Restriction enzyme

Definition: A restriction enzyme (or restriction endonuclease) is an enzyme that cuts double-stranded or single stranded DNA at specific recognition nucleotide sequences known as restriction sites. Such enzymes, found in bacteria and archaea, are thought to have evolved to provide a defense mechanism against invading viruses. Inside a bacterial host, the restriction enzymes selectively cut up foreign DNA in a process called restriction; host DNA is methylated by a modification enzyme (a methylase) to protect it from the restriction enzymes activity. Collectively, these two processes form the restriction modification system. To cut the DNA, a restriction enzyme makes two incisions, once through each sugar-phosphate backbone (i.e. each strand) of the DNA double helix. Source: The natural source of this enzymes are found in bacteria and archaea. After isolating the first restriction enzyme, HindII, in 1970, and the subsequent discovery and characterization of numerous restriction endonucleases, the 1978 Nobel Prize for Physiology or Medicine was awarded to Daniel Nathans, Werner Arber, and Hamilton O. Smith. Their discovery led to the development of recombinant DNA technology that allowed, for example, the large scale production of human insulin for diabetics using E. coli bacteria. Over 3000 restriction enzymes have been studied in detail, and more than 600 of these are available commercially and are routinely used for DNA modification and manipulation in laboratories. Specificity: Restriction enzymes recognize a specific sequence of nucleotides and produce a double-stranded cut in the DNA. While recognition sequences vary between 4 and 8 nucleotides, many of them are palindromic, which correspond to nitrogenous base sequences that read the same backwards and forwards. In theory, there are 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 backwards on the same DNA strand (i.e., single stranded) as in GTAATG. The inverted repeat palindrome is also a sequence that reads the same forward and backwards, but the forward and backward sequences are found in complementary DNA strands (i.e., double stranded) as in GTATAC (Notice that GTATAC is complementary to CATATG). The inverted repeat is more common and has greater biological importance than the mirror-like. EcoRI digestion produces "sticky" ends,

whereas SmaI restriction enzyme cleavage produces "blunt" ends

Recognition sequences in DNA differ for each restriction enzyme, producing differences in the length, sequence and strand orientation (5' end or the 3' end) of a sticky-end "overhang" of an enzyme restriction. Different restriction enzymes that recognize the same sequence are known as neoschizomers. These often cleave in a different locales of the sequence; however, different enzymes that recognize and cleave in the same location are known as an isoschizomer. Nomenclature: Since their discovery in the 1970s, more than 100 different restriction enzymes have been identified in different bacteria. Each enzyme is named after the bacterium from which it was isolated using a naming system based on bacterial genus, species and strain. For example, EcoRI name Abbreviation Meaning Description E Escherichia genus co coli species R RY13 strain I First identified order of identification in the bacterium Types Restriction endonucleases are categorized into three or four general groups (Types I, II and III) based on their composition and enzyme cofactor requirements, the nature of their target sequence, and the position of their DNA cleavage site relative to the target sequence.[18][19][20] There are four classes of restriction endonucleases: types I, II,III and IV. All types of enzymes recognise specific short DNA sequences and carry out the endonucleolytic cleavage of DNA to give specific double-stranded fragments with terminal 5'-phosphates. They differ in their recognition sequence, subunit composition, cleavage position, and cofactor requirements, [21][22] as summarised below: Type I ype I restriction enzymes were the first to be identified and are characteristic of two different strains (K-12 and B) of E. coli.[23] These enzymes cut at a site that differs, and is some distance (at least 1000 bp) away, from their recognition site. The recognition site is asymmetrical and is composed of two portionsone containing 34

