Enzymes extensvely used for Dna modification purposes.

1. DNA MODIFYING ENZYMES
ARIJIT PANI I M.Sc I Bangalore University

2. DNA : The Hereditary Material
 Nucleic acids play an important role in the storage and expression of
genetic information. They are divided into two major classes:
 Deoxyribonucleic acid (DNA): Functions solely in information storage
 Ribonucleic acids (RNAs) : Involved in most steps of gene expression
and protein biosynthesis.
 All nucleic acids are made up from nucleotide components, which consist
of a nitrogenous base, a sugar and a phosphate residue.
 Using the methods of X- Ray diffraction, Rosalind Franklin and Maurice
Wilkins showed that DNA produces a characteristic X- Ray diffraction pattern
from which James Watson and Francis Crick deduced the double helical
model of DNA structure.

3. DNA STRUCTURE
The Watson and Crick structure of DNA has following features:-
1) Two anti-parallel polynucleotide strands that wind about a common axis
with a right handed twist to form a double helix.
2) The diameter of a double helix will be around 20 Å.
3) Each base is hydrogen bonded to a base on opposite strand (A with T and
G with C) to form base pair.
4) The ideal B DNA helix has 10 base pairs per turn.
5) The helix has a pitch (the distance raised by common axis in a complete
turn) of 34 Å. So the per base pair raise in common axis will be 3.4 Å.
6) The double helix has major and minor grooves.

4. What are DNA-Binding Proteins?
 DNA-binding proteins are proteins composed of DNA-binding domains such
as zinc finger, the helix-turn-helix, and the leucine zipper etc and thus have a
specific or general affinity for double stranded DNA.
 Sequence-specific DNA-binding proteins generally interact with the major
groove of B-DNA, because it exposes more functional groups that identify a base
pair.
 DNA-binding proteins include transcription factors, polymerases, nucleases,
histones etc.

5. DNA MODIFICATION
DNA modification
occurs during
Replication
Transcription
Repair
Epigenetic
Regulations
Genetic
Recombination

6. DNA MANIPULATIVE ENZYMES
DNA
Manipulative
Enzymes
Nucleases
Exonucleases Endonucleases
Ligases Polymerases
Modifying
Enzymes
DNase RNase
Polynucleotide
Kinases
Alkaline
Phosphatases
Topoisomerases

7.  NUCLEASES
 Nucleases cleave the phosphodiester bonds between monomers of
nucleic acids.
 Nucleases produce single and double stranded breaks in their target
molecules.
 In living organisms, they are essential machinery for many aspects of DNA
repair. Defects in certain nucleases can cause genetic instability.
 There are two primary classes of nucleases:
o Exonucleases digest nucleic acids from the ends.
o Endonucleases act on regions in the middle of target molecules.

8. EXONUCLEASES
Exonuclease is a nuclease enzyme which cleaves chemical bonds
between nucleotides at the 3’ or 5’ ends of the nucleic acid chains.
Exonucleases are important in DNA repairing, genetic
recombination, prevention of the occurrence of mutations,
genome stabilization etc.
Bal 31 is isolated from a marine bacterium Alteromonas
espejiana. It is a Ca2+ dependent enzyme that degrades the
nucleotides from both the strands of dsDNA molecule.
In E coli, there are 17 different exonucleases present
including DNA polymerase I, II and III.
Exonuclease III of E. coli digests only one strand of the dsDNA
molecule. It removes the nucleotide from the 3' terminus of the
strand leaving 5' overhangs

9. ENDONUCLEASES
Endonucleases are enzymes that cleave the phosphodiester
bond within a polynucleotide chain by recognizing specific
sequences,
Most of them are dimeric enzymes composed of two protein
subunits. Two protein subunits wrap the double-stranded DNA
and separately cleave both strands from both sides.
S1 nuclease is an endonuclease isolated from Aspergillus oryzae.
It is a heat stable enzyme. It cleaves only single stranded DNA.
Another type of endonuclease called DNase I is a non–specific
enzyme. It is able to cleave both single and double stranded DNAs
by cleaving any of the internal phosphodiester bonds.
Another class of endonucleases, the restriction endonucleases
cleaves DNA at specific sites having specific palindromic
sequences.

10. APPLICATIONS
 They are used in the process of molecular cloning.
 Exonuclease III is used for generating single stranded templates.
 They help to distinguish gene alleles by recognizing single nucleotide
polymorphisms (SNPs).
 They are used in DNA fingerprinting.

