It includes all the enzymes list...

1. ENZYMES IN GENETIC
ENGINEERING & IN
CENTRAL DOGMA
PM .Yeswanth

2. CONTENT
▪ INTRODUCTION
▪ NUCLEASES
▪ RESTRICTION ENDONUCLEASES
▪ LIGASES
▪ POLYMERASES
▪ MODIFYING ENZYMES
▪ ENZYMES INVOLVED IN CENTRAL DOGMA

3. INTRODUCTION
▪ Enzymes are the biocatalyst which are protein in nature fasten the chemical
reaction without undergoing any change itself in the reaction.
▪ The term ‘enzyme’ was introduced by Kuhne in 1878.
▪ In 1890’s Fisher proposed “Lock and Key” model of enzyme action.
▪ In 1926,J.B.Sumner first is0lated the crystallized form of “Urease”
enzyme.
▪ In 1951,Pauling and Corey discovered the α-helix and β-sheet structures of
enzymes.

4. NUCLEASES
Nucleases are the group of enzymes which cuts DNA at specific sites.
They are of two types:- 1) Exo-nucleases
2)Endo-nucleases
EXONUCLEASES:-
These enzymes act upon genome and digest the base pairs on 5' or 3' ends
of single stranded DNA, these are of following:
a) λ Exonuclease
b) Exonuclease III

5. a) λ Exonuclease:-This enzyme removes nucleotide from 5’ end of double
stranded DNA.
b)Exonuclease III :- The Enzyme removes nucleotide from 3’ end of the double
stranded DNA fragment.
ENDONUCLEASES:-
They act upon genetic material and cleave the double stranded DNA
at any point except the ends.
examples:-
a) S1-Nuclease- It degrades the single stranded DNA OR Single stranded
protrusion of double stranded DNA with cohesive ends.
b)Bal 31 nuclease-it degrade simultaenously both 3’ and 5’ strands of DNA
and they possess blunt end at both termini.

6. Restriction Endonucleases:-
These are the group of ezymes which cuts the DNA within its inner site & are
also called as Molecular Scissors.
The are first isolated from the Ecoli cell.
They are of three types :-Type 1,2,3.
▪ Type I:-
These are the complex nucleases which functions simultaenously as endonucleases
and methylase and they require Mg++ and ATP as cofactor.
The recognition site will be of 15bp long and cleavage site is about 1000bp away from
the recognition site.
ex:ECOR1,SAL1,ECOK,ECOB etc.

7. •Type II:-
They have separate activity for endonucleases,methylation of DNA and requires
mg++ as cofactor.
This enzyme cleaves the DNA molecule 100bp away from the recognition site and
these sites have palindromic sequence.
ex: HIND III,HSU-I, XmaI, SmaI,Bam HI etc.
•Type III:-
This enzyme has both nuclease as well as methylation activity and requires both
ATP and Mg++ as cofactor.
It cleaves the DNA 25-27bp away from the recognition site at non-palindromic
sequence.
ex :HpaII, MboII, Fok I etc.

8. DNA LIGASE:-
DNA ligase catalyses the formation of phosphodiester bond between the 5’
phosphate of one strand of DNA or RNA and the 3’-hydroxyl of another.
This enzyme is used to covalently link or ligate fragments of DNA together
•The DNA ligase used in molecular cloning differ in their abilities to ligate noncanonical
substrate, such as blunt ended duplex DNA:RNA hybrid or ssDNAs.
Types of DNA Ligase:1)BacteriophageT4 DNA ligase
2)E.coli DNA Ligase
3)Taq DNA Ligase
4)T4 DNA Ligase

9. 1)BacteriophageT4 DNA Ligase (ATP):-
•The most widely used DNA ligase is derived from theT4 bacteriophage.
• It is a monomeric polypeptide
• MW 68KDa is encoded by bacteriophage gene30.
• It has broder specificity and repairs single stranded Nicks in duplex DNA, RNA or
DNA:RNA hybrids.

10. 2)E.Coli DNA ligase:-
• It is derived from E.coli cell and requires NAD+ ascofacter.
• It is a monomeric enzyme of MW 74KDa which catalyzes the formation of the
phosphodiester bond in duplex DNA containing cohesive ends.
•This enzyme has narrower substrate specificity, making it a useful tool in specific
application.

11. 3)Taq DNA ligase [NAD+]:-
The gene encoding thermostable ligases have been identified from several
thermophilic bacteria.
Several of this ligase have been cloned and expressed to high levels in E.coli
• It is uses in the detection of mutation as thermostable DNA ligase retain their
activities after exposure to higher temp for multiple rounds
• it is uses in DNA amplificaton reaction to detect mutation in mammalian DNA.

