DNA replication is the process by which a cell makes an exact copy of its DNA. In prokaryotes like E. coli, replication occurs through a theta mechanism originating from a single origin of replication site called oriC. It is semi-conservative, semi-discontinuous, and bidirectional. Initiation involves melting of the DNA duplex by DnaA protein, followed by binding of helicase, primase, and polymerase to form the prepriming and primase complexes. Elongation then extends the RNA primers to synthesize leading and lagging strands, with lagging strands synthesized discontinuously as Okazaki fragments. Termination occurs at specific terminator sequences aided by Tus protein.

1. P R E S E N T E D B Y
P R I Y A N C K A A R O R A
A S S I S T A N T P R O F E S S O R
D E P A R T M E N T O F B I O T E C H N O L O G Y
F A C U L T Y O F E N G I N E E R I N G A N D T E C H N O L O G Y
R A M A U N I V E R S I T Y , K A N P U R , U T T A R P R A D E S H
Replication of DNA

2. Introduction
 It is an enzymatic process in which the DNA makes its
own copy. It replicates in order to carry out the process
of cell division.
Properties of replication-
 It take place from 5’ to 3’ direction.
 Occurs in semi-discontinuous fashion i.e. in one strand
the replication takes place continuously (leading strand),
where as in the other strand, replication occurs
discontinuously that means in a form of small fragment
termed as okazaki fragments, forming a lagging strand.
 Occurs in semi-conservative manner, i.e. each strand in a
DNA double helix acts as template for the synthesis of
new, complementary strand.

3. Replication
The figure is depicting replication is semi-conservative, semi-discontinuous, bi-
directional and takes place from 5’ to 3’ direction.

4. Mechanism
1. Initiation
 Replication starts at specific site called origin of replication. This is
the site from where every time replication begins.
 In Prokaryotes, it is referred as ‘oriC’ and it is approximately 245bp
in length and is rich in AT sequences, comprising of 9 nucleotide
motif of 5 repeats and 13 nucleotide motif of 3 repeats.
 These regions are postulated to help melt the DNA duplex in the oriC
region for initiation of DNA replication.

5.  A common type of replication that takes place in
circular DNA, such as that found in E. coli and other
bacteria, is called theta replication because it
generates a structure that resembles the Greek letter
theta (θ).
 In prokaryotes, there is a presence of single copy of
oriC, where as, in Eukaryotes, there a presence of
multiple copies of origin of replication (For example-
In yeast approximately, 300 copies are present and
in Humans, 20,000 copies of origin of replication are
present.)

6. Theta mode of replication in prokaryotes
Single origin of
replication called
oriC
Bi-directional movement of
replication fork
Theta mode of replication

7. Steps of Initiation
1. dnaA protein binds to the '9-mer' region in oriC and forming
a multimeric complex with 10-20 protein subunits (i.e. at a
single oriC region there will be bound 10-20 dnaA protein
molecules).Binding requires ATP.
2. dnaA protein binds to 9 nucleotide motive repeats and
forms barrel like structure, upon which the DNA gets
wrapped. This generates the torsional stress, which is
responsible to melt the DNA at the region of 13 nucleotide
motif repeats.
3. Two dnaB protein binds to single stranded DNA of the oriC
DNA segment, one at each end. dnaC helps in recruitment of
dnaB at site in fork. The dnaB is a helicase enzyme which
helps in further unwinding of DNA, in both the direction.
4. This stage represents prepriming complex.

8.  Separated strands in the oriC region are prevented from reannealing by
the binding of single-stranded binding protein (ssb protein).
 Primase binds to dnaB protein at oriC and forms a primosome. In
prokaryotes, dnaG protein acts as a primase.
 The primase within the primosome complex provides RNA primers for
synthesis of both strands of duplex DNA.
 Primase lays down tracks of few complimentary nucleotides (7-10).

9. Initiation
Prepriming complex Primosome complex

10. Elongation
 With the action of helicase, the strand ahead of the replication fork starts to
overwind. The enzyme Topoisomerase, releases the supercoiling in the DNA
strand and maintains the topology of the DNA by binding to the DNA strand and
breaking the phosphate bond in either one or both strands.
 After synthesis of the 9-12 mer RNA primer, DNA Pol III holoenzyme enters the
replication fork and is able to utilize the RNA as a primer for DNA synthesis.
 The addition of nucleotide or the extension of RNA primer is done using DNA
polymerase enzyme. In Prokaryotes, DNA Polymerase III is used in extension of
primer to form daughter DNA strand.

11.  At each growing fork, one strand called leading strand is synthesized continuously
from single primer on the leading strand template and grows in 5’-3’ direction.
 As the synthesis can be proceeded in only 5’-3’ direction, the synthesis of daughter
strand of 5’-3’ template develops gaps in between, forming the lagging strand.
 Various primers binds at various points in the lagging stand.
• Primase can bind to the Pol III complex, but the arrangement of the DNA
strand as it passes through the Pol III/primase complex is quite unique, in
lagging strand. It forms a loop structure such that primase and the Pol III
active site can accomplish discontinuous synthesis of the lagging template
strand even though the general direction of the Pol III complex is opposite to
the require direction of DNA synthesis.

12. Loop of DNA Polymerase III activity

13.  After primase makes another primer on the lagging template, the adjacent Pol
III active site can extend the primer (incorporating dNTP's) by utilizing the
same loop structure and feeding the template through in the direction shown.
• The lagging strand loop cannot be fed through the Pol III complex forever, and
after a nascent DNA strand is synthesized the loop is released and a new one is
formed using the opened template DNA further up the fork:
• As synthesis continues:
 there will be a single continuous DNA strand on the leading strand
 there will be a series of short fragments on the lagging strand,
containing both RNA and DNA, called Okazaki fragments.

