Enzymes Used for Generation of a Recombinant DNA

The following points highlight the eight main enzymes used for generation of a recombinant DNA. The enzymes are: 1. Restriction Endonucleases 2. Alkaline Phosphatases 3. Reverse Transcriptase 4. Polynucleotide Kinase 5. DNA Ligase 6. Nucleases 7. Terminal Deoxynucleotide Transferases (TDNT) 8. DNA Polymerase.

Enzyme Type # 1.

Restriction Endonucleases:

A commonly used tool in molecular biology is restriction endonucleases. Restriction endonu­cleases, otherwise known as restriction en­zymes, are molecular scissors that can cut double-stranded DNA at a specific base-pair sequence.

Each type of restriction enzyme re­cognizes a characteristic sequence of nucle­otides that is known as its recognition site. Researchers can use these enzymes to cut DNA in a predictable and precise manner.

Discovery of Restriction Endonucleases:

Scientific discoveries often have their origin in seemingly unimportant observation that receive little attention by researchers before their general significance is appreciated. In case of genetic engineering, the original obser­vation was that bacteria use enzymes to defend themselves against viruses.

Most orga­nisms eventually evolve means of defending themselves from predators and parasites, and bacteria are no exception. Among the natural enemies of bacteria are bacteriophages, viruses that infect bacteria and multiply within them.

At some point, they cause the bacterial cells to burst, releasing thousands more viruses. Through natural selection, some types of bac­teria have acquired powerful weapons against these viruses; they contain enzymes called re­striction endonucleases that fragment the vi­ral DNA as soon as it enters the bacterial cell.

Many restriction endonucleases recognize spe­cific nucleotide sequences in a DNA strand, bind to the DNA at those sequences, and cleave the DNA at a particular place within the re­cognition sequence. Why don’t restriction en­donucleases cleave the bacterial cells’ own DNA as well as that of the viruses?

The ans­wer to this question is that bacteria modify their own DNA, using other enzymes known as methylases to add methyl (—CH₃) groups to some of the nucleotides in the bacterial DNA. When nucleotides within a restriction endonuclease’s recognition sequence have been methylated, the endonuclease cannot bind to that sequence.

Consequently, the bacterial DNA is protected from being degraded at that site. Viral DNA, on the other hand, has not been methylated and, therefore, is not pro­tected from enzymatic cleavage.

Types of Restriction Endonucleases:

All restriction enzymes fall into one of three classes, basing upon their molecular structure and need for specific co-factors.

I. Class I Endonucleases:

These have a mo­lecular weight around 300,000 Daltons, Eire composed of non-identical subunits, and require Mg²⁺, ATP (adenosine triphos­phate), and SAM (S-adenosylmethionine) as cofactors for activity. Not used in RDT experiments.

II. Class II Endonucleases:

These are much smaller, with molecular weights in the range of 20,000 to 100,000 Daltons. They have identical sub-units and require only Mg²⁺ as a cofactor (Nathans and Smith, 1975). Only class II endonucleases are used in RDT experiments due to their site spe­cific cleavage action.

III. Class III Endonucleases:

These are large molecules, with a molecular weight of around 200,000 Daltons, composed of non- identical sub-units. These enzymes differ from enzymes of other two classes in that they require both Mg²⁺ and ATP but not SAM as co-factors. Class III endonucleases are the rarest of three types. Not used in RDT experiments.

Nomenclature:

As a large number of restriction enzymes have been discovered, a uniform nomenclature sys­tem is adopted to avoid confusion.

This nomenclature was first proposed by Smith and Nattens in 1973.

1. The first letter of restriction enzymes (RE) should be from first letter of the species name of organism from which the enzyme is isolated.

The letter should be written in capitals and italics, e.g., RE from E. coli will have E as starting letter.

2. The second and third letters of RE should be from the first and second letters of ge­nus name of the organism. The letter should be written in lower case and should be in italics, e.g., RE from E coli will have Eco as starting words.

3. If the RE is isolated from particular strain of an organism, then that should be writ­ten as fourth letter. It should be in capi­tals and not in italics. For example, RE from E. coli R strain will be written as Eco R.

4. If the RE isolated is the first of its kind from that particular organism, then the number I should be given. If already two REs are isolated, then number III should be given for new restriction enzymes. The number should be written in roman, e.g., the first E. coli RE should be written as Eco RI whereas the third restriction en­zyme isolated from E. coli R strains should be written as Eco RIII.

Recognition Sequences:

The recognition sequences for class II endo­nucleases form palindromes with rotational symmetry. In a palindrome, the base se­quence in the second half of a DNA strand is the mirror image of sequence in its first half (Fig. 4.2). But in a palindrome with rotational symmetry, the base sequence in the first half of one strand of a DNA double helix is the mir­ror image of second half of its complementary strand (Fig 4.3).

Thus in such palindromes, the base sequence in both the strands of a DNA duplex reads the same when read from the same end (either 5′ or 3′) of both the strands.

