Restriction Endonucleases Student Copy

RESTRICTION ENDONUCLEASES AND LIGATION 4.2 Enzymes for cutting DNA—restriction endonucleases  Gene cloning requires that DNA molecules be cut in a very precise and reproducible fashion. This is illustrated by the way in which the vector is cut during construction of a recombina...

RESTRICTION ENDONUCLEASES AND LIGATION 4.2 Enzymes for cutting DNA—restriction endonucleases  Gene cloning requires that DNA molecules be cut in a very precise and reproducible fashion. This is illustrated by the way in which the vector is cut during construction of a recombinant DNA molecule (Figure 4.7a).  Each vector molecule must be cleaved at a single position, to open up the circle so that new DNA can be inserted: a molecule that is cut more than once will be broken into two or more separate fragments and will be of no use as a cloning vector. Furthermore, each vector molecule must be cut at exactly the same position on the circle—as will become apparent in later chapters, random cleavage is not satisfactory. It should be clear that a very special type of nuclease is needed to carry out this manipulation. Often it is also necessary to cleave the DNA that is to be cloned (Figure 4.7b). There are two reasons for this. First, if the aim is to clone a single gene, which may consist of only 2 or 3 kb of DNA, then that gene will have to be cut out of the large (often greater than 80 kb) DNA molecules produced by skilfull use of the preparative techniques described in Chapter 3. Second, large DNA molecules may have to be broken down simply to produce fragments small enough to be carried by the vector. Most cloning vectors exhibit a preference for DNA fragments that fall into a particular size range: most plasmid-based vectors, for example, are very inefficient at cloning DNA molecules more than 8 kb in length. Purified restriction endonucleases allow the molecular biologist to cut DNA molecules in the precise, reproducible manner required for gene cloning. The discovery of these enzymes, which led to Nobel Prizes for W. Arber, H. Smith, and D. Nathans in 1978, was one of the key breakthroughs in the development of genetic engineering. CLASSIFICATION OF RESTRICTION ENDONUCLEASE Three different classes of restriction endonuclease are recognized, each distinguished by a slightly different mode of action. Types I and III are rather complex and have only a limited role in genetic engineering. Type II restriction endonucleases, on the other hand, are the cutting enzymes that are so important in gene cloning. Type II restriction endonucleases cut DNA at specific nucleotide sequences The central feature of type II restriction endonucleases (which will be referred to simply as “restriction endonucleases” from now on) is that each enzyme has a specific recognition sequence at which it cuts a DNA molecule. A particular enzyme cleaves DNA at the recognition sequence and nowhere else. For example, the restriction endonuclease called PvuI (isolated from Proteus vulgaris) cuts DNA only at the hexanucleotide CGATCG. In contrast, a second enzyme from the same bacterium, called PvuII, cuts at a different hexanucleotide, in this case CAGCTG. Many restriction endonucleases recognize hexanucleotide target sites, but others cut at four, five, eight, or even longer nucleotide sequences. Sau3A (from Staphylococcus aureus strain 3A) recognizes GATC, and AluI (Arthrobacter luteus) cuts at AGCT. There are also examples of restriction endonucleases with degenerate recognition sequences, meaning that they cut DNA at any one of a family of related sites. HinfI (Haemophilus influenzae strain Rf), for instance, recognizes GANTC, so cuts at GAATC, GATTC, GAGTC, and GACTC. Blunt ends and sticky ends The exact nature of the cut produced by a restriction endonuclease is of considerable importance in the design of a gene cloning experiment. Many restriction endonucleases make a simple double-stranded cut in the middle of the recognition sequence (Figure 4.9a), resulting in a blunt end or flush end. PvuII and AluI are examples of blunt end cutters. Other restriction endonucleases cut DNA in a slightly different way. With these enzymes the two DNA strands are not cut at exactly the same position. Instead the cleavage is staggered, usually by two or four nucleotides, so that the resulting DNA fragments have short single-stranded overhangs at each end (Figure 4.9b). These are called sticky or cohesive ends, as base pairing between them can stick the DNA molecule back together again (recall that sticky ends were encountered on p. 20 during the description of e phage replication). One important feature of sticky end enzymes is that restriction