DNA modifying enzymes

The slides on the DNA modifying enzymes

1. DNA MODIFYING ENZYMES
PRESENTED BY
DASTHAGIRI PASHA S

2. 1.DNA modifying enzymes
2.Polymerase
3.DNase
4.RNase
5.Polynucleotide kinase
6.Alkaline phosphatase
7.Nucleases
CONTENT

6. APPLICATIONS

7. DNA modifying enzymes --
❖DNAmodifyingenzymesareinvolvedin
geneticengineering.
❖RestrictionenzymesandDNAligase
representthecuttingandjoining
functioninDNAmanipulation.
❖Theseenzymes areinvolvedinthe
degradation,synthesisandalternationof
thenucleicacid

8. POLYMERASES
❖DNA polymerase are enzymes that synthsis a new
strand of DNA complementary to an existing DNA or
RNA template .
❖Polymerases can function only if the template
possess a double stranded region that act as a
primer for initiation of polymerization.
❖The polymerase and nuclease activities of DNA
polymerase 1 are controlled by different parts of
enzyme molecule.
❖These enzymes attaches to a short single -stranded
region in a mainly double stranded DNA molecule ,
and then synthesis a completely new strand ,
degrading the existing strand as it proceed

9. The polymerase can be
classified into 3 types they are
a)DNA dependent DNA polymerase that
copies DNA from DNA .
b)RNA dependent RNA polymerase that
synthesis DNA from RNA
c)DNA dependant RNA polymerase that
produces RNA from DNA.

10. DEOXY RIBONUCLEASE
❏A nuclease enzyme that can catalyse the hydrolytic cleavage
of phosphodiester bond in the DNA backbone are known as
Deoxy ribonuclease.
❏These enzymes are broadly classified as endo and exo
deoxyribonuclease
❏DNase does not have speciefic recognition site and cleave
DNA sequence at random location
❏DNase are wide variety which known different substrate
specificities ,chemical mechanism , and biological functions.

11. ➢They are two types of DNase -
➢DNase 1
➢DNase 2
○1.DNase 1 :-
○They which cleave double stranded DNA or single
stranded DNA.
○The major products are 5’-phosphorylated,bi,tri, and
tetranucleotides.
○The presence of manganese ions ,DNase 1 hydrolyze each
strand of duplex DNA producing single stranded Nick's
in the DNA backbone ,generating various random
cleavages.

12. Applications of DNase 1
➢Eliminating DNA contamination
from preparation of RNA.
➢Analyzing the DNA -protien
interaction via DNA foot printing.
➢Nicking DNA prior to radio labeling
by Nick translation.

13. DNase2 :-
●It's a non speciefic endonucleases
pH is (4.5-5.5)
●Dnase 2 initially introduce multiple
single stranded Nick's in DNA
backbone .
●Generating single stranded Nick's by
producing of acid soluble nucleotide
and oligonucleotide .

14. Applications of DNase 2 :-
❖DNA fragmentation
❖Molecular weight marker
❖Cell apoptosis assays etc...

15. Ribonuclease (RNase )

16. ●Nuclease that can catalyse hydrolysis of
ribonucleotide from either single
stranded or double stranded RNA
sequence are called ribonucleotide .
●RNA is important for RNA maturation
and processing .
●RNase A and RNase H play important
role in initial defence mechanism against
RNA viral infection.

17. Applications of RNase :-
●Eliminating or reducing RNA
contamination in preparation of plasmid
DNA.
●Mapping mutation in DNA or RNA by
mismatch cleavage .RNase will cleave
the RNA in RNA-DNA hybrid at sites of
single base mismatch & the cleavage
product can be analysed.
●Useful of RNA sequencing.

18. POLYNUCLEOTIDE KINASE
➔PNK is a tetramer with phosphatase
activity at 3’ end and kinase at 5’ end
with tunnellike active site.
➔PNK catelysed the transfer of a p04
group from gamma position of ATP to
the 5’end of either DNA or RNA .
➔Basic residue of active site of PNK
8nteract with -vely charged phosphate
of the DNA .

21. APPLICATIONS OF PNK
❏The linkers and adapter are
phosphorylated along with the
fragment of DNA before
ligation,which requires a 5’
phophate .this include products of
PCR ,which are generated by using
non phosphorylated primer
❏PNK is also used for radiolabelling
oligonucleotides

22. ALKALINE PHOSPHATASE
●It is homodimer enzyme
●Optal pH is about 10
●Alkaline phosphate is closed to metal
ions that is mg & zn .
●Human body has present 4 isoforms
●The genes encode tissue specific
isoform are present on chromosome -2

23. They are 3 Alkaline phosphate are
used in gene manipulation
❖Bacterial alkaline phosphate
(BAP)
❖Calf intestinal alkaline
phosphate (CIP)
❖Shrimp alkaline phophate (SAP)

24. APPLICATIONS OF ALKALINE
PHOSPHATASE

25. NUCLEASES

26. APPLICATIONS OF NUCLEASES

27. CONCLUSION

28. References
➢Paul A .genetic engineering.in.genetic from gene to
genome
➢Garden j e principle of genetics wily India pvt.ltd
new delhi
➢Old R W and primrose H B (1980) principle og gene
manipulation.
➢http//www.onlinebiologynotes.com