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:---
1. Nucleases
2. Ligases
3. Polymerases
4. 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:-
i. Exonucleases
ii. 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.
A. Bal31:- It removes nucleotides from both strands of
a double-stranded molecule.
B. E.coli exonuclease III:- It removes nucleotides only
from 3’ end of a double-stranded molecule.
C. Lambda exonuclease:- It removes nucleotides only
from 5’ end of a double-stranded molecule.

8. ii. Endonucleases
• They are able to break internal phosphodiester bonds
within a DNA molecule.
I. S1 endonuclease:- It cleaves only single-stranded DNA,
including single-stranded nicks in mainly double-
stranded DNA.
II. 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.

12. 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.

14. 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:---
a. DNA Pol I
b. DNA Pol II
c. DNA Pol III
d. Reverse Transcriptase

16. 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:---
I. Alkaline phosphatase:- It removes the phosphate group
present at the 5’ terminus of a DNA molecule.
II. T₄ polynucleotide kinase:- It adds phosphate groups to the
5’ terminus.
III. Terminal deoxynucleotidyl transferase:- It adds one or more
deoxyribonucleotides to the 3’ terminus of a single- &
double-stranded DNA molecule. This is called Tailing.
IV. Topoisomerases:- It introduce or remove supercoils from
covalently closed circular DNA.