Paper C14 Unit 2: Molecular cloning- tools and strategies DNA MODIFYING ENZYMES AND THEIR APPLICATIONS
1.DNA ligase 2.Reverse transcriptase 3.Restriction endonuclease 4.Terminal transcriptase 5.Nuclease 6.DNA polymerase 7.Ribonuclease-H 8.Alkaline phosphatase 9.Polynucleotide kinase
1. DNA ligase: •DNA ligase is isolated from E.coli and Bacteriophage commercially and used in recombinant DNA technology. •The enzyme DNA ligase joins the DNA fragments with cloning vector.
2. Reverse transcriptase: •RT is used to synthesize complementary strand (cDNA) from mRNA template. •It is also known as RNA dependent DNA polymerase •It is isolated from retrovirus
3. Restriction endonuclease: •Restriction endonuclease enzyme recognize and cut DNA strand at specific sequence called restriction site. •This enzyme is isolated from wide variety of microorganisms. Endonuclease enzyme degrades foreign genome when enter inside microbial cell but the host cell own DNA is protected from its endonuclease by methylation of bases at restriction site. There are 3 types of restriction endonuclease:
Type I Restriction endonuclease: •It has both methylation and endonuclease activity. •It require ATP to cut the DNA •It cuts DNA about 1000bp away from its restriction site eg. EcoKI
Type II Restriction endonuclease: •It does not require ATP to cut DNA •It cuts DNA at restriction site itself eg. EcoRI, Hind III
Type III Restriction endonuclease: •It requires ATP to cut DNA •It cuts DNA about 25bp away from restriction site. eg. EcoPI
4. Terminal transferase: •It is the enzyme that converts blunt end of DNA fragments into sticky end. •If the restriction enzyme cuts DNA forming blunt ends, then efficiency of ligation is very low. So the enzyme terminal transferase converts bunt end into sticky end. •Terminal transferase enzyme synthesize short sequence of complementary nucleotide at free ends of DNA, so that blunt end is converted into sticky end.
5. Nuclease: •The enzyme nucleases hydrolyses the phosphodiester bond on DNA strand creating 3’-OH group and 5’-P group. •It usually cut DNA on either side of distortion caused by thymine dimers or intercalating agents •The gap is filled by DNA polymerase and strand is joined by DNA ligase •Nucelase are of two types; endonuclease and exonuclease
6. DNA polymerase: •DNA polymerase is a complex enzyme which synthesize nucleotide complementary to template strand. •It adds nucleotide to free 3′ OH end and help in elongation of strand •It also helps to fill gap in double stranded DNA. •DNA polymerase-I isolated from E. coli is commonly used in gene cloning •Taq polymerase isolated from Thermus aquaticus is used in PCR.
7. Ribonuclease-H (RNase H): •RNase-H removes mRNA from DNA-RNA heteroduplex and that mRNA is used to synthesize cDNA •It is isolated from retrovirus
8. Alkaline phosphatase: •The enzyme Alkaline phosphatase helps in removal of terminal phosphate group from 5′ end •It prevents self annealing of vector DNA soon after cut open by restriction endonuclease
9. Polynucleotide kinase: •It adds phosphate group from ATP molecule to terminal 5’end after dephosphorylation by alkaline phosphatase. Enzyme Type # 1. DNA Ligase: Recombinant DNA experiments require the joining of two different DNA segments or frag ments 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 # 2. Reverse Transcriptase: Many times we do not get our gene of interest rather its mRNA. In this case reverse trans criptase 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 tran scription. 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 3. Restriction endonuclease Notes provided earlier.
Enzyme Type # 4. 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 # 5. 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 non specific 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 # 6. 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. The native DNA Pol I has been successfully used to remove 3′ protruding DNA ends (in the absence of dNTPs), or to fill in cohesive ends (in the presence of dNTPs) before addition of molecular linkers. However, the 5′→3′ exonuclease activity of DNA Pol I makes it unsuitable for all applications that require polymerization activity alone (e.g. to fill in cohesive ends before addition of linkers, or to copy single-stranded DNA in the dideoxy method for sequencing). Fortunately, it was discovered that proteolytic digestion of E. Coli DNA Pol I (109 kDa) generates two fragments (76 and 36 kDa). The large fragment, also known as DNA Pol IK or Klenow fragment (named after its inventor, (Klenow and Henningsen 1970)), contains the 5′→3′ polymerase and 3′→5′ exonuclease (proofreading) activities of DNA Pol I, while the small fragment exhibits the 5′→3′ exonuclease activity alone. Since then, recombinant sources of Klenow significantly improved the functional quality of this fragment by eliminating contaminations due to the presence of residual native enzyme in proteolytically treated preparations.
Enzyme Type # 7. Alkaline Phosphatases: Alkaline phosphatase is a glycoprotein with two identical subunits. The cohesive ends of broken plasmids, instead of joining with for eign DNA, join the cohesive end of the same DNA molecules and get re-circularized. To over come 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 in crease the frequency of production of recombi nant DNA molecules. The nicks between two 3′ ends fragment and vector DNA are repaired inside the bacterial cells during the transfor mation. Enzyme Type # 8. 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 pyrophos phate. It is widely used in molecular biology and genetic engineering to add radio-labelled phosphates. In RDT experiments mostly T4 polynucleotide kinase is used.
References: 1.http://www.biotechnologynotes.com 2.https://www.onlinebiologynotes.com 3.Principles of gene manipulation and genomics.,Primrose S.B.,Twyman R.M.