12. 4)T4 RNA Ligase :-
•T4 RNA ligase is the only phage RNA ligase that has been extensively characterized
and used in genetic engineering.
•This enzyme catalyzed the phosphodiester bond formation of RNA molecule with
hydrolysis of ATP to PPI
• It is monomeric enzyme with 373 deduced aminoacid residues is a product of theT4
gene 63.

13. Polymerases:-
This is an enzyme that synthesizes long chains of polymers or nucleic
acids. It is of two types 1)Prokaryotic
2)Eukaryotic polymerases.
PROKARYOTES EUKARYOTES
DNA polymerase I More than 15
DNA polymerase II DNA polymerases α,δ, and ϵ
DNA polymerase III DNA polymerase γ
DNA polymerases IV
DNA polymeraseV

14. Prokaryotic
DNA Polymerases I:-
This enzyme is chiefly involved DNA repair and used for invitro DNA replication.it
has three main activity,they are of
•The 5’ to 3’ polymerase activity is responsible for primer extension or DNA synthesis.
•The 5’ to 3’ exonuclease activity is involved in excision of DNA strands during DNA repair,
it can remove ~10 bases at a time.
•The 3’ to 5’ exonuclease activity is responsible for proof reading and only one nucleotide is
removed at a time.
DNA Polymerases II:-
This enzyme functions in DNA repair. It has 5’ to 3’ polymerase and 3’ to 5’
exonuclease activities, and uses as template only such DNA duplexes that have short gaps.
DNA Polymerase III:-
This enzyme is responsible for DNA replication in vivo.
•It has 5’ to 3’ polymerase and 3’ to 5’ exonuclease activities.
•It catalyzes DNA synthesis at very high rates.

15. Eukaryotic polymerses:-
Function
α β ε γ δ
Nuclear
replication
initiation of
both
strands and
priming
Nuclear
Repair.
Nuclear
Replication
of lagging
strand.
Mitochondri
al
Replication
of mt DNA.
Nuclear
Replication
elongation
of leading
strands.

16. Modifying Enzymes:-
These enzymes modifiesTHE DNA by the addition or removal of specific group they
are of..
1)Alkaline Phosphatase:
These are isolated from Ecoli and removes phosphate group from 5’ termini of
DNA.
2)T4 Polypeptide kinase:-
These enzymes catalyse the transfer of phosphate group ofATP to
5’ terminus of the dephosphorylated DNA or RNA.
3)Restriction Enzymes:-
These enzymes will cut the DNA at the specific site and so for called as
molecular scissor.

17. 4)ReverseTranscriptase:-
This enzyme is used to produce cDNA copies of mRNA.
5)DNA ligase:-
This enzyme is used to seal the nicks in recombinant DNA or joining the cut
DNA fragments.
6)S1 Nuclease:-
This enzyme is used for the removal of single stranded protrusion from 3’ or
5’ ends.
7)Klenow fragment of Ecoli DNA polymerase I:-
This enzyme used to make protruding ends double sranded by extending
the shorter.

18. 8)Lambda exonuclease:-
it is used for removal of nucleotides from 5‘ end.
9)Ecoli exonuclease III:-
It is used to removal of nucleotide from 3’ end.
10)Exonuclease Bal31:-
it is used for making of DNA fragments with blunt ends.
11)Terminal deoxynucleotidyl transferase:-
it is used for the addition of single stranded sequence to 3’ end of
blunt ended fragments.
12)Linker and Adaptors:-
it used for modification of cut ends of the DNA fragment.

19. Enzymes involved in the Central Dogma:-
1)DNA helicase:-
Also known as helix destabilizing enzyme,unwinds theDNA helix at the
replication fork,
2)DNA Polymease:-
it builds a new duplex DNA strand by adding nucleotide in the 5’ to 3’ direction,
Also performs proof-reading and error correction.
3)Single Strand Binding(SSB) Proteins:-
Binds to ssDNA and prevents the DNA double helix from re-annealing after
DNA helicase unwinds it thus maintaining the strand separation.
4)Topoisomerases:-
Relaxes the DNA from its super –coiled nature.
5)DNA Ligase:-
It re-anneals the semi-conservavtive strands and joins Okazaki fragments of
the lagging strand.

20. 6)Primase:-
provides a starting point of RNA or DNA for DNA polymerase to begin synthesis
of the new DNA strand.
7)RNA Polymerase (Eukaryotes):-
a)RNA polymerase I:
It is responsible for the synthesis of precursors foe the large ribosomal
RNAs.
b)RNA Polymerase II:
It synthesizes the precursors for mRNAs and small nuclear RNAs.
c)RNA Polymerase III:
It participates in the formation of tRNAs and small RNAs.