endonucleases with different recognition sequences may produce the same sticky ends. BamHI (recognition sequence GGATCC) and BglII (AGATCT) are examples—both produce GATC sticky ends (Figure 4.9c). The same sticky end is also produced by Sau3A, which recognizes only the tetranucleotide GATC. Fragments of DNA produced by cleavage with either of these enzymes can be joined to each other, as each fragment carries a complementary sticky end. Ligation – joining DNA molecules together The final step in construction of a recombinant DNA molecule is the joining together of the vector molecule and the DNA to be cloned. This process is referred to as ligation, and the enzyme that catalyzes the reaction is called DNA ligase. Sticky ends increase the efficiency of ligation  Ligation of complementary sticky ends is much more efficient. This is because compatible sticky ends can base pair with one another by hydrogen bonding (Figure 4.20b), forming a relatively stable structure for the enzyme to work on.  If the phosphodiester bonds are not synthesized fairly quickly then the sticky ends fall apart again.  These transient, base-paired structures do, however, increase the efficiency of ligation by increasing the length of time the ends are in contact with one another. Putting sticky ends onto a blunt-ended molecule There are three methods can be used to put the correct sticky ends onto the blunt DNA fragments. 1) Linkers  The first of these methods involves the use of linkers. These are short pieces of doublestranded DNA, of known nucleotide sequence, that are synthesized in the test tube.  It is blunt-ended, but contains a restriction site, BamHI in the example shown. DNA ligase can attach linkers to the ends of larger blunt ended DNA molecules.  More than one linker will attach to each end of the DNA molecule, producing the chain structure shown in Figure 4.21b.  Digestion with BamHI cleaves the chains at the recognition sequences, producing a large number of cleaved linkers and the original DNA fragment, now carrying BamHI sticky ends.   This modified fragment is ready for ligation into a cloning vector restricted with BamHI. Disadvantages of using linkers Consider what would happen if the blunt-ended molecule shown in Figure 4.21b contained one or more BamHI recognition sequences. If this was the case, the restriction step needed to cleave the linkers and produce the sticky ends would also cleave the blunt-ended molecule (Figure 4.22). The resulting fragments will have the correct sticky ends, but that is no consolation if the gene contained in the blunt-ended fragment has now been broken into pieces. 2) Adaptors  The second method of attaching sticky ends to a blunt-ended molecule is designed to avoid the problem encountered by linkers.  Adaptors, like linkers, are short synthetic oligonucleotides. But unlike linkers, an adaptor is synthesized so that it already has one sticky end (Figure 4.23a).  The idea is of course to ligate the blunt end of the adaptor to the blunt ends of the DNA fragment, to produce a new molecule with sticky ends. Disadvantages of adaptors  This may appear to be a simple method but in practice a new problem arises. The sticky ends of individual adaptor molecules could base pair with each other to form dimers (Figure 4.23b), so that the new DNA molecule is still blunt-ended (Figure 4.23c).  The sticky ends could be recreated by digestion with a restriction endonuclease, but that would defeat the purpose of using adaptors in the first place. How to overcome the disadvantages of using adaptors  The answer to the problem lies in the precise chemical structure of the ends of the adaptor molecule.  Normally the two ends of typical double stranded DNA are chemically distinct, a fact that is clear from a careful examination of the polymeric structure of DNA (Figure 4.24a). One end, referred to as the 5’terminus, carries a phosphate group (5-P); the other, the 3’terminus, has a hydroxyl group (3-OH). In the double helix the two strands are antiparallel (Figure 4.24b), so each end of a double-stranded molecule consists of one 5-P terminus and one 3-OH terminus. Ligation takes place between the 5′-P and 3-OH ends (Figure 4.24c).  Adaptor molecules are synthesized so that the blunt end is the same as “natural” DNA, but the sticky end is different. The 3-OH terminus of the sticky end is the same as usual, but the 5-P terminus is modified: it lacks the phosphate group, and is in fact a 5-OH terminus (Figure 4.25a). DNA ligase is unable to form a phosphodiester bridge between 5-OH and 3-OH ends.  The result is that, although base pairing is always occurring between the sticky ends of adaptor molecules, the association is never stabilized by ligation (Figure 4.25b).  