Enzymes-Part II.pdf

M2- Part II Tools Enzymes A K A Mandal A K A Mandal 1  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...

M2- Part II Tools Enzymes A K A Mandal A K A Mandal 1  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 Several types of ligases are used A K A Mandal 2  Types of ligases E. coli DNA ligase Encoded by the lig gene DNA ligase in E. coli, as well as most prokaryotes, uses energy gained by cleaving nicotinamide adenine dinucleotide (NAD) to create the phosphodiester bond It does not ligate blunt-ended DNA except under conditions of molecular crowding with polyethylene glycol, and cannot join RNA to DNA efficiently A K A Mandal 3  Types of ligases T4 DNA ligase The DNA ligase from bacteriophage T4 is the ligase most- commonly used in the laboratory It can ligate cohesive ends of DNA, oligonucleotides, as well as RNA and RNA-DNA hybrids, but not single-stranded nucleic acids. It can also ligate blunt-ended DNA with much greater efficiency than E. coli DNA ligase. Unlike E. coli DNA ligase, T4 DNA ligase cannot utilize NAD and it has an absolute requirement for ATP as a cofactor Applications – Joining blunt-ended double-stranded DNA. – Joining cohesive-ended double-stranded DNA – Sealing nicks on a DNA or RNA strand annealed to a DNA or RNA complementary strand A K A Mandal 4  Types of ligases Mammalian ligases In mammals, there are four specific types of ligase DNA ligase I: ligates the nascent DNA of the lagging strand after the Ribonuclease H has removed the RNA primer from the Okazaki fragments DNA ligase III: complexes with DNA repair protein XRCC1 to aid in sealing DNA during the process of nucleotide excision repair and recombinant fragments DNA ligase IV: complexes with XRCC4. It catalyzes the final step in the non-homologous end joining DNA double-strand break repair pathway DNA ligase from eukaryotes and some microbes uses adenosine triphosphate (ATP) rather than NAD A K A Mandal 5  Types of ligases Thermostable ligases Ligases from various thermophilic bacteria have been cloned and sequenced, and are available commercially for use in ligase amplification reaction because of their thermostable properties(e.g. Taq DNA ligase) A K A Mandal 6  The mode of action of DNA ligase All living cells produce DNA ligases, but the enzyme used in genetic engineering is usually purified from E. coli bacteria that have been infected with T4 phage Within the cell the enzyme carries out the very important function of repairing any discontinuities that may arise in one of the strands of a double-stranded molecule A discontinuity is quite simply a position where a phosphodiester bond between adjacent nucleotides is missing (contrast this with a nick, where one or more nucleotides are absent) Although discontinuities may arise by chance breakage of the cell’s DNA molecules, they are also a natural result of processes such as DNA replication and recombination Ligases therefore play several vital roles in the cell A K A Mandal 7  In the test tube, purified DNA ligases, as well as repairing single-strand discontinuities, can also join together individual DNA molecules or the two ends of the same molecule The chemical reaction involved in ligating two molecules is exactly the same as discontinuity repair, except that two phosphodiester bonds must be made, one for each strand A K A Mandal 8  Bacteriophage T4 DNA ligase is the ligase most commonly used in the construction of recombinant DNA molecules for molecular biology applications. It is able to ligate DNA fragments having either complementary cohesive or blunt ends, and has an absolute requirement for ATP as a cofactor; it cannot use NAD E. coli DNA ligase (which, like most prokaryotic DNA ligases uses NAD as a cofactor instead of ATP) can sometimes be used in place of T4 DNA ligase for ligation of single-strand breaks or joining of DNA molecules with cohesive termini. However, unlike T4 DNA ligase, the E. coli enzyme does not show blunt- ended ligation activity, except under conditions of molecular crowding withA K APEG Mandal 8000 9  For this reason T4 DNAligase has become more widely used in DNA manipulations For intermolecular ligations it is important that at least one of the DNA molecules possesses a 5′- phosphate at either end of the dsDNA in order to form a phosphodiester bond A K A Mandal 10 Ligation: the final step in construction of a recombinant DNA molecule A K A Mandal 11 A K A Mandal 12  Sticky ends increase the efficiency of ligation The ligation reaction in the figure below shows two blunt-ended fragments being joined together. Although this reaction can be carried out in the test tube, it is not very efficient. This is because the ligase is unable to “catch hold” of the molecule to be ligated, and has to wait for chance associations to bring the ends together. If possible, blunt end ligation should be performed at high DNA concentrations, to increase the chances of the ends of the molecules coming together in the correct way A K A Mandal 13  In contrast, ligation of complementary sticky ends is much more efficient. This is because compatible sticky ends can base pair with one another by hydrogen bonding , 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. A K A Mandal 14 The different joining reactions catalysed by DNA ligase: (a) ligation of blunt-ended molecules; (b) ligation of sticky-ended molecules A K A Mandal 15  Putting sticky ends onto a blunt- ended molecule Compatible sticky ends are desirable on the DNA molecules to be ligated together in a gene cloning experiment Often these sticky ends can be provided by digesting both the vector and the DNA to be cloned with the same restriction endonuclease, or with different enzymes that produce the same sticky end, but it is not always possible to do this A common situation is where the vector molecule has sticky ends, but the DNA fragments to be cloned are blunt-ended. Under these circumstances one of three methods can be used to put the correct sticky ends onto the DNA fragments A K A Mandal 16 Putting sticky ends onto a blunt- ended molecule Linker Adaptor Homopolymer tailing A K A Mandal 17 Linkers The first of these methods involves the use of linkers. These are short pieces of double stranded 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. Although a blunt end ligation, this particular reaction can be performed very efficiently because synthetic oligonucleotides, such as linkers, can