14 restriction enzymes, Biotechnologia, Genetyka molekularna

//-->.pos {position:absolute; z-index: 0; left: 0px; top: 0px;}Digestion of DNA WithRestriction EndonucleasesDigestion of DNA with restriction endonucleases is the first step in many gene manipulationprojects. These enzymes are part of the system that carries out restriction and modification. It appearsthat their main role is to protect cells from invasion by foreign DNA’s, especially bacteriophage DNA.Restriction endonucleases recognize specific 4-base (tetramer), 5-base (pentamer), or 6-base (hexamer)sites located on the incoming DNA, and make double-stranded cuts. The sites are short enough that theycan be found randomly in the DNA of any organism, including the organism that produces therestriction endonuclease. To protect its own sites, then, the producing organism has a methyl transferasethat recognizes and methylates the same site that the endonuclease cuts. Methyl transferases are oftenreferred to as methylases butmethyl transferaseis a better description of the mode of action of theseenzymes, and is therefore the preferred term. This process is called modification; methylation preventsrestriction. Thus, an organism would have its own sites protected while incoming DNA would lack theappropriate methylation and therefore be vulnerable. The accepted abbreviations for restrictionendonucleases and methyltransferases are REase and MTase, respectivelyNaming EnzymesThere is a uniform system for naming restriction endonucleases and their corresponding methyltrahsferases, based on the genus and species of the source organism, the particular strain or serotype, andthe order of discovery. By convention, the first letter of the genus name and the first two letters of thespecies name are used to derive the basic enzyme name. Thus Escherichia coli yields Eco (becausegenus and species names are italicized, it was originally the custom to italicize the enzyme name [Eco]but recent nomenclature recommendations have dispensed with this convention). Then comes adesignation, if any, of the particular strain or serotype (sometimes an enzyme is encoded by a plasmidand the plasmid designation is used). A common REase from E. coli comes from an R factor. Finally, aRoman numeral is applied to indicate the order of discovery. Thus the first restriction enzyme from E.coli carrying an R factor is Eco R I. Some others are:HindIIISmaIBamHIKpnIthe third enzyme fromHaemophilus influenzaestrain dthe first enzymeSerratia marcesensthe first enzyme fromBacillus amyloliquifaciensstrain Hthe first enzyme fromKlebsiella pneumoniaeThe names of REases are distinguished from the names of MTases by placing an “R.” or an “M.” infront of the name. Thus m.EcoRI is the corresponding methyl transferase for the restrictionendonuclease R.EcoRI. Typically, though, most people only use the endonucleases, so the “R.” tends tobe dropped unless both endonucleases and methyl transferases are used in the same work.9Restriction EnzymesRestriction SitesRecombinant DNA technology is based upon the fact that many enzymes produce staggered cuts leavingcomplementary single-stranded tails. Being complementary, the single stranded tails can be made toform hydrogen bonds with one another and the cohering fragments can then be ligated together. Sincethe tails are based solely on the restriction sequence, it is possible to ligate DNA’s from two differentspecies if they have been cut with the same enzyme. The ability of restriction endonucleases to producecohesive single-stranded tails depends upon the symmetry of the restriction site and the way that theparticular enzyme cuts relative to the symmetry.The two strands of DNA are said to be anti-parallel. That is, one strand runs 5’→3’ and the other runs3’→5’. This produces a structural symmetry called rotational or dyad symmetry. In dyad symmetry,one can rotate DNA 180o and obtain the same structure:axis of rotation5'3'axis of rotation5’------------G A AT T C ------------ 3’3’------------C T TA A G------------- 5’3'5'For restriction sites, not only does the overall structure possess dyad symmetry, but also the DNAsequence itself possesses dyad symmetry. For example, the restriction enzyme EcoRI recognizes thesite:•When this hexameric sequence is rotated about its axis, not only is the structural polarity maintained, butalso the identical sequence is obtained. This symmetry of sequence is due to the unique nature of thebase sequence in which the second three bases are the complement, in reverse order, of the first three:A B C C’ B’ A’One strand therefore is the reverse order of the other. Such an arrangement is often referred to as apalindrome. In literature, a palindrome is a phrase that reads the same forwards and backwards. Anexample of a literary palindrome is when Adam, in the Garden of Eden, introduced himself to Eve:Madam, I’m AdamThe restriction site is not a true palindrome, of course, because the reverse is on the opposite strand.10Restriction EnzymesCleavageDuring restriction, the endonuclease must cut each of the strands to generate a double-strand