INTRODUCTION HISTORY ENZYMES AND PROTEINS INVOLVED IN PROKARYOTIC DNA REPLICATION DNA polymerases Types and function Additional enzymes Helicase , SSBP, Topoisomerase, Primase , Ligase , Events and function of enzymes CONCLUSION REFERENCES

1. ENZYME INVOLVE IN PROKARYOTIC DNA
REPLICATION
By
KAUSHAL KUMAR SAHU
Assistant Professor (Ad Hoc)
Department of Biotechnology
Govt. Digvijay Autonomous P. G. College
Raj-Nandgaon ( C. G. )

2. SYNOPSIS
 INTRODUCTION
 HISTORY
 ENZYMES AND PROTEINS INVOLVED
IN PROKARYOTIC DNA REPLICATION
 DNA polymerases
Types and function
 Additional enzymes
Helicase
SSBP
Topoisomerase
Primase
Ligase
 Events and function of enzymes
 CONCLUSION
 REFERENCES

3. INTRODUCTION
• Replication can be broadly defined as genome
duplication, an essential process for the
propagation of cellular genomes .
• The genome to be duplicated is the parental
genome, and the copies are daughter genomes.
• DNA replication is a biological process that occurs
in all living organisms and copies their DNA; it is
the basis for biological inheritance.

4. HISTORY
• 1953 – Watson and crick presented the now famous
double helix model of DNA.
• 1957 – Kornberg discovered an enzyme (in E . coli) that
was responsible for the of DNA replication.
• 1958 – Meselson and Stahl demonstrated that DNA
replication in a semi conservative manner.
• 1968 – Okazaki et al have suggested that only one strand,
the 3’→5’or continuous strand, is continuously replicated.

5. ENZYMES AND PROTEINS INVOLVED IN
PROKARYOTIC DNA REPLICATION
DNA POLYMERASE
First isolation of an enzyme involved in DNA replication
in 1955.
• Arthur Kornberg won the 1959 Nobel Prize.
• Kornberg was believed to be the only DNA
polymerase in E. coli. However, mutations in this gene
(polA1) were not lethal, that mean other DNA
polymerases must exist in E. coli.

6. ROLES OF DNA
POLYMERASES
 All DNA polymerases link dNTPs into DNA chains .
 An incoming nucleotide is attached by its 5’-phosphate group
to the 3’-OH of the growing DNA chain. Energy comes from
the dNTP releasing two phosphates.
 The incoming nucleotide is selected by its ability to hydrogen
bond with the complementary base in the template strand.
The process is fast and accurate.
 DNA polymerases synthesize only from 5’ to 3’.

8. TYPES AND PROPERTIES OF PROKARYOTIC DNA
POLYMERASES
DNA POLYMERASE I
.
 DNA Polymerase I (or Pol I) is an enzyme that participates in
the process of DNA replication.
 Discovered by Arther Kornberg in 1956.
 It was the first known DNA polymerase in E. coli and many
other bacteria.
 The gene that encodes Pol I is known as polA.
 The E. coli form of the enzyme is composed of 928 amino acid.
.

9. • POL I POSSESSES FOUR ENZYMATIC ACTIVITIES:
• A 5’3’ (forward) DNA-Dependent DNA polymerase activity,
requiring a 3’primer site and a template strand
• A 3' -> 5' (reverse) exonuclease activity that mediates
proofreading.
• It has 5’3’ exonuclease activity to remove nucleotides from
5’ end of DNA or from an RNA primer
• A 5’3’ (forward) RNA-Dependent DNA polymerase activity.
Pol I operates on RNA templates with considerably lower
efficiency (0.1–0.4%) than it does DNA templates, and this
activity is probably of only limited biological significance.

10. • In the replication process, DNA Polymerase I
removes the RNA primer from the lagging strand
and fills in the necessary nucleotide between the
okazaki fragment in 5' -> 3' direction.
• it only adds nucleotides that correctly base pair
with an existing DNA strand acting as a template.
• DNA ligase then joins the various fragments
together into a continuous strand ofDNA.

11. PROOFREADING
DNA polymerase proofreads the bases to
search for incorrect nucleotides
Mismatched bases are excised by the 3’ to
5’ exonuclease activity of the enzyme, and
the correct base is inserted in its place

12. The 3'-to-5' exonuclease activity of the proofreading function

14. DNA POLYMERASE II
• DNA polymerase II (also known as DNA Pol II or Pol II) is a
prokaryotic DNA replication involved in DNA repair.
• [1]
• The enzyme is 90 kDa in size and is coded by thepolB gene.
• DNA Pol II can synthesize DNA new base pairs at an
average rate of between 40 and 50 nucleotides/second.
• . Pol II differs from pol I in that it lacks a 5'->3' exonuclease
activity.

15. DNA polymerase III
• DNA polymerase III holoenzyme is the primary
enzyme complex involved in prokaryotic DNA
replication.
• It was discovered by Thomas Kornberg and Malcolm
Gefter in 1970.
• DNA Pol III holoenzyme also has proofreading
capabilities that correct replication mistakes by
means of exonuclease activity working 3'→5'.

16. From the RNA primer, the new DNA strands
are elongated by the action of DNA
polymerase III
Since new DNA is made only in the 5’ to 3’
direction, chain growth is continuous on one
strand and discontinuous on the other strand

17. • DNA Pol III enzymes, comprising α, ε
and θ subunits.
– the α subunit (encoded by the dnaE
gene) has the polymerase activity.
– the ε subunit (dnaQ) has 3'→5'
exonuclease activity.
– the θ subunit (holE) stimulates the ε
subunit's proofreading.

18. DNA polymerase IV
It is encoded by the dinB gene, and is used in DNA
repair.
DNA polymerase V
It is encoded by umuDC, and is used in DNA
repair.
Polymerase Iv and v does not play direct role in DNA
replication.

19. ADDITIONAL ENZYMES/PROTEINS
• DNA HELICASE-
DNA helicases unwind the double helix in
advance of the replication fork.
It is a hexameric protein.

20. Single-stranded binding proteins
(SSBs)
• It stabilize single-stranded DNA.
• Its binding is in sequence independent manner.
• It bind the ssDNA formed by helicase, preventing
reannealing.

21. PRIMASE
• Primase is a specialized RNA polymerase which forms a short
RNA primers on an ssDNA template.
• These are DNA-dependent RNA polymerase enzymes that
initiate DNA synthesis,
Do not require specific DNA sequence.
• DNA Pol can extend both RNA and DNA primers annealed to
DNA template .
• Later the RNA primer is removed

22. TOPOISOMERASES
Topoisomerase removes
supercoils produced by DNA
unwinding at the replication fork

23. DNA LIGASE
• Gaps between fragments are ligated by DNA ligase.
• Action of DNA ligase in sealing the gap between adjacent DNA
fragments to form a longer, covalently continuous chain.

24. Events and function of enzymes during DNA
replication in E.coli

27. CONCLUSION
• DNA replication is a biological process that occurs
in all living organisms and copies their DNA; it is
the basis for biological inheritance.
• DNA replication can also be performed in vitro.
• The polymerase chain reaction (PCR), a common
laboratory technique, employs such artificial
synthesis.

28. REFERENCES
• J.D. WATSON – Molecular biology of gene (6th
edition)
• GERALD KARP – cell and molecular biology (5th
edition)
• NELSON AND COX – principles of biochemistry (4th
edition)
• NET SOURCE
• www.wikipedia.com