Adaptors can therefore be ligated to a blunt-ended DNA molecule but not to themselves.  After the adaptors have been attached, the abnormal 5-OH terminus is converted to the natural 5-P form by treatment with the enzyme polynucleotide kinase , producing a sticky-ended fragment that can be inserted into an appropriate vector. 3) Producing sticky ends by homopolymer tailing  The technique of homopolymer tailing offers a radically different approach to the production of sticky ends on a blunt-ended DNA molecule.  A homopolymer is simply a polymer in which all the subunits are the same.  A DNA strand made up entirely of, say, deoxyguanosine is an example of a homopolymer, and is referred to as polydeoxyguanosine or poly(dG).  Tailing involves using the enzyme terminal deoxynucleotidyl transferase to add a series of nucleotides onto the 3-OH termini of a double-stranded DNA molecule.  If this reaction is carried out in the presence of just one deoxyribonucleotide, a homopolymer tail is produced (Figure 4.26a).  Of course, to be able to ligate together two tailed molecules, the homopolymers must be complementary. Frequently polydeoxycytosine (poly(dC)) tails are attached to the vector and poly(dG) to the DNA to be cloned.  Base pairing between the two occurs when the DNA molecules are mixed (Figure 4.26b).  In practice, the poly(dG) and poly(dC) tails are not usually exactly the same length, and the base-paired recombinant molecules that result have nicks as well as discontinuities (Figure 4.26c). Repair is therefore a two-step process, using Klenow polymerase to fill in the nicks followed by DNA ligase to synthesize the final phosphodiester bonds.  This repair reaction does not always have to be performed in the test tube. If the complementary homopolymer tails are longer than about 20 nucleotides, then quite stable base-paired associations are formed.  A recombinant DNA molecule, held together by base pairing although not completely ligated, is often stable enough to be introduced into the host cell in the next stage of  the cloning experiment (see Figure 1.1). Once inside the host, the cell’s own DNA polymerase and DNA ligase repair the recombinant  DNA molecule, completing the construction begun in the test tube. Blunt end ligation with a DNA topoisomerase  A more sophisticated, but easier and generally more efficient way of carrying out blunt end ligation, is to use a special type of enzyme called a DNA topoisomerase.  In the cell,DNA topoisomerases are involved in processes that require turns of the double helix to be removed or added to a double-stranded DNA molecule. Turns are removed during DNA replication in order to unwind the helix and enable each polynucleotide to be replicated, and are added to newly synthesized circular molecules to introduce supercoiling.  DNA topoisomerases are able to separate the two strands of a DNA molecule without actually rotating the double helix. They achieve this feat by causing transient single- or double-stranded breakages in the DNA backbone (Figure 4.27).  DNA topoisomerases therefore have both nuclease and ligase activities. To carry out blunt end ligation with a topoisomerase, a special type of cloning vector is needed. This is a plasmid that has been linearized by the nuclease activity of the DNA topoisomerase enzyme from vaccinia virus. The vaccinia topoisomerase cuts DNA at the sequence CCCTT, which is present just once in the plasmid. After cutting the plasmid, topoisomerase enzymes remain covalently bound to the resulting blunt ends. The reaction can be stopped at this point, enabling the vector to be stored until it is needed. Cleavage by the topoisomerase results in 5-OH and 3-P termini (Figure 4.28a). If the blunt- ended molecules to be cloned have been produced from a larger molecule by cutting with a restriction enzyme, then they will have 5-P and 3-OH ends. Before mixing these molecules with the vector, their terminal phosphates must be removed to give 5-OH ends that can ligate to the 3-P termini of the vector. The molecules are therefore treated with alkaline phosphatase (Figure 4.28b). Adding the phosphatased molecules to the vector reactivates the bound topoisomerases, which proceed to the ligation phase of their reaction. Ligation occurs between the 3-P ends of the vectors and the 5-OH ends of the phosphatased molecules. The blunt-ended molecules therefore become inserted into the vectors. Only one strand is ligated at each junction point (Figure 4.28c), but this is not a problem because the discontinuities will be repaired by cellular enzymes after the recombinant molecules have been introduced into the host bacteria.