This is a brief account of the process of DNA replication in prokaryotes, including the prerequisites and the process.

1. SYED MUHAMMAD KHAN (BS HONS. ZOOLOGY)
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DNA REPLICATION IN PROKARYOTES
DNA has two important functions: (1) Heterocatalytic function: When DNA directs the
synthesis of molecules other than itself, i.e. synthesis of RNA, proteins, etc. (2) Autocatalytic
function: When DNA directs the synthesis of itself, i.e. DNA replication.
MODELS OF DNA REPLICATION:
The following are the models of DNA replication, the most authentic one is the
semiconservative model but other models also exist (they are not that widely accepted):
Semiconservative Replication: According to this model, each of the two double helices or
duplexes of the newly synthesized DNA conserves only one of the parent polynucleotide
strands (Semiconservative – half conservative). According to Watson-Crick’s model of DNA
structure and replication, once DNA replication is initiated, the two original polynucleotide
strands of the duplex or helix will unwind, at least locally, so that each can serve as a
template for a new strand. This means that both duplexes that result from replication are
hybrid (containing an old strand derived from the original molecule and a new strand which
has been formed during the replication process).
Conservative Replication: According to this model, both strands of the parent double helix
would be conserved and the new DNA molecule would consist of two newly synthesized
strands.
Dispersive Replication: According to this model, replication involves fragmentation of the
parent double helix and the intermixing of pieces of the parent strands with newly
synthesized pieces, hence forming the two new double helices.
MESELSON & STAHL’S EXPERIMENT:
M. Meselson and F.W. Stahl (1958) verified the semiconservative nature of DNA replication
via their experiment. They cultured Escherichia coli cells in a medium in which the nitrogen
was 15N (a ‘heavy’ isotope of nitrogen, but not a radioisotope) instead of commonly
occurring and lighter 14N. Gradually, the nitrogenous bases of DNA in new cells contained
15N (where 14N normally occurs), i.e. the DNA molecules were denser. DNA in which the
nitrogen atoms are heavy (15N) can be distinguished from DNA containing light nitrogen
(14N), because, during centrifugation, the two different DNAs band at different density
positions in the centrifuge tube, i.e. the DNA made up of 15N would band lower than 14N
containing DNA.
E. coli cells grown for some time in the presence of 15N-medium were washed free of the
medium and transferred to 14N-containing medium and allowed to continue to grow for
specific lengths of time (i.e., for various numbers of generation time). DNA isolated from

2. SYED MUHAMMAD KHAN (BS HONS. ZOOLOGY)
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cells grown for one generation of time in the 14N medium had a density intermediate to that
of the DNA from cells grown only in 15N-containing medium and that of DNA from cells
grown only in 14N-containing medium, i.e. this hybrid DNA consisted of DNA molecules in
which one strand contained 15N and the other contained 14N.
When the cells were kept in the 14N-medium for two generations of time, two DNA bands
were formed: (1) one at the same density position as the DNA from cells grown exclusively
in 14N medium and (2) the other of intermediate density. Subsequent generations produced
greater numbers of DNA molecules that banded at the ‘‘light’’ (14N-containing DNA) position
in the density gradient. These results prove the model of semiconservative replication.
Figure: Meselson & Stahl’s Experiment.
ENZYMES OF DNA METABOLISM:
Nucleases, polymerases, and ligases are enzymes of DNA metabolism in both prokaryotes
and eukaryotes.

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NUCLEASES:
Nuclease enzymes act to hydrolyze or break down a polynucleotide chain into its
component nucleotides. The nuclease enzymes may be of the following two kinds:
(a) Exonucleases: Exonucleases are nuclease enzymes which begin their attack from a free
end of a polynucleotide. Depending on the specificity of the enzyme, an exonuclease will
either begin at a free 3'-OH end of a polynucleotide or a free 5'-P end. In both cases the
enzyme travels along the chain in a stepwise manner, liberating single nucleoside
monophosphate molecules and eventually digesting the entire polymer.
(b) Endonucleases: Endonuclease enzymes attack one of the two sides of phosphodiester
linkages, but they react only with those bonds that occur within the interior of a
polynucleotide chain.
POLYMERASES/REPLICASES:
A polymerase enzyme catalyzes the formation of a polymer, i.e. DNA polymerases are
involved in the formation of DNA – a polymer. DNA polymerase adds nucleotides
(triphosphates) to the DNA chain (that contains monophosphates). Phosphate of a
nucleoside triphosphate molecule forms a 3', 5' phosphodiester bond with a free 3'-OH in
the growing polynucleotide chain, and a molecule or pyrophosphate (P~P) is simultaneously
released. Pyrophosphate contains a ‘‘high-energy’’ bond, meaning that when the released
pyrophosphate is hydrolyzed into two phosphate molecules, energy is liberated which drives
the polymerization process forward. The resultant polymerization will always proceed in a
net 5´→3´ direction.
There are three types of DNA polymerases in prokaryotes, DNA polymerase I and II are
meant for DNA repair, and DNA polymerase III is meant for actual DNA replication.
 DNA Polymerase I: It is also called the Kornberg enzyme (after its discoverer – Arthur
Kornberg). It is a DNA repair enzyme. It is mainly involved in removing RNA primers from
Okazaki or precursor fragments and filling the resultant gaps, and it can also remove
thymine dimers produced due to UV-irradiation and fill the gap (these are called the
proofreading or editing functions of DNA polymerase I). It has five active sites, namely:
(1) template site, (2) primer site, (3) 5´ → 3´ cleavage or exonuclease site, (4) nucleoside
triphosphate site, and (5) 5´ → 3´ cleavage site (or 5´ → 3´ exonuclease site).
 DNA Polymerase II: It resembles DNA polymerase I and is also a repair enzyme.
 DNA Polymerase III: DNA polymerase-III plays an essential role in DNA replication. It is a
multimeric (having multiple subunits) enzyme or holoenzyme having ten subunits. The
core enzyme comprises three subunits: α, β, and θ. The remaining seven subunits