11.  LIGASES
 Ligases are enzymes that join the nucleic acid molecules together.
 DNA ligase catalyses the formation of a phosphodiester bond
between the 5' phosphate of one strand and the 3' hydroxyl group of
another.
 In nature the function of DNA ligase is to repair single strand breaks
(discontinuities) that arise as a result of DNA replication and/or
recombination.

12. LIGASES
1. NH2 group of the ligase enzyme initiates a nucleophilic attack
towards the ἀ-phosphate of ATP.
2. Results in release of PPi.
3. The 5’ phosphate group of the nick attacks the phosphate of the
adenylated ligase and itself gets activated.
4. The 3’ OH group of the nick now attacks the activated 5’ phosphate
group to release the AMP.
5. The nick gets ligated.

13.  DNA ligases are used with restriction enzymes to insert DNA
fragments, often genes, into plasmids.
 Helps to perform blunt end as well as sticky end ligation.
 DNA ligase I is used in in-silico drug design to identify
ligase inhibitors as possible therapeutic agents to treat cancer.
 Some ligases are used as targets for the development of new
antibacterial drugs.
APPLICATIONS

14.  DNA POLYMERASE
DNA polymerases are enzymes that catalyze the synthesis of a new DNA strand
from a pre-existing strand.
The enzyme adds deoxyribonucleotides to the free 3’-OH of the chain
undergoing elongation. The direction of synthesis is 5’-3’.
It has three major requirements for its activity:
 a template strand
 a primer with a free 3’-OH group
 four dNTPs
 some cofactors like Mg2+ ions
Most common DNA polymerases used in recombinant DNA technology
o DNA Polymerase I of E. coli
o Klenow Fragment
o Thermostable DNA Polymerase
o Reverse Transciptase

15. DNA POLYMERASE
 E. coli DNA polymerase I is an enzyme that has both DNA polymerase
as well as DNA nuclease activity.
Different domains of the E. coli Pol I are responsible for different
catalytic activities.
The C-terminal is responsible for the polymerase activity
The N-terminal of the enzyme catalyses the 5'-3'exonuclease activity.
The central region of the enzyme is responsible for 3'-5' exonuclease
activity that can remove any misread nucleotide and hence acts as a
proofreading mechanism.
 Pol I is devoid of 5’-3’ exonuclease activity (which is unsuitable for
many applications in RDT) is called a klenow fragment.

16. DNA POLYMERASE
Reverse transcriptase (RT) is a RNA dependent DNA polymerase found in
RNA viruses also called as retroviruses.
This enzyme is involved in the replication of retroviruses, where the RNA
genome is first converted into DNA and then integrated into the host.
RT uses mRNA template instead of DNA for synthesizing new DNA strand
(cDNA).
RT also shows RNAse H activity that degrades the RNA molecule from a
DNA-RNA hybrid.

17. DNA POLYMERASE
Thermostable DNA polymerases are a class of DNA
polymerases that remain functional even at high
temperatures i.e, they are resistant to denaturation by
heat treatment. They are isolated from the bacterium
Thermus aquaticus that lives in hot springs. The
enzyme isolated from this bacterium is known as
‘Taq Polymerase’.
Pfu DNA Polymerase is a thermostable enzyme
isolated from Pyrococcus furiosus. In addition to
5´to 3´ DNA polymerase activity, it also
possesses 3´ to 5´ exonuclease (proofreading)
activity. Pfu DNA Polymerase exhibits the lowest
error rate and is useful for polymerization
reactions requiring high-fidelity synthesis.

18. APPLICATIONS
 Taq polymerase is used in the polymerase chain reaction
(PCR) technique which is used to amplify DNA fragments.
 Formation of a double stranded cDNA from the mRNA
molecule using RT finds applications in genetic engineering.
 Klenow fragment is predominantly used in DNA sequencing.

19.  ALKALINE PHOSPHATASES
Alkaline phosphatase is purified from either E. Coli or higher
organisms (e.g. calf intestine).
It is used for removal of 5′-phosphate groups from nucleic
acids in order to prevent recircularization of DNA vectors in
cloning experiments.
Now, this vector can be joined with the foreign DNA and the
nicks formed can be ligated using ligase.
This vector containing the insert is suitable for transformation.

