ENZYMES INVOLVED

INVOLVED IN
IN
ENZYMES
GENE
GENEMANIPULATION
MANIPULATION
Submitted to Dr.Asif Zahoor
Submitted by SUMMIYA AKRAM
Roll # 15240
BS.Microbiolgy
5th semester

Enzymes

These are the type of substances which

are basically the catalyst in order to
carry out the chemical processes.

Genetic manipulation

It is also called genetic engineering.

In this the biotechniological manipulation
of organism genome is done.

Enzymes involved in genes

manipulation

There are the followig enzymes which

play role in genetic manipulation,divided
into following different classes;
Enzymes involved in cutting.
Enzymes that act on termini.
Enzymes used in joining.
Enzymes for synthesis and modification.

Enzymes that act on

termini

these enzymes require termini of RNA or

DNA in order to perform its function.
Terminal deoxynucleotidyl transferase.
Alkaline phosphatase.
Kinase.

functions

Terminal deoxynucleotidyl tranferase

adds nucleotides to any available 3
terminus of DNA.
Phosphatase group from 5 end of DNA or
RNA is removed by alkaline phosphatase.
Kinase play a key role in addition of free
phosphate group to varied substrates e.g
DNA or RNA.

Enzymes involved in
cutting
Cutting is regulated by nucleases
Breakage of phosphodiaster bond to
degrade nucleic acid is carried out by
nucleases
There are two types of nuclease:
Endonucleases.
Exonucleases.

Endonuclease

Cutting of DNA within its strand is

performed by the group of enzyme known
as endonuclease.
Endonuclease is of three types:
Restriction enzymes.
Deoxyribonuclease I(DNase1).
S1 nuclease.

Restriction enzymes

Bacteria are the main source of restriction

enzymes that produce them in order to response
to bacteriophages.
Restriction enzymes are of three types:
Type 1:cleavage of DNA at random length from
recognition site.
Type 2:short palindromic sequence of 4-8 base
pairs is recognized by this type.it is also
important to cleave and recognize DNA at same
site.
Type 3:after recognition site 20-30 base pairs of
DNA are cleaved by this type.

Deoxyribonuclease

E.coli that is the carrier of MBP clone of

bovine pancreatic DNase 1 gives
deoxyribonuclease.
Basically it cuts sites of single or double
stranded DNA at random sites.
S1 nuclease:Aspergillus oryzae is a
sourse of s1 nuclease.
Cutting of ssRNA or ssDNA is carried
out by s1 nuclease.

Enzymes for synthesis and

modification
Modification and synthesis of DNA or RNA is
carried out by following:
Polymerase
Reverse transcriptase
Polymerase:Nucleic acid is synthesized by
polymerase.
These are the only enzymes that are
template dependent.e.g includes:
DNApolymerase-I.
T7 DNA polymerase.
Taq DNA polymerase.

DNA polymerase-I

Synthesis of DNA is 5-3 is catalysed by

template dependent DNA polymerase
that is DNA polymerase I.
T7 DNA polymerase is isolated from that
E.coli strain which is infected with
bacteriophage t7.It polymerizes in 5-3
direction.
Taq DNA polymerase:It has been
extracted from Thermus aquaticus.It
reads from 5-3.

DNA polymerase-II

Extracted from E.coli;damage portion of

DNA is repaired by DNA polymerase-II.
DNA polymerase-III is also obtained by
E.coli exonuclease proof reading of DNA
strand from 5-3 is done with DNA
polymerase-III.
RNA polymerase: E.coli infected from
bacteriophage is the source of RNA
polymerase.preparation of RNA
transcriptase from the DNA fragments
that are clonned by RNA polymerase.

Reverse transcriptase

Moloney murine leukemia virus is a carrier of

reverse transcriptase.Complementary DNA from
mRNA molecule is generated by this enzyme.
Enzymes used in joining:joining includes DNA
ligase and RNA ligase.

DNA ligase:It

is isolated from E.coli.

T4 DNA ligase

It acts as a catalyst in phosphodiester

bond formation in DNA or RNA duplex.
It play a key role in repairing of nicks in
DNA or RNA or RNA/DNA hybrids.

RNA ligase

Source:Its origin is also from E.coli

strain that contain T4RNA ligase 1
gene.
T4RNA ligase:It catalyse the ATP
dependent intra and intermolecular
formation of phosphodiester bond
between 5 phosphate and 3 hydroxyl
termini of oligonucleotide ssRNA or
ssDNA.