Genetic Engineering Lecture 2 Restriction Enzymes PDF

 Restriction Enzymes Restriction Enzyme Introduction:  Restriction enzymes are molecular scissors discovered in a wide variety of prokaryotes (i.e. bacteria in 1962)  Restriction enzymes cut double stranded DNA molecules at specific points.  Most bac...

 Restriction Enzymes Restriction Enzyme Introduction:  Restriction enzymes are molecular scissors discovered in a wide variety of prokaryotes (i.e. bacteria in 1962)  Restriction enzymes cut double stranded DNA molecules at specific points.  Most bacteria use restriction enzymes as a defense mechanism against invading viruses (bacteriophages) and restrict the growth of bacteriophages in bacterial cell by selective cleavage of the viral DNA upon entry.  Restriction enzymes also called Restriction endonuclease o Endo: inside, nuclease: cuts nucleic acid o Recognizes a short and specific DNA sequence and cuts it from inside. o This specific DNA sequence is called recognition sequence  More than 3000 restriction enzymes have been studied in detail till now, and more than 600 of them are available commercially.  An important tool for genetic engineering. Restriction enzyme definition: Restriction enzyme is endonuclease synthesis by bacteria that recognizes a specific nucleotide sequence and cuts the double stranded DNA only at that specific site, i.e., restriction site. Genetic Engineering BIOC0402 Lecture 2 Dr. Fatema S Alatawi 1  Restriction Enzymes Recognition Sequences (restriction site) Recognition sequences is a specific sequence of nucleotides (between four and eight bases) that recognized by restriction enzyme to makes cut in both strands of the double-stranded DNA.  The majority of recognition sequence are palindromic. For example, read the same in the opposite direction (eg. madam, race car…) 5’ GAATTC 3’ 3’ CTTAAG 5’  Palindrome: When read in the 5' to 3' direction, the sequence on the “top” strand is identical to that of the “bottom” strand. 5’-GGATCC-3’ Bam H1 3’-CCTAGG-5’ Isoschizomers and Neochischizomers Isoschizomers: Different restriction enzymes that have the same recognition sequence and cut in the same site or location. For example, SmaI and XmaI are isoschizomer pair. Genetic Engineering BIOC0402 Lecture 2 Dr. Fatema S Alatawi 2  Restriction Enzymes Neoschizomers: Different restriction enzymes that that have the same recognition sequence but cleave at different location. For example, ApaI and Bsp120I are neoschizomer pair. Why don’t bacteria destroy their own DNA with their restriction enzymes?  Bacteria protect their self DNA from restriction digestion by methylation of its recognition site.  Methylation is adding a methyl group (CH3) to DNA by Methylases which add methyl groups to adenine or cytosine bases within the recognition site thereby modifying the site and protecting the DNA.  Therefore, the restriction enzyme within a cell doesn’t destroy its own DNA. Genetic Engineering BIOC0402 Lecture 2 Dr. Fatema S Alatawi 3  Restriction Enzymes Restriction Modification System  Restriction-modified (R-M) system have two process: 1. Endonuclease activity: cuts foreign DNA at the recognition site 2. Methyltransferase activity: protects host DNA from cleavage by the restriction enzyme. Methylate one of the bases in each strand.  Restriction enzyme and its cognate modification system constitute the R-M system Genetic Engineering BIOC0402 Lecture 2 Dr. Fatema S Alatawi 4  Restriction Enzymes Cleavage pattern by restriction enzymes:  Once restriction enzyme recognizes restriction site, it can generate either sticky ends or blunt ends depending upon type of restriction enzyme. Sticky End Cutters  Most restriction enzymes make staggered cuts  Staggered cuts produce single stranded “sticky-ends”  DNA from different sources can be spliced easily because of sticky-end overhangs. Hind III Eco RI Blunt End Cutters  Some restriction enzymes cut DNA at opposite base  They leave blunt ended DNA fragments,  These are called blunt end cutters. Genetic Engineering BIOC0402 Lecture 2 Dr. Fatema S Alatawi 5  Restriction Enzymes Table: Some restriction enzymes, their recognition sequence and cleavage pattern Nomenclature  Smith and Nathans (1973) proposed enzyme naming scheme  three-letter acronym for each enzyme derived from the source organism  First letter from genus  Next two letters represent species  Additional letter or number represent the strain or serotypes Genetic Engineering BIOC0402 Lecture 2 Dr. Fatema S Alatawi 6  Restriction Enzymes  For example. the enzyme HindII was isolated from Haemophilus influenzae serotype d.  Named for bacterial genus, species, strain, and type Example: EcoR1 Genus: Escherichia Species: coli Strain: R Order discovered: 1 Classification of Restriction Enzymes Restriction enzymes are classified according to their structure, recognition site, cofactor, and activator. They divide into four types (Types I, II, III, and IV): 1. Type I restriction enzyme: - It cuts at sites far from a recognition site and Genetic Engineering BIOC0402 Lecture 2 Dr. Fatema S Alatawi 7  Restriction Enzymes - requires both ATP and S-adenosyl-L-methionine (AdoMet) as cofactor to function. - It has a feature of both restriction digestion and methylase. 2. Type II restriction enzymes: - The most commonly available and used restriction enzymes - It cleaves within or at short-specific distances from a recognition site (palindromic in nature) - mostly requires magnesium to function. - It has only feature of restriction digestion independent of methylase. 3. Type III restriction enzymes: - It recognizes two separate non-palindromic and inversely oriented sequences and cleaves at 20–30 base pairs away from a recognition site. - It contains more than one subunit. - requires ATP for restriction digestion and S-adenosyl- Lmethionine for DNA methylation. 4. Type IV restriction enzymes: - It recognizes modified DNA: o methylated, hydroxyl-methylated, and glucosyl- hydroxy-methylated. Genetic Engineering BIOC0402 Lecture 2 Dr. Fatema S Alatawi 8  Restriction Enzymes - Examples are McrBC and Mrr systems of E. coli. Table: Some types of restriction enzymes General Mechanism of restriction enzyme: 1. Scanning  Restriction enzyme scans the length of the DNA. 2. Recognition Sequence  It binds to the DNA molecule when it recognizes a specific sequence. Genetic Engineering BIOC0402 Lecture 2 Dr. Fatema S Alatawi 9  Restriction Enzymes 3. Cleavage  Makes one cut in each of the sugar phosphate backbones of the double helix by hydrolyzing the phoshphodiester bond.  Specifically, the bond between the 3’ O atom and the P atom is broken. - Direct hydrolysis at the phosphorous atom  3’OH and 5’ PO43- is produced.  Mg2+ is required for the catalytic activity of the enzyme. Genetic Engineering BIOC0402 Lecture 2 Dr. Fatema S Alatawi 10  Restriction Enzymes  It holds the water molecule in a position where it can attack the phosphoryl group and also helps polarize the water molecule towards deprotonation. Genetic Engineering BIOC0402 Lecture 2 Dr. Fatema S Alatawi 11