20. APPLICATIONS
Removing 5’ phosphates from plasmid and
bacteriophage vectors that have been cut with
restriction enzyme.
Removing 5’ phosphates from from fragments
of DNA before labeling with radioactive
phosphate.

21.  POLYNUCLEOTIDE KINASES
 It is a product of the T4 bacteriophage, and commercial
preparations are usually products of the cloned phage gene
expressed in E. coli.
 The enzymatic activity of PNK is utilized in two types of
reactions:
 PNK transfers the gamma phosphate from ATP to the 5'
end of a polynucleotide (DNA or RNA). The target
nucleotide is lacking a 5' phosphate either because it has
been dephosphorylated or has been synthesized chemically.
 In the "exchange reaction", target DNA or RNA that has
a 5' phosphate is incubated with an excess of ADP. PNK will
first transfer the phosphate from the nucleic acid onto an
ADP, forming ATP and leaving a dephosphorylated target.
PNK will then perform a forward reaction and transfer a
phosphate from ATP onto the target nucleic acid.

22.  RIBONUCLEASE (RNase)
Ribonuclease (commonly abbreviated RNase) is a type of
nuclease that catalyzes the degradation of RNA into smaller
components by cleaving the phosphodiester bond between
adjacent ribonucleoside.
 RNase A which is obtained from bovine pancreas cleaves next to
uracil and cytosine.
RNase T1 derived from Aspergillus oryzae cleaves next to
guanosine.
Ribonuclease H (RNase H) specifically degrades RNA in RNA/
DNA hybrids.

23. APPLICATIONS
Some of the major use of RNase A are:
Eliminating or reducing RNA contamination in
preparations of plasmid DNA.
Mapping mutations in DNA or RNA by mismatch
cleavage. RNase will cleave the RNA in RNA:DNA
hybrids at sites of single base mismatches, and the
cleavage products can be analyzed.
Useful in RNA sequencing.

24.  TOPOISOMERASES
During DNA replication and transcription, DNA becomes
overwound ahead of a replication fork.
If left unattended, this torsion would eventually stop the ability of
DNA or RNA polymerases involved in these processes to continue
down the DNA strand.
Topoisomerases are enzymes that participate in the overwinding or
underwinding of DNA.
Topoisomerases bind to double-stranded DNA and cut the phosphate
backbone of either one or both the DNA strands.
This intermediate break allows the DNA to be untangled or
unwound, and, at the end, the DNA backbone is resealed again.
Supercoiling of DNA

25. TOPOISOMERASES
Topoisomerases are separated into two types
depending on the number of strands cut in
one round of action:
A type I topoisomerase cuts one strand of a
DNA double helix, relaxation occurs, and
then the cut strand is re-ligated. Cutting one
strand allows the part of the molecule on
one side of the cut to rotate around the
uncut strand, thereby reducing stress in the
DNA.
They do not require ATP for hydrolysis.
Type IA Type IB Type IC
•All cells
•Relaxes only –ve
supercoiled DNA.
•E. Coli
•Topoisomerase I
•Topoisomerase III
•Eubacteria & Archaea
•Reverse gyrase
•Eukaryotes & many
prokaryotes
•Relax both –ve & +ve
supercoils.
•Human
•Topoisomerase I
•Archaea
•relaxes +ve and -ve
supercoils with a rate
similar to or higher
than other
topoisomerases.
•Archaea
•Topoisomerase V

26. TOPOISOMERASES
A type II topoisomerase cuts both strands of one DNA
double helix, passes another unbroken DNA helix through
it, and then re-ligates the cut strands. Type II
topoisomerases utilize ATP hydrolysis and are subdivided
into two subclasses which possess similar structure and
mechanisms.
Type IIA Type IIB
•Bacteria
•DNA Gyrase
•Topoisomerase IV
•Eukaryotes
•Topoisomerase II
•Only one member that
occurs in archaea.
•Topoisomerase VI

28. PREVIOUS YEAR QUESTIONS
2013
1. Ligases. [3 marks]
2014
1. T4 PNK. [5 marks]
2015
1. Ligases. [5 marks]

29. REFERENCES
BOOKS
1. Molecular biology of the cell, Bruce Alberts.
2. Gene Biotechnologies, S. N Jogdand.
WEBSITES
1. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1083870/
2. https://en.wikipedia.org/wiki/DNA
3. http://www.nature.com/nbt/journal/v15/n5/abs/nbt0597-427.html?foxtrotcallback=true

30. THANK YOU