Restriction enzymes

Bacterial methylation and restriction enzymes functions.

1. RESTRICTION ENZYMES Presented by: JOYDEEP PAL STUDENT CODE: BWU/BBT/18/036 TOPIC: Restriction Enzymes and their role SUBJECT: BIOINFORMATICS REG: NO: 18013000519 of 2018-19 ROLL NO: 18010310021 DATE: 20.05.21

2. NUCLEASE ENZYMES: Nuclease enzyme… Two types of Nuclease enzyme are: Exonucleases and Endonucleases. Exonucleases: To remove nucleotides from the end of a DNA molecule. Endonucleases: Able to break internal phosphodiester bond.

3. Restriction Enzymes: The enzymes are also called “ MOLECULAR SCISSORS ”. Found in a wide variety of prokaryotes. First concept of R.E. was postulated in 1960s by W. Arber and first RE was isolated in 1970s by Nathans and Smith named HindII . Most of bacteria are used Restriction enzymes as a defense against bacteriophage. Restriction enzymes are used for either cleaving the phage DNA or modification itself. Example of R.E: EcoRI , BamHI , HindII etc.. Fig: Viral genome is entering into the bacterial cell.

4. HOW THE R.E. CLEAVES? The  sticky ends , have unpaired DNA nucleotides on either 5'- or 3'- strand, which are known as overhangs. A straight cut of  restriction enzymes  generates  blunt ends , where both strands terminate in a base pair. Sequence of EcoRI : 5’ GAATTC 3’ , it is PALINDROMIC sequence. 5’ GAATTC 3’ 3’ CTTAAG 5’ Fig: The DNA sequences are digested by the EcoRI and SmaI restriction enzymes.

5. Recognition Site: The DNA sequence to which restriction enzymes can bind. The site of the DNA sequence where it is cleaved by the restriction enzyme. The recognition sequences can also be classified by the number of bases in its recognition site, usually between 4 and 8 bases. Many of them are palindromic. FIG: Recognition site of BamHI Fig: A palindromic recognition site reads the same on the reverse strand as it does on the forward strand when both are read in the same orientation.

6. TYPES OF RESTRICTION ENZYMES: Naturally occurring restriction endonucleases are categorized into four groups (Types I, II III, and IIs): Type I enzymes cleave DNA at random sites more than 1kb from a recognition site; ATP required. Ex: EcoKI , EcoK12 etc.. Type II enzymes cleave DNA within a recognition site, do not require ATP, most require divalent cation (Mg2++). Ex: EcoRI , BamHI etc.. Type III restriction enzymes (e.g., EcoP15) recognize two separate non-palindromic sequences that are inversely oriented. Ex: EcoP15 etc.. Type IIs enzymes cleavage occurs on one side of recognition sequence up to 20bp away. Ex: FauI , HphI etc...

7. RESTRICTION MODIFICATION: Restriction-modification (R-M) systems as defense mechanisms. R-M systems recognize the methylation status of incoming foreign DNA, e.g., phage genomes.  Methylated sequences are recognized as self (bacterial genome), while recognition sequences on the incoming DNA lacking methylation are recognized as nonself and are cleaved by the restriction endonuclease ( REase ).  The methylation status at the genomic recognition sites is maintained by the cognate methyltransferase ( MTase ) of the R-M system. The combination of restriction endonuclease and methylase enzyme termed as RESTRICTION MODIFICATION(RM) SYSTEM. Fig: Bacterial cell is protecting as self methylated sequence from phage DNA.

8. METHYLATION: In  bacteria ,  DNA methylation  is used as a signal for the regulation of a specific  DNA -protein interaction. Typical sites of methylation include the N6 position of Adenine, the N4 position of Cytosine or the C5 position of Cytosine residue. Dam methylase is responsible for N-residue of Adenine and Dcm methylase is responsible for methylation of Cytosine.

9. Nomenclature of R.E: Since their discovery in the 1970s, many restriction enzymes have been identified. More than 3500 different Type II restriction enzymes have been characterized. U sing a naming system based on bacterial  GENUS,   SPECIES  and STRAIN . E.C. number of EcoRI enzyme is 3.1.23.13. HindII enzyme: Genus: Haemophilus Species: influenzae; Strain: Rd and second identified. Derivation of the EcoRI name Abbreviation Meaning Description E Escherichia genus co coli specific species R RY13 strain I First identified order of identification in the bacterium

10. APPLICATIONS OF RESTRICTION ENZYMES: They are used to assist insertion of genes into  plasmid vectors  during gene cloning and protein production experiments. R estriction enzymes are used to digest genomic DNA for gene analysis by Southern Blot. Allows for the large scale production of human insulin for diabetes using E.coli, as well as Hepatitis B and HPV vaccine.

11. Acknowledgement: I would like to thank our respected faculty KRISHNENDU SIR for guide and support me to work in this kind of interesting topic.