Page 1 :
ENZYMES IN GENETIC ENGINEERING: RESTRICTION, NUCLEASES: EXO & ENDO NUCLEASES, , 2-1.1 Introduction:, , A restriction enzyme is a nuclease enzyme that cleaves DNA sequence at a, random or specific recognition sites known as restriction sites. In bacteria, restriction, enzymes form a combined system (restriction + modification system) with modification, enzymes that methylate the bacterial DNA. Methylation of bacterial DNA at the, , recognition sequence typically protects the own DNA of the bacteria from being cleaved, , by restriction enzyme., , There are two different kinds of restriction enzymes:, (1) Exonucleases catalyses hydrolysis of terminal nucleotides from the end of DNA or, RNA molecule either 5’to 3’ direction or 3’ to 5’ direction. Example: exonuclease I,, exonuclease II etc., (2) Endonucleases can recognize specific base sequence (restriction site) within DNA or, , RNA molecule and cleave internal phosphodiester bonds within a DNA molecule., Example: EcoRI, Hind III, BamHI etc., , 2-1.2History:, , In 1970 the first restriction endonuclease enzyme HindII was isolated. For the, subsequent discovery and characterization of numerous restriction endonucleases, in, 1978 Daniel Nathans, Werner Arber, and Hamilton O. Smith awarded for Nobel Prize for, , Physiology or Medicine. Since then, restriction enzymes have been used as an essential, , tool in recombinant DNA technology.
Page 2 :
2-1.3 Restriction Endonuclease Nomenclature:, , Restriction endonucleases are named according to the organism in which they, were discovered, using a system of letters and numbers. For example, HindIII, (pronounced “hindee-three”) was discovered in Haemophilus influenza (strain d). The, Roman numerals are used to identify specific enzymes from bacteria that contain multiple, , restriction enzymes indicating the order in which restriction enzymes were discovered in, , a particular strain., , Hindlll, , First letter of the genus name ¢, (Haemophilus) and the first, two letters of the species, name (influenza)., , is the 4” is for the 3 enzyme discovered in, straintype that organism, , 2-1.4 Classification of Restriction Endonucleases:, There are three major classes of restriction endonucleases based on the types of, , sequences recognized, the nature of the cut made in the DNA, and the enzyme structure:, , e Type I restriction enzymes, e Type Il restriction enzymes, e Type III restriction enzymes, , 2-1.4.1 Type I restriction enzymes:, , e These enzymes have both restriction and modification activities. Restriction, depends upon the methylation status of the target DNA., , e Cleavage occurs approximately 1000 bp away from the recognition site., , e The recognition site is asymmetrical and is composed of two specific portions in, , which one portion contain 3-4 nucleotides while another portion contain 4-5
Page 3 :
2-1.4.3 Type III restriction enzymes:, , e These enzymes recognize and methylate the same DNA sequence but, cleave 24-26 bp away., , e They have two different subunits, in which one subunit (M) is responsible, for recognition and modification of DNA sequence and other subunit (R), , has nuclease action., , ¢ Mg*ions, ATP are needed for DNA cleavage and process of cleavage is, stimulated by SAM., © Cleave only one strand. Two recognition sites in opposite orientation are, , necessary to break the DNA duplex., , , , , , , , , , , , , , , , Property Type I RE Type II RE Type III RE, Abundance Less common than | Most common Rare, Type Il, Recognition site Cut both strands at a | Cut both strands at} Cleavage of one, non- specific | a specific, usually | strand, only 24-26, location > 1000 bp | palindromic bp downstream of, away from | recognition site (4-8 | the 3° recognition, recognition site bp) site, Restriction and Single Separate nuclease Separate enzymes, modification multifunctional and methylase sharing a, enzyme common subunit, Nuclease subunit Heterotrimer Homodimer Heterodimer, structure, Cofactors ATP, Mg2+,SAM__| Mg2+ Mg2+ (SAM), DNA cleavage Two recognition Single recognition | Two recognition, requirements sites in any site sites in a, orientation head-to-head, , , , , , orientation
Page 5 :
Once the target restriction site is located, the recognition process (coupling), , triggers large conformational changes of the enzyme and the DNA, which leads to, activation of the catalytic center., , Catalysis results in hydrolysis of phosphodiester bond and product release., , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , a, rear, eoartgd DNA, a Tiihecibncttmlacal: o, Restriction, endonuclease, Bam HI, ma er *, G@eATCC Non specific binding, cG TA GG, ‘ Liccil cenbestlaudksl ¢ } sliding, pes a5 ee, & =, T qT, etn + oo Specific binding, 7 ot £F S, a ae Loh s, \ Coupling, , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , 4, , , , , , , , , , , , Fig 2-1.4.2: Structures of free, nonspecific, and specific DNA-bound forms of BamHI., , The two dimers are shown in brown, the DNA backbone is in green and the bases in gray., , BamHI becomes progressively more closed around the DNA as it goes from the, nonspecific to specific DNA binding mode.
