Question

Write about different types of dna modifying enzyme.

Answer 1

In the Recombinant DNA technology, we are manipulating the genome of an organism (bacteria, viruses, plants, etc.) for constructing the Recombinant DNA molecule. The manipulation process requires a lot of enzymes. The major task of the manipulation of the DNA involves cutting and ligation of the vector DNA and the gene of interest. The enzymes used in the DNA recombinant DNA technology are:

Nucleases

Ligases

Polymerases

Reverse transcriptase (for cDNA)

Alkaline phosphatase

Polynucleotide kinase

Terminal deoxynucleotidyl transferase

Nucleases:

Nucleases are the enzymes which degrade the nucleic acid (here DNA). They act on the DNA by breaking the phosphodiester bond. There are two major types of Nuclease enzyme, namely, Exonuclease and Endonuclease.

Exonucleases, as the name suggests, removes the nucleotides one at a time from the end of the DNA molecule. On the counterpart, Endonucleases aid break internal phosphodiester bonds within a DNA molecule.

Different exonuclease has different activities. Some exonucleases such as Bal31 would act on both the DNA strands at one end whereas some exonucleases such as exonuclease III from E. Coli acts only on one strand, producing overhand structure (single-stranded DNA). Thus, if we provide more time to the Bal31, the DNA would become shorter and shorter with Reverse.

Endonucleases are also classified in the same manner. Some endonuclease such as S1 endonuclease which is isolated from Aspergillus oryzae cleaves only single strands of any DNA molecule. Some endonuclease such as DNase I cleaves both the strands of the DNA (double-stranded and single-stranded). However, DNase I is not specific. The restriction endonuclease is a class of enzyme produced by the bacteria which identifies the particular sequence and cleaves explicitly. Example of restriction endonuclease is EcoRI, BamHI, PvuI, etc.

Ligases

The DNA ligases have the function of sealing the DNA nicks. In the cell, if the phosphodiester bond between two nucleotides of the DNA molecule is not formed (Okazaki fragment) or damaged, then DNA ligases are recruited to repair.

These enzymes aid in the formation of the phosphodiester bond between the two nucleotides.

DNA polymerase:

In genetic engineering, different types of DNA polymerase enzymes are used. One enzyme attaches to the short single-stranded regions (or nick) of double-stranded regions to completely synthesis the DNA. It also degrades the RNA primer and thus, has both polymerase and exonuclease activity.

In the DNA polymerase I, the first 323 amino acids shows the nuclease activity. If this segment is removed, the polymerase enzyme would not have nuclease activity and is known as Klenow fragment.

For PCR, the DNA polymerase required needs to be thermostable. The cycle of PCR involves increase and decrease of temperature for the annealing and denaturation process. Thus, the DNA polymerase should withstand the fluctuation of the temperature. The bacteria living in the hydrothermal vents and the hot springs have the amazing thermostable enzymes and proteins. Taq polymerase is isolated from the Thermus aquaticus and used in the PCR. It does not denature at 94°C.

Reverse transcriptase:

For the production of the DNA from the mRNA, we need a special enzyme called Reverse transcriptase. These enzymes are generally found in viruses which uses it to convert their RNA (genome) to DNA. This enzyme is extensively used in the study of transcriptome and production of cDNA library. It uses RNA as template and synthesis cDNA out of it.

Other DNA modifying enzymes:

Alkaline Phosphatase: This enzyme removes phosphate group at the 5′ end of the DNA. It is used in the modification of the adaptor[1]molecules so as to prevent ligation of two adaptors with each other.

Polynucleotide kinase: It would have the reverse effect of the alkaline phosphatase. It adds the phosphate group to the 5′ end of the DNA. Once the adaptors are added to the gene of interest, they are being modified back in order to ligate it to the vector DNA.

Terminal deoxynucleotidyl transferase: It adds ribonucleotides to the 3′ terminus of the DNA. It is used in Homopolymer tailing process for ligating the gene of interest to the vector DNA.