15. Primer removal
 How are these RNA/DNA fragments converted into one
long continuous DNA strand? The RNA could be
removed by a polymerase which has 5'->3' exonuclease
activity, however, DNA Polymerase III lacks this
activity.
 This activity is carried out by DNA polymerase I in case
of prokaryotes.
 DNA polymerase I digests the RNA primer using its 5’-3’
exonuclease activity and extends the gaps by its 5’-3’
polymerization activity.
 DNA Pol I leaves and DNA ligase then joins these
discontinuous DNA fragments to form a continuous DNA
duplex on the lagging strand.

16. Overview of Initiation and Elongation of DNA replication on
Prokaryotes
General Overview of a DNA Replication Fork. At the origin of replication, topoisomerase II relaxes the
supercoiled chromosome. Two replication forks are formed by the opening of the double-stranded DNA at the origin,
and helicase separates the DNA strands, which are coated by single-stranded binding proteins to keep the strands
separated. DNA replication occurs in both directions. An RNA primer complementary to the parental strand is
synthesized by RNA primase and is elongated by DNA polymerase III through the addition of nucleotides to the 3′-
OH end. On the leading strand, DNA is synthesized continuously, whereas on the lagging strand, DNA is synthesized
in short stretches called Okazaki fragments. RNA primers within the lagging strand are removed by theexonuclease
activity of DNA polymerase I, and the Okazaki fragments are joined by DNA ligase.

17. Summary of steps in E. coli DNA Synthesis
 dnaA protein melts duplex in oriC region.
 dnaB (helicase), along with dnaC and ATP binds to replication fork (dnaC protein exits).1 (Pre-
priming complex)
 Single strand binding protein (ssb protein) binds to separated strands of DNA and prevents
reannealing.
 Primase complexes with helicase, creates RNA primers (pppAC(N)7-10) on the strands of the
open duplex2 (Primase+helicase constitute the Primosome).
 After making the RNA primers, DNA pol III holoenzyme comes in and extends the RNA
primer (laying down dNTP's) on the leading strand.
 As the replication fork opens up (via helicase + ATP action) leading strand synthesis is an
uninterrupted process, the lagging strand experiences a gap.
 The gap region of the lagging strand can wind around one active site unit of the Pol III complex,
and bound Primase initiates an RNA primer in the gap region3.
 On the lagging strand, Pol III extends the RNA primer with dNTP's as the lagging template
strand is looped through the Pol III complex
 After synthesis of a nascent fragment the lagging strand loop is released and the single strand
region further up near the replication fork is subsequently looped through the Pol III complex.
 Steps 7-9 are repeated.
 Meanwhile, Pol I removes the RNA primer regions of the Okazaki fragments via 5' to 3'
exonuclease activity ( nick translation
 Pol I exits and ligase joints the DNA fragments (on lagging strand).

18. Termination
 Proper termination of DNA replication is important for genome stability. E.
coli replication terminates in the region opposite oriC.
 In case of prokaryotes, termination occurs at particular sequence called
Ter-sequence, assisted by the protein called Tus protein (Terminus
Utilization Substance).
 Ter sequence has multiple copies of about 23 bp.
 When bound to the terminator sequence, a tus protein allows replication
fork to pass if the fork is moving in one direction, but blocks progress if the
fork is moving is moving in opposite direction i.e. This protein may act to
prevent helicase from unwinding DNA (will therefore halt pol III and pol
I action). Thus they permit movement of helicase only in one direction and
doesn’t permit its direction in other direction.

19. Termination
There is a formation of catenated daughter DNA in prokaryotes, which is resolved by
enzyme decatenase, which is Topoisomerase IV.

20. List of proteins and enzymes which will bind
at replication fork
 dna A- Initiator protein. Melts/denatures DNA at oriC, exposing two template ssDNA
strands.
 dna B- It is a helicase. It unwinds the DNA helix at the front end of each replication fork
during replication.
 dna C- It a helicase loader. It loads the dnaB helicase onto ssDNA template strand.
 dnaG- Primase. Synthesizes RNA primers used to initiate DNA synthesis.
 ssbp- Single stranded binding protein. Binds with single stranded regions of DNA in
replication fork and prevents the strand from rejoining.
 DNA gyrase- acts as topoisomerase. It regulates DNA supercoiling by catalyzing the
winding and unwinding of DNA strands. They do this by making the incision that breaks
the DNA backbone, so they can pass the DNA strand through one another,
relaxing/coiling the DNA before resealing the breaks.
 DNA ligase- fixes nicks in the DNA backbone during DNA replication, DNA damage,
and DNA repair processes. It forms phosphodiester bond between two nucleotides.
 β- clamp- It acts as a sliding clamp in prokaryotes replication. The sliding clamp is a
ring-shaped protein that encircles duplex DNA, binds to the DNA polymerase and tethers
it to the DNA template, preventing its dissociation and providing high processivity.
 γ clamp loader- Clamp loaders are so called for their action in loading ring-shaped
sliding clamps onto DNA.

21. Prokaryotic DNA polymerases and their
functions
Type of DNA
Polymerase
5’-3’
polymerizing
activity
5’-3’
exonuclease
activity
(Primer
removal)
3’-5’
exonuclease
activity (Proof
reading
activity)
Function
DNA
Polymerase I
Yes Yes Yes Removal of
primer, DNA
repair, gap filing
DNA
Polymerase II
Yes No Yes DNA repair
DNA
Polymerase III
Yes No Yes Main replicating
enzyme
DNA
Polymerase IV
Yes No No Translesion
DNA repair
DNA
Polymerase V
Yes No No Translesion
DNA repair