This document provides an overview of DNA replication and the associated proteins involved. It defines DNA replication as the process by which DNA forms an exact replica of itself. Several key points are made: DNA replication is semi-conservative in nature, as proven by Meselson-Stahl experiments; it requires many proteins including DNA polymerases, helicases, primase, ligase and topoisomerases; in E. coli, replication initiates at specific origin sites and terminates at terminator sequences; eukaryotic replication similarly involves unwinding, priming, and synthesis but uses different DNA polymerases than prokaryotes.

1. DNA REPLICATION
AND ITS
ASSOCIATED
PROTEINS
Mr. Rizaul Ahmed
Asst. Professor
Dept of Zoology
University of Science and
Technology, Meghalaya.

2. CONTENTS
DEFINITION
SIGNIFICANCE OF REPLICATION
STRUCTURE OF DNA
NATURE OF DNA REPLICATION
VARIOUS PROTEINS INVOLVED IN REPLICATION
DNA POLYMERASE
ORIGIN OF REPLICATION
MECHANISM
REPLICATION IN E.coli
EUKARYOTIC REPLICATION

3. Why NOT DNA synthesis?
Original Replica

4. DEFINITION
The most critical issue of life is the faithful
and timely duplication of the genetic material
i.e.- genes. Genes are encoded in the DNA.
The DNA replication is the process by virtue
of which DNA forms its exact replica.
Replication occurs in the synthetic phase or S-
phase of the cell cycle before the cell enter
into the M- phase. The replication process has
been studied since 1958

5. SIGNIFICANCE OF REPLICATION
When cells divide, the
new cells produce must
have a full set of genes
as a result it is very
necessary to replicate
the DNA before the cell
divides. Actually, it is
the basis of life.

6. STRUCTURE OF DNA
DNA is a linear, unbranched polymer
in which monomeric subunits called
nucleotides are linked in any order.
Each nucleotide comprises of three
components- 2, -deoxyribose sugar
A nitrogenous base and
A phosphate group.

8. A molecule made up of just the
sugar and nitrogenous base is
called a nucleoside while
addition of phosphate group
converts it into a nucleotide.
A polynucleotide is formed due
to the formation of the
phosphodiester bond between
the 5’ and 3’ carbon of two
consecutive nucleotide.
Polynucleotide has a chemical
direction of 5’ to 3’ or 3’ to 5’.

9. Various assumptions are made regarding the nature of
replication. Three models have been proposed in this
regard. They are –
NATURE OF REPLICATION
Conservativ
e
Semiconserva
tive
Dispersive

10. The possibilities were as follows:

11. SEMICONSERVATIVE NATURE
Each of the parent DNA strands
serve as the template for the
synthesis of a new strand. Thus
producing two DNA molecules each
with one new and other parental
strand. This is the semi conservative
nature first proposed by Watson and
Crick and proved by Mathew
Meselson and Franklin Stahl in 1957

12. Meselson-Stahl Experiment
Frank Stahl Matthew Meselson

16. VARIOUS PROTEINS INVOLVED IN DNA REPLICATION
DNA replication requires
multiple number of protein.
Some of them are multisubunit
protein complexes. These
include-
Dna A
Dna B or Helicase
Dna C
SSB Protein
Topoisomerase or Gyrase
Primase
DNA Polymerases
DNA Ligase

17. DNA POLYMERASE
Arthur Kornberg isolated the
first DNA Polymerase enzyme
that carry out replication in E.
coli in the year 1957. It is the
DNA Polymerase I.
Later, two other polymerases
were discovered i.e.
polymerase II and polymerase
III.