Mechanism of Action of Restriction Endo­nucleases:

EcoRI can be taken as an example of class II endonucleases and its works can be seen. When the restriction endonuclease encounters its respective restriction site sequence (5′ GAATTC 3′), it cleaves each backbone between the G and the closest A base residues.

Once the cuts have been made, the resulting frag­ments are held together only by relatively weak hydrogen bonds that hold the comple­mentary bases to each other. The weakness of these bonds allows the DNA fragments to sepa­rate from each other. Each resulting fragment has a protruding 5′ end composed of unpaired bases (Fig. 4.4).

Other enzymes also create cuts in the DNA backbone in the same manner, which results in protruding 3′ ends. Protru­ding ends—both 3′ and 5’—are sometimes called ‘sticky ends’ because they tend to bond with complementary sequences of bases.

In other words, if an unpaired length of bases (5′ A A T T 3′) encounters another unpaired length with the sequence (3′ T T A A 5′) they will bond to each other—they are ‘sticky’ for each other. Ligase enzymes are then used to join the phosphate backbones of two molecules.

The cellular origin, or even the species origin, of the sticky ends does not affect their sticki­ness. Any pair of complementary sequences will tend to bond, even if one of the sequences comes from a length of human DNA, and the other comes from a length of bacterial DNA.

In fact, it is this quality of stickiness that al­lows the production of recombinant DNA mol­ecules (molecules which are composed of DNA from different sources and have given birth to a powerful technology and industry) the ge­netic engineering or the recombinant DNA.

Examples of some other restriction en­zymes, their mode of cutting and generation of 5′ overhangs and 3′ overhangs, are illus­trated in (Fig. 4.5). Sticky ends (also called cohesive ends or overhanging heads) are use­ful for DNA cloning because complementary sequences anneal and can be ligated directly by DNA ligase.

If two different DNA samples cleaved with the same type of restriction en­zymes are mixed together in the presence of DNA ligase, a recombinant DNA molecule can be generated. This is possible because of the presence of same type of sticky ends.

The complementary sequences of sticky ends from the unrelated DNA samples will anneal to­gether and are finally joined by the DNA li­gase enzyme to form the recombinant DNA molecule.

Some restriction enzymes, on the other hand, cut both the strands of a DNA molecule at the same site so that the resulting termini or ends have blunt or flush ends (Fig. 4.6) in which the two strands end at the same point. The blunt ends also can effectively be utilized as recombinants followed by some end point modifications.

As the list of restriction enzymes grew and their recognition sequences were identified, it was found in some cases that more than one enzyme could recognize the same sequence. RJ Roberts conferred the term isoschizomer (same cutter) on restriction enzymes that recognized the same DNA sequence.

Star Activity:

Sometimes restriction enzymes recognize and cleave the DNA strand at the recognition site with asymmetrical palindromic sequence; for example, Bam HI cuts at the sequence GA TCC, but under extreme conditions, as in low ionic strength it will cleave in any of the following sequences NGA TCC, GPOA TCC, GGNTCC. Such an activity of the restriction endonucleases is called star activity.

Difficulties Associated with Restriction Digestion:

There are certain limitations for restriction endonucleases.

Those are as follows:

1. Different enzymes can generate the same ends. For example, Sau3A1 and BamHI produce GATC−. If these two enzymes are used then they will form the same ends which will not ligate later.

2. Restriction endonuclease preparation should be free from nucleases, otherwise the ends produced by theses enzymes can be degraded by exonucleases.

3. Most restriction endonucleases are very stable when stored at -20°C in the recom­mended storage buffer. Exposure to tem­perature above -20°C can decrease the ac­tivity of these enzymes.

4. The specificity of some restriction endonu­clease is affected by the type of buffer used.

5. Secondary structures in DNA often inter­fere with recognition or cleavage by endo­nuclease.

Works of Reference for Restriction Diges­tion:

Nowadays we get a complete online database for every available restriction endonuclease. REBASE provides a current review of restriction enzymes, whether and where they can be obtained, a list of publications concern­ing the enzymes, and much more. At this site, you can search DNA sequences for their open reading frames and cleavage sites can be searched out.

Uses of Restriction Endonucleases:

Restriction enzymes have been used for se­quence analysis, cloning and amplifying DNA. DNA from animal viruses bacteriophages con­tains 5,000 to 50,000 base pairs. It is impor­tant to know the primary structure of DNA, i.e., the sequence of bases, for decoding the in­formation stored in genes, for understanding gene structure and regulation at molecular level.

The discovery of restriction enzymes was a major breakthrough in sequence analysis of DNA. By using combinations of different re­striction enzymes it is possible to hydrolyse large DNA molecules into fragments less than 300 base pairs in length.

These fragments can then be used for sequence analysis and are arranged into a physical map of the chromo­some. This is a slow and laborious process. The mapping of entire 5,000 base pair DNA of the virus SV40 into some 100 fragments has taken several years. Complete sequence analysis of the fragments would take much longer.

