DNA modifying enzymes play important roles in genetic engineering and manipulating DNA. These enzymes include polymerases, DNase, RNase, polynucleotide kinase, alkaline phosphatase, and nucleases. Polymerases are involved in DNA synthesis, DNase and RNase degrade DNA and RNA, respectively, and polynucleotide kinase and alkaline phosphatase modify the phosphate groups on DNA and RNA. These enzymes are used in applications such as eliminating contamination, mapping mutations, DNA footprinting, radiolabeling, and DNA fragmentation.

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 --
❖ DNA modifying enzymes are involved in
genetic engineering.
❖ Restriction enzymes and DNA ligase
represent the cutting and joining
function in DNA manipulation.
❖ These enzymes are involved in the
degradation, synthesis and alternation of
the nucleic acid

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 strandedregion 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 dependentDNA polymerase that
copies DNA from DNA .
b)RNA dependentRNA polymerase that
synthesis DNA from RNA
c) DNA dependant RNA polymerase that
produces RNA from DNA.

10. DEOXY RIBONUCLEASE
❏ A nucleaseenzymethat can catalyse the hydrolytic cleavage
of phosphodiesterbond in the DNA backbone are known as
Deoxy ribonuclease.
❏ These enzymes are broadly classified as endo and exo
deoxyribonuclease
❏ DNase does not have specieficrecognition site and cleave
DNA sequenceat random location
❏ DNase are wide variety which known different substrate
specificities,chemicalmechanism , and biological functions.

11. ➢ They are two types of DNase -
➢ DNase1
➢ DNase2
○ 1.DNase1 :-
○ They which cleavedouble strandedDNA or single
strandedDNA.
○ The major products are 5’- phosphorylated,bi,tri, and
tetranucleotides.
○ The presence of manganeseions ,DNase1 hydrolyze each
strand of duplex DNA producingsingle strandedNick's
in the DNA backbone,generating variousrandom
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. DNase 2 :-
● 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