nucleotides, and another containing 45 nucleotidesseparated by a spacer of about 68 nucleotides. Several enzyme cofactors, including S-Adenosyl methionine (AdoMet), hydrolyzed adenosine triphosphate (ATP), and magnesium (Mg2+) ions, are required for their activity. Type I restriction enzymes possess three subunits called HsdR, HsdM, and HsdS; HsdR is required for restriction; HsdM is necessary for adding methyl groups to host DNA (methyltransferase activity) and HsdS is important for specificity of cut site recognition in addition to its methyltransferase activity. Type II Typical type II restriction enzymes differ from type I restriction enzymes in several ways. They are a dimer of only one type of subunit; their recognition sites are usually undivided and palindromic and 48 nucleotides in length, they recognize and cleave DNA at the same site, and they do not use ATP or AdoMet for their activity they usually require only Mg2+ as a cofactor.[15] These are the most commonly available and used restriction enzymes. Type III Type III restriction enzymes (e.g. EcoP15) recognize two separate non-palindromic sequences that are inversely oriented. They cut DNA about 20-30 base pairs after the recognition site.[25] These enzymes contain more than one subunit and require AdoMet and ATP cofactors for their roles in DNA methylation and restriction, respectively. Examples: See, Lehninger Principles of Biochemistry-3rd edition (page 1122) by David L. Nelson and Michael M. Cox. Target sequence (cut at *) 5' -->3' Ava I Anabaena variabilis C* C/T C G A/G G Bam HI Bacillus amyloliquefaciens G* G A T C C Bgl II Bacillus globigii A* G A T C T Eco RI Escherichia coli RY 13 G* A A T T C Eco RII Escherichia coli R245 * C C A/T G G Hae III Haemophilus aegyptius GG*CC Hha I Haemophilus haemolyticus GCG*C Hind III Haemophilus inflenzae Rd A* A G C T T Hpa I Haemophilus parainflenzae GTT*AAC Kpn I Klebsiella pneumoniae GGTAC*C Mbo I Moraxella bovis *G A T C Mbo I Moraxella bovis *G A T C Pst I Providencia stuartii CTGCA*G Sma I Serratia marcescens CCC*GGG SstI Streptomyces stanford GAGCT*C Sal I Streptomyces albus G G*TCGAC Taq I Thermophilus aquaticus T*CGA Xma I Xanthamonas malvacearum C*CCGGG DNA digestion by Restriction enzyme: When a molecule of DNA is double stranded, as DNA usually is, the two strands run in opposite directions. Therefore, one end of the molecule will have the 3' end of strand 1 and the 5' end of strand 2, and vice versa in the other end. However, the fact that the molecule is two stranded allows numerous different variations Enzyme Organism from which derived Blunt ends The simplest DNA end of a double stranded molecule is called a blunt end. In a blunt-ended molecule both strands terminate in a base pair. Blunt ends are not always desired in biotechnology since when using a DNA ligase to join two molecules into one, the yield is significantly lower with blunt ends. When performing subcloning, it also has the disadvantage of potentially inserting the insert DNA in the opposite orientation desired. On the other hand, blunt ends are always compatible with each other. Here is an example of a small piece of blunt-ended DNA: 5'-CTGATCTGACTGATGCGTATGCTAGT-3' 3'-GACTAGACTGACTACGCATACGATCA-5'