cut.Cleavage is the result of hydrolysis, a reaction in which water is added across a bond, thereby breakingit. In this case, the water is added across the phosphodiester bond, cleaving the two adjacentnucleotides. Cleavage (at least by restriction endonucleases) yields 5’-phosphate and 3’-hydroxyltermini. By contrast, Nucleotides are joined by condensation reactions, in which phosphodiester bondsare formed by splitting out a water molecule. DNA ligases are enzymes that function via condensation.Because each of the strands are identical to each other both in sequence and structure (remember, thestrands are the same, but antiparallel), the cuts are made in the same spot on each strand, relative to theaxis of rotation. This creates a staggered cut, leaving overhanging single-stranded tails on each end. Thecuts made by EcoRI are typical.EcoRI cleavage can also be written using the shorthand notation for DNA structure. The shorthandnotation used in the figure below is explained in Appendix II.11Restriction EnzymesIn the EcoRI example, the cuts were made to the left of the axis of rotation, producing 5’overhangs. Other enzymes, however, cleave to the left of the axis, producing 3’ overhangs, or on theaxis, producing “blunt” ends. Three examples, HindIII, KpnI, and SmaI are shown below:Clearly, ends created by HindIII and KpnI are complementary and can permit ligation. But blunt endssuch as formed by SmaI, under the right circumstances, can also be ligated. Moreover, it is possible totreat the cut ends with a variety of secondary enzymes to provide lots of flexibility with respect tosubsequent cloning steps. The type of enzyme used and the type of modification possible depends on thenature of the cut relative to the axis, and on whether the 3’OH end is recessed or over-hanging.Isoschizomers and Compatible EnzymesOccasionally, several restriction endonucleases may recognize the exact same sequence. The firstenzyme discovered to recognize a particular sequence is known as theprototype.When additionalenzymes are discovered that recognize the same sequence, they are calledisoschizomers.If the newenzyme recognizes the same sequence but cleaves it differently, then it is known as aneoschizomer.Inthe case of the sequence CCCGGG:EnzymeXmaICfr9ISmaISequenceCCCGGGCCCGGGCCCGGGNomenclatureprototypeisoschizomerneoschizomerSometimes there is overlap in the recognition sites for different enzymes. For example, the site forBamHI, GGATCC shares the middle four bases with the site for BglII, AGATCT, and the entiretetrameric sequence of Sau3A,GATC. It is thus possible to ligate one DNA cut with BamHI toanother DNA cut with BglII. Such enzymes are said to becompatible.Since the outside bases for eachenzyme are different, the result would be a hybrid sequence that cannot be cut by either BamHI or BglII.The central GATC, however, would be regenerated and could be cut by Sau 3A. Similarly, DNA cutwith Sau3A could be ligated to DNA cut with either BamHI or BglII. Since Sau3A recognizes a four-base site, the adjacent bases are random. Thus there is a one in four probability that the fusion of aSau3A site to a BamHI site will regenerate the BamHI sequence. The probability of finding a tetrameric12Restriction Enzymessequence such as Sau3A in any random piece of DNA is much greater than finding a hexamericsequence. Thus an enzyme like Sau3A will cut DNA much more frequently than will BamHI.Enzyme StructureCleavage SiteRecognition SequenceReactionRequirementsATPS-AdenosylMethionineType Iup to 1000 basepairs awayasymmetric & discontinuousEcoK = AAC(N)6GTGCEcoB = TGA(N)8TGCTcontinuous & symmetricEcoRI = GAATTCType IIwithin recognitionsequencecontinuous & asymmetricBbvCI = CCTCAGCdiscontinuous & symmetricBglI = GCCNNNNNCCGup to 20 bp away on3’ sidecontinuous & asymmetricFokI = GGATG(N)9CCTAC(N)13continuous & symmetricAcuI = CTGAAG(N)16GACTTC(N)14Type IIGoutside sequencediscontinuous & symmetricBcgI =10(N)CGA(N)6TGC(N)1212Mg2+Type IISMg2+Mg2+(N)GCT(N)6ACG(N)10Type III24 – 26 bp away on3’ sidecontinuous & asymmetricEcoP15I = CAGCAG(N)25GTCGTC(N)27Stimulated byATPS-AdenosylMethionineType IVUsing Restriction EnzymesEach restriction enzyme has its own optimal set of reaction conditions, which can be found on theinformation sheet provided by the supplier. A number of companies produce high-quality restrictionenzymes. The most important reaction condition variables are the ionic strength (i.e. salt concentration)of the reaction buffer and the temperature of digestion. Of the two, reaction temperature is often mostcritical. The ionic strength is less stringent and it is therefore permissible to broadly categorizerestriction enzymes as requiring high, medium, or low salt. On page 16 is a list of formulas for thesebuffers. Page 17 gives the temperature and buffer requirements for some of the common restrictionenzymes as well as their recognition sequences and sites of cutting. There are a few exceptions to thisgeneral categorization. We will discuss the conditions for using these enzymes as they come up.13

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