19. POLYMERASE III IS THE MAIN ENZYME IN E. coli
DNA polymerase III is the main
enzyme for polymerization. It is
a holoenzyme complex
comprising of about 10
subunits. Core enzyme formed
by alpha, epsilon and theta
subunits. Alpha and epsilon are
related to polymerization and
proofreading function.
Thomas Kornberg

20. Subunits of DNA Polymerase III
Protein Subunit Gene Size (kDa)
α dnaE 129.9 DNA synthesis
ε dnaQ 27.5 3′ → 5′ proofreading exonuclease
θ holE 8.6 Stimulates ε
τ dnaX 71.1
Coordinates both halves of Pol III
holoenzyme by linking DnaB with
Pol III, also interacts with primase
γ dnaX 47.5
Subunit of γ complex, β clamp
loading, binds ATP
δ holA 38.7
Subunit of γ complex, interacts
with β
δ′ holB 36.9
Subunit of γ complex, cofactor of
γ ATPase
χ holC 16.6
Subunit of γ complex, interacts
with SSB
ψ holD 15.2
Subunit of γ complex, links χ and
γ
β dnaN 40.6 Processivity, sliding clamp

23. EUKARYOTIC DNA POLYMERASES
Eukaryotes have five classes DNA Polymerases. They are-
1. ALPHA
2. BETA
3. GAMMA
4. DELTA
5. EPSILONSS
The main replicating enzyme is DNA Polymerase Delta.
DNA Polymerase alpha primes the replication process

25. PRIMER-In most cases
primer is a
oligosaccharide of
RNA. It has a free 3’ -
OH group to which a
nucleotide can be
added. This is called
“primer terminus”.
TEMPLATE-It is the
parental strand. The
polymerization
reaction is guided by
template according to
the base-pairing rules
predicted by Watson
and Crick.

26. MECHANISM
Replication occurs in three phases-
Initiation-It involves recognition of the origin.
Elongation-It concerns the events that causes copy of
parent polynucleotides
Termination-The point at which replication process is
halted or stopped.

27. ORIGIN OF REPLICATION
Initiation of replication does
not occur randomly. It always
occurs at definite position or
positions on DNA.
In case of E.Coli it is a 245bp
sequence called “Ori C”.
In yeast replication originates
at ARS or Autonomously
Replicating Sequences.

28. INITIATION OF REPLICATION IN E. coli

30. TOPOLOGICAL PROBLEM
Unwinding of DNA double
helix create a torsional stress
on the non melting portion of
DNA. This is removed by
“topoisomerase or gyrase”
enzyme.
Two kinds of topoisomerases
found .They are-
topoisomerase I and
topoisomerase II.

31. ELONGATION PHASE
Elongation phase
involves two major
operations.-
1. Synthesis of
leading strand
2. Synthesis of
lagging strand

33. DNA replication is Semi-discontinous in Nature

34. In Bacteria Same
strand acts as
both Leading and
Lagging Strand.

35. Maturation of Okazaki Fragment

36. Figure 3.7 Action of DNA ligase in sealing the nick between adjacent DNA
fragments (e.g., Okazaki fragments) to form a longer, covalently continuous
chain. The DNA ligase catalyzes the formation of a phosphodiester bond
between the 3’-OH and the 5’-phosphate groups on either side of a nick, sealing

37. TERMINATION
E. coli has seven terminator sequence . Each one
acting as the recognition site for a sequence
specific DNA binding protein complex called ‘Tus &
Ter’.
Tus allows the replication fork to pass if it is moving
in one direction but blocks its movement in opposite
direction. All this occurs when it is bind to a
termination sequence.

40. Elizabeth H. Blackburn

41. Figure 3.15 Synthesis of telomeric DNA by
telomerase. The example is of Tetrahymena
telomeres. The process is described in the text.
(a) The starting point is the chromosome end
with5’ gap left after primer removal.
(b) Binding of telomerase to the overhanging
telomere repeat at the end of the chromosome.
(c) Synthesis of three-nucleotide DNA segment at
chromosome end, using the RNA template of
telomerase.
(d) The telomerase moves so that the RNA
template can bind to the newly synthesized TTG
in a different way.
(e) Telomerase catalyzes the synthesis of a new
telomere repeat, using the RNA template. The
process recurs, to add more telomere repeats.
(f) After telomerase has left, new DNA is made on
the template, starting with an RNA primer.
(g) After the primer is removed, the result is a
longer chromosome than at the start, with a new
5’ gap.

42. REPLICATION IN EUKARYOTES
Mechanism-
1.Helicases unwind the
strand.
2.Polynucleotides are
separated by RPA.
3.Polymerase alpha has
the priming activity. But
processivity is less
4.Pol.alpha is replaced by
pol.delta.

44. THANK YOU