Dna manipulative enzymes

ALL THE DNA MANIPULATIVE ENZYMES BY RAHUL GAUTAM

1. DNA Manipulative Enzymes

2. The basis of recombinant DNA technology is the ability to manipulate DNA molecules in the test tube . Almost all DNA manipulative techniques make use of purified enzymes. Within the cell, these enzymes participate in essential processes such as DNA replication & transcription.

3. Types of DNA Manipulative Enzymes DNA manipulative enzymes can be grouped into four broad classes depending on the type of reaction that they catalyze:--- Nucleases Ligases Polymerases Modifying enzymes

4. 1. Nucleases Nucleases degrade DNA molecules by breaking the phosphodiester bonds that link one nucleotide to the next in a DNA strand. Some nucleases are specific for DNA & some for RNA. There are two different kinds of nucleases:- Exonucleases Endonucleases

5. i. Exonucleases They remove nucleotides one at a time from the end of a DNA molecule. The main distinction b/w different exonucleases lies in the number of strands that are degraded when a double-stranded molecule is attacked. Bal31:- It removes nucleotides from both strands of a double-stranded molecule. E.coli exonuclease III:- I t removes nucleotides only from 3’ end of a double-stranded molecule . Lambda exonuclease:- It removes nucleotides only from 5’ end of a double-stranded molecule .

6. ii. Endonucleases They are able to break internal phosphodiester bonds within a DNA molecule. S1 endonuclease:- It cleaves only single-stranded DNA, including single-stranded nicks in mainly double-stranded DNA . Deoxyribonuclease I( DNase I):- It cleaves both single- & double- stranded DNA. The special group of enzymes called Restriction Endonucleases cleave double-stranded DNA only at a specific recognition sites. It is also called molecular scissor. These enzymes are found in bacteria & archaea, & provide a defense mechanism against invading viruses.

7. 2. Ligases DNA ligase is used to seal the nicks that remain in the DNA molecule by forming a phosphodiester bond. It is also called molecular glue. E.g., T₄ polynucleotide ligase is prepared from E.coli cells infected with T₄ phage. It is involved in the replication of phage DNA & is encoded by the T₄ bacteriophage. The reaction requires energy which is provided by adding ATP to the reaction mixture.

8. 3. Polymerases DNA polymerases are enzymes that synthesize a new strand of DNA complementary to an existing DNA or RNA template. Four types of DNA polymerases are used in genetic engineering:--- DNA Pol I DNA Pol II DNA Pol III Reverse Transcriptase

9. 4. DNA Modifying Enzymes There are numerous enzymes that modify DNA molecules by addition or removal of specific chemical groups. They are also called End-modification enzymes. The most important are as follows:--- Alkaline phosphatase:- It removes the phosphate group present at the 5’ terminus of a DNA molecule. T₄ polynucleotide kinase:- It adds phosphate groups to the 5’ terminus. Terminal deoxynucleotidyl transferase:- It adds one or more deoxyribonucleotides to the 3’ terminus of a single- & double-stranded DNA molecule. This is called Tailing. Topoisomerases:- It introduce or remove supercoils from covalently closed circular DNA.