restriction endonucleases are named as follows: 1st alphabet represents the genus of the organism from which the enzyme is isolated. 2nd and 3rd alphabet represents the species of the organism. 4th alphabet represents the strain. The roman number represents the order of isolation or discovery of the enzyme. For example, EcoRI is the restriction enzyme, derived from  Escherichia coli,  strain R and is the first to be isolated.

7. TYPE 1 These restriction enzymes cut the DNA far from the recognition sequences . However, they do not produce discrete restriction fragments, hence, are of not much practical value. These are complex, multi-subunit restriction and modification enzymes. They were initially thought to be rare, but through genomic analysis, they are found to be common and are of considerable biochemical interest

8. MODE OF ACTION OF TYPE 1 1. Recognition and Binding : Type I enzymes recognize specific, asymmetric DNA sequences typically 4-6 base pairs in length. 2 . DNA Translocation : After binding, the enzyme translocates along the DNA, consuming ATP in the process. 3.Cleavage: Cleavage occurs at random sites, often hundreds to thousands of base pairs away from the recognition site. This translocation and cleavage mechanism results in unpredictable cut sites.

9. TYPE 2 These enzymes cut at specific positions closer to or within the restriction sites. Discrete restriction fragments and gel banding patterns are observed. They are exclusively used for DNA analysis and gene cloning in the laboratories. These are a family of unrelated proteins. They are named after the bacterial species from which they are isolated. For eg ., EcoRI is isolated from bacterial species  E.coli . The restriction enzymes generate two different types of cuts . Blunt ends are produced when they cut the DNA at the centre of the recognition sequence, and sticky ends produce an overhang.

10. MODE OF ACTION OF TYPE 2 1 . Recognition and Binding : Type II enzymes recognize palindromic DNA sequences, which are often 4-8 base pairs long. 2. Cleavage : These enzymes cleave the DNA within or very close to their recognition sites, producing predictable and reproducible DNA fragments. Cleavage results in either blunt ends or sticky ends (overhangs), depending on the enzyme . :** EcoRI recognizes the sequence 5'-GAATTC-3' and cleaves between G and A, creating sticky ends.

11. TYPE 3 These are multi-functional  proteins  with two subunits- Res and Mod. It is a modification methyltransferase . The DNA sequence specific for the system is recognized by the Mod subunit . - Composed of two subunits: one for recognition and one for cleavage. - Require ATP and SAM, but do not hydrolyze ATP for cleavage. - Recognize specific DNA sequences and cleave a short distance away

12. MODE OF ACTION OF TYPE 3 1. Recognition and Binding :Type III enzymes recognize specific sequences that are typically asymmetric. 2. Cleavage : These enzymes cleave DNA at a fixed distance (usually 20-30 base pairs) away from the recognition site, typically on one strand only. Cleavage is usually less frequent and less predictable compared to Type II enzymes.   Example :** EcoP15I recognizes the sequence 5'-CAGCAG-3' and cleaves 25 base pairs downstream from the recognition site.