Overhangs and sticky ends Non-blunt ends are created by various overhangs. An overhang is a stretch of unpaired nucleotides in the end of a DNA molecule. These unpaired nucleotides can be in either strand, creating either 3' or 5' overhangs. These overhangs are in most cases palindromic. The simplest case of an overhang is a single nucleotide. This is most often adenosine and is created as a 3' overhang by some DNA polymerases. Most commonly this is used in cloning PCR products created by such an enzyme. The product is joined with a linear DNA molecule with 3' thymine overhangs. Since adenine and thymine form a base pair, this facilitates the joining of the two molecules by a ligase, yielding a circular molecule. Here is an example of an A-overhang: 5'-ATCTGACTA-3' 3'-TAGACTGA-5' Longer overhangs are called cohesive ends or sticky ends. They are most often created by restriction endonucleases when they cut DNA. Very often they cut the two DNA strands four base pairs from each other, creating a four-base 5' overhang in one molecule and a complementary 5' overhang in the other. These ends are called cohesive since they are easily joined back together by a ligase. Also, since different restriction endonucleases usually create different overhangs, it is possible to cut a piece of DNA with two different enzymes and then join it with another DNA molecule with ends created by the same enzymes. Since the overhangs have to be complementary in order for the ligase to work, the two molecules can only join in one orientation. This is often highly desirable in molecular biology. For example, these two "sticky" ends are compatible: 5'-ATCTGACT + GATGCGTATGCT-3' 3'-TAGACTGACTACG CATACGA-5' They can form complementary base pairs in the overhang region: GATGCGTATGCT-3' 5'-ATCTGACT CATACGA-5' 3'-TAGACTGACTACG R- M system Restriction-Modification system occurs in many bacterial species and constitute a defense mechanism against the introduction of foreign DNA into the cell. They consist of two components : 1. Restriction endonuclease : which recognizes a short, symmetrical DNA sequence and hydrolyse DNA backbone in each strand at a specific sites within that sequence - so that the foreign DNA will hence be degraded to relatively short fragments. 2. Methylase : Adds a methyl group to a C or A base within the same recognition sequences in the cellular DNA - This modification renders the host DNA resistant to degradation by the endonuclease. Types of restriction modification system There are three kinds of restriction modification system: type I, type II and type III, all with restriction enzyme activity and a methylase activity. They were named in the order of discovery, although the type II system is the most common. Type I systems are the most complex, consisting of three polypeptides: R (restriction), M (modification), and S (specificity). The resulting complex can both cleave and methylate DNA. Both reactions require ATP, and cleavage often occurs a considerable distance from the recognition site. The S subunit determines the specificity of both restriction and methylation. Cleavage occurs at variable distances from the recognition sequence, so discrete bands are not easily visualized by gel electrophoresis. Type II systems are the simplest and the most prevalent. Instead of working as a complex, the methyltransferase and endonuclease are encoded as two separate proteins and act independently (there is no specificity protein). Both proteins recognize the same recognition site, and therefore compete for activity. The methyltransferase acts as a monomer, methylating the duplex one strand at a time. The endonuclease acts as a homodimer, which facilitates the cleavage of both strands. Cleavage occurs at a defined position close to or

within the recognition sequence, thus producing discrete fragments during gel electrophoresis. For this reason, Type II systems are used in labs for DNA analysis and gene cloning. Type III systems have R and M proteins that form a complex of modification and cleavage. The M protein, however, can methylate on its own. Methylation also only occurs on one strand of the DNA unlike most other known mechanisms. The heterodimer formed by the R and M proteins competes with itself by modifying and restricting the same reaction. This results in incomplete digestion. DNA methylation: DNA methylation involves the addition of a methyl group to the 5 position of the cytosine pyrimidine ring or the number 6 nitrogen of the adenine purine ring (cytosine and adenine are two of the four bases of DNA). This modification can be inherited through cell division. In bacteria:Adenine or cytosine methylation is part of the restriction modification system of many bacteria, in which specific DNA sequences are methylated periodically throughout the genome. A methylase is the enzyme that recognizes a specific sequence and methylates one of the bases in or near that sequence. Foreign DNAs (which are not methylated in this manner) that are introduced into the cell are degraded by sequence-specific restriction enzymes and cleaved. Bacterial genomic DNA is not recognized by these restriction enzymes. The methylation of native DNA acts as a sort of primitive immune system, allowing the bacteria to protect themselves from infection by bacteriophage. E. coli DNA adenine methyltransferase (Dam) is an enzyme of ~32 kDa that does not belong to a restriction/modification system. The target recognition sequence for E. coli Dam is GATC, as the methylation occurs at the N6 position of the adenine in this sequence (G meATC). The three base pairs flanking each side of this site also influence DNADam binding. Dam plays several key roles in bacterial processes, including mismatch repair, the timing of DNA replication, and gene expression. As a result of DNA replication, the status of GATC sites in the E. coli genome changes from fully methylated to hemimethylated. This is because adenine introduced into the new DNA strand is unmethylated. Re-methylation occurs within two to four seconds, during which time replication errors in the new strand are repaired. Methylation, or its absence, is the marker that allows the repair apparatus of the cell to differentiate between the template and nascent strands.