be made in very large amounts and added into the ligation mixture at a high concentration A K A Mandal 18  More than one linker will attach to each end of the DNA molecule, producing the chain structure However, 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 A K A Mandal 19 Linkers and their use: (a) the structure of a typical linker; (b) the attachment of linkers to a blunt- ended molecule A K A Mandal 20  One potential drawback with the use of linkers – Consider what would happen if the blunt-ended molecule 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 – 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 A K A Mandal 21 A possible problem with the use of linkers A K A Mandal 22 Adaptors The second method of attaching sticky ends to a blunt-ended molecule is designed to avoid this problem. Adaptors, like linkers, are short synthetic oligonucleotides. But unlike linkers, an adaptor is synthesized so that it already has one sticky end 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 A K A Mandal 23 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, so that the new DNA molecule is still blunt-ended – 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 A K A Mandal 24  The answer to the problem lies in the precise chemical structure of the ends of the adaptor molecule Normally the two ends of a polynucleotide strand are chemically distinct 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, 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 A K A Mandal 25  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 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 A K A Mandal 26  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 A K A Mandal 27 Adaptors and the potential problem with their use. (a) A typical adaptor. (b) Two adaptors could ligate to one another to produce a molecule similar to a linker, so that (c) after ligation of adaptors a blunt-ended molecule is still blunt-ended and the restriction step is still needed A K A Mandal 28 The distinction between the 5′ and 3′ termini of a polynucleotid e. A K A Mandal 29 The use of adaptors: (a) the actual structure of an adaptor, showing the modified 5′-OH terminus; (b) conversion of blunt ends to sticky ends through the attachment of adaptors. A K A Mandal 30 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) A K A Mandal 31  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 A K A Mandal 32  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 A K A Mandal 33  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 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 34  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. 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 35 Homopolymer tailing: (a) synthesis of a homopolymer tail; (b) construction of a recombinant DNA molecule from a tailed vector plus tailed insert DNA; (c) repair of the recombinant DNA molecule. A K A Mandal 36  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 super coiling A K A Mandal 37  Blunt end ligation with a DNA topoisomerase 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. DNA topoisomerases therefore have both nuclease and ligase activities A K A Mandal 38  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. A K A Mandal 39  Cleavage by the topoisomerase results in 5′-OH and 3′-P termini 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 A K A Mandal 40  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 poin, 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 A K A Mandal 41 The mode of action of a Type 1 DNA topoisomerase, which removes or adds turns to a double helix by making a transient break in one of the strands. A K A Mandal 42 Blunt end ligation with a DNA topoisomerase. (a) Cleavage of the vector with the topoisomerase leaves blunt ends with 5′-OH and 3′-P termini. (b) The molecule to be cloned must therefore be treated with alkaline phosphatase to convert its 5′-P ends into 5′-OH termini. (c) The topoisomerase ligates the 3′-P and 5′-OH ends, creating a double-stranded molecule with two discontinuities, which are repaired by cellular enzymes after introduction into the host bacteria. A K A Mandal 43  Special methods are often required if DNA produced by PCR amplification is to be cloned Many of the strategies for cloning DNA fragments do not work well with PCR products. The reason for this is that the polymerases used in the PCR have a terminal transferase activity For example, the Taq polymerase adds a single 3′ A overhang to each end of the PCR product Thus PCR products cannot be blunt-end-ligated unless the ends are first polished (blunted) A K A Mandal 44  A DNA polymerase like Klenow can be used to fill in the ends. Alternatively, Pfu DNA polymerase can be used to remove extended bases with its 3′ to 5′ exonuclease activity However, even when the PCR fragments are polished, blunt- end-ligating them into a vector still may be very inefficient One solution to this problem is to use T/A cloning In this method, the PCR fragment is ligated to a vector DNA molecule with a single 3′ deoxythymidylate extension A K A Mandal 45  TA Cloning Technology Introduction to TA Cloning TA Cloning is one of the most popular methods of cloning the amplified PCR product using Taq and other polymerases. These polymerases lack 5'-3' proofreading activity and are capable of adding adenosine triphosphate residue to the 3' ends of the double stranded PCR product. Such PCR amplified product can be cloned in a linearized vector with complementary 3' T overhangs. A K A Mandal 46 TA Cloning Functioning TA cloning is brought about by the terminal transferase activity of certain type of DNA polymerase such as the Taq polymerase. This enzyme adds a single, 3'-A overhang to each end of the PCR product. As a result, the PCR product can be directly cloned into a linearized cloning vector that have single base 3'-T overhangs on each end. Such vectors are called T- vectors. The PCR product with A overhang, is mixed with this vector in high proportion. The complementary overhangs of a "T" vector and the PCR product hybridize. The result is a recombinant DNA, the recombination being brought about by DNA ligase. A K A Mandal 47  References Primrose SB and Twyman R M. Principles of Gene manipulation and Genomics, Seventh edition, Blackwell Scientific Publications, 2006. Terence A. Brown. Gene cloning and DNA analysis: an introduction, Wiley-Blackwell, 2006. 49