The DNA fragments produced by restriction endo­nucleases can covalently be linked in vitro to linear plasmid DNA or to lambda phage DNA. The recombinant DNA species produced can be inserted into E. coli by transformation.

Each transformed cell can then be grown as a sepa­rate clone. By this method a complex genome can be broken down into thousands or millions of pieces, and each piece is isolated to form a separate clone.

Enzyme Type # 2.

Alkaline Phosphatases:

Alkaline phosphatase is a glycoprotein with two identical subunits. The cohesive ends of broken plasmids, instead of joining with for­eign DNA, join the cohesive end of the same DNA molecules and get re-circularized. To over­come this problem the restricted plasmid is treated with an enzyme, alkaline phosphatase, that digests the terminal phosphoryl group.

The restriction fragments of the foreign DNA to be cloned are not treated with alkaline phosphatase.

Therefore, the 5′ end of foreign DNA fragment can covalently join to 3′ end of the plasmid. The recombinant DNA thus obtained has a nick with 3′ and 5′ P hydroxy ends. Li­gase will only join 3′ and 5′ ends of recombi­nant DNA together if the 5′ end is phosphorylated.

Thus, alkaline phosphatase and ligase prevent re-circularization of the vector and in­crease the frequency of production of recombi­nant DNA molecules. The nicks between two 3′ ends fragment and vector DNA are repaired inside the bacterial cells during the transfor­mation.

Enzyme Type # 3.

Reverse Transcriptase:

Many times we do not get our gene of interest rather its mRNA. In this case reverse trans­criptase enzyme can be used to prepare a double stranded DNA (our gene of interest) from the available single-stranded mRNA (template) by a process called reverse tran­scription.

Reverse transcriptase enzyme is also called RNA dependent DNA polymerase. These enzymes are present in most of the RNA tu­mour viruses and retroviruses.

Enzyme Type # 4.

Polynucleotide Kinase:

Kinase is the group of enzyme, which adds a free pyrophosphate (PO₄) to a wide variety of substrates like proteins, DNA and RNA. It uses ATP as cofactor and adds a phosphate by breaking the ATP into ADP and pyrophos­phate. It is widely used in molecular biology and genetic engineering to add radio-labelled phosphates. In RDT experiments mostly T₄ polynucleotide kinase is used.

Enzyme Type # 5.

DNA Ligase:

Recombinant DNA experiments require the joining of two different DNA segments or frag­ments in vitro. The ends generated by some RE will be either cohesive (sticky) or blunt. The cohesive ends will anneal (join) themselves by forming hydrogen bonds. But the segments annealed thus are weak and do not withstand experimental conditions.

To get a stable joining, the DNA should be joined by using an en­zyme called ligase. In the case of blunt ends we use linker or adaptors for successful liga­tion.

There are two types of DNA ligases:

(a) T4 DNA Ligase:

Naturally coded by T4 bacteriophage. The catalytic activity of the enzyme requires the presence of ATP as cofactor and Mg⁺⁺. This is predominantly used in RDT experiments.

(b) NAD⁺ dependent DNA Ligase:

Natu­rally found in E. coli. Uses NAD+ as a co-factor and only found in bacteria.

Mechanism of Action:

The cofactor is first spited (ATP→ AMP + 2Pi) and then AMP binds to the enzyme to form the enzyme-AMP complex. This complex then binds to the nick or break (with 5′ −PO₄ and 3′ −OH) and makes a covalent bond in the phosphodiester chain. The ligase reaction is carried out at 4°C for better results.

Enzyme Type # 6.

Nucleases:

Nucleases are group of enzymes which cleave or cut the genetic material (DNA or RNA). These enzymes are further classified into two types based upon the substrate on which they act. Nucleases which act on or cut the DNA are classified as DNases, whereas those which act on the RNA are called RNases.

DNases are further classified into two types based upon the position where they act. DNases that act on the ends or terminal regions of DNA are called exonucleases and those that act at a non­specific region in the centre of the DNA are called endonucleases.

Exonucleases require a DNA strand with at least two 5′ and 3′ ends. They cannot act on DNA which is circular. Endonucleases can act on circular DNA and do not require any free DNA ends (i.e., 5 or 3 end). Exonucleases release nucleotides, whereas endonucleases release short segments of DNA. The frequently used nucleases in the experiments of RDT are Exonuclease III and bacteriophage exonuclease.

Enzyme Type # 7.

Terminal Deoxynucleotide Transferases (TDNT):

This is a polymerase which adds nucleotides at 3′-OH end (like Klenow fragment) but does not require any complementary sequence and does not copy any DNA sequence (unlike Klenow fragment). Terminal deoxynucleotide transferase (TDNT) adds nucleotide whatever comes into its active site and it does not show any preference for any nucleotide.

Enzyme Type # 8.

DNA Polymerase:

These are mostly used when we are carrying out the cloning of the recombinant DNA in the prokaryotic host cells like E. coli. Then we fill the gaps in duplexes by stepwise addition of nucleotide to 3′ ends.