4. SYED MUHAMMAD KHAN (BS HONS. ZOOLOGY)
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increase processivity (processivity means rapidity and efficiency with which a DNA
polymerase extends growing chain).
LIGASES:
DNA ligase enzymes catalyze phosphodiester bond formation between free 3´-OH and free
5´-P groups of a nick of DNA – they join DNA fragments together.
RNA PRIMERS & DNA REPLICATION:
Primers are short RNA segments that are complementary to a short DNA sequence, to which
they bind, they are necessary for the functioning of DNA polymerases. DNA polymerase
can’t initiate the synthesis of DNA without the availability of a primer RNA strand. Before
actual DNA replication starts, short RNA segments, called RNA primers or simply primers,
have to be synthesized by DNA primase enzyme (utilizing ribonucleoside triphosphates).
This RNA primer is synthesized by copying a particular base sequence from one DNA strand
and differs from a typical RNA molecule in that after the synthesis the primer remains
hydrogen-bonded to the DNA template. The primers are about 10 nucleotides long in
eukaryotes and they are made at intervals on the lagging strand where they are elongated
by the DNA polymerase enzyme to begin each Okazaki fragment. These RNA primers are
later excised (cut) and filled with DNA with the help of a DNA repair system in eukaryotes
(or DNA polymerase I in prokaryotes).
REPLICONS:
Replicons are discrete units in which DNA replication in prokaryotes and eukaryotes is done.
The number of replicons may vary in a genome; there is just one in bacteria (E.coli) and 500
in yeast to several thousand in plants and animals (eukaryotes have more replicons than
prokaryotes). In the E. coli, there is a single replicon with an origin and termination sites
(finishing sites).
PROTEINS INVOLVED IN THE OPENING OF DNA HELIX:
Helicases, SSB proteins, and Topoisomerases are involved in the opening of DNA helix, their
details are as follows:
DNA HELICASES:
DNA helicases (DNA B Proteins) are ATP dependent unwinding enzymes that promote
separation of the two parental strands and establish replication forks – for DNA replication.
SINGLE STRAND DNA BINDING PROTEINS (SSBPs):
SSBPs prevent the single DNA strands from rewinding about one another (or forming
double-stranded hairpin loops in each single strands), during replication. SSB proteins bind

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to exposed DNA strands without covering the bases, which, therefore, remain available for
the templating process (to be used as templates).
TOPOISOMERASES (DNA GYRASES):
Topoisomerases are enzymes that change the topology of DNA by changing its linking
number (Lk) by introducing a nick/cut in the DNA. There are two types of topoisomerases:
Topoisomerase I and Topoisomerase II. Topoisomerase I changes Lk in units of one by
breaking a single strand of DNA and allowing the duplex to unwind. Whereas,
Topoisomerase II changes Lk by units of two by breaking both strands, creating a gate
through which a second segment of helix is passed.
REPLISOME & PRIMOSOME:
Replisome is a multienzyme complex formed by the association of DNA polymerases, RNA
primases, and helicases, which carries out the synthesis of leading and lagging strands in a
coordinated fashion.
Primosome is the association of the primase molecule and DNA helicase at a replication fork
to form a replication machine, on the lagging strand, which moves with the fork,
synthesizing RNA primers.
MECHANISM OF DNA REPLICATION IN PROKARYOTES
DNA replication in prokaryotes involves two main steps: (1) Initiation and (2) Elongation,
their details are as follows:
1) INITIATION:
This process comprises three steps: (1) recognition of the origin (O), (2) opening of DNA
duplex to generate a region of single-stranded DNA, and (3) capture of Helicase (DNA B
protein).
The opening requires negatively supercoiled DNA and initiator proteins. Helicase (DNA B
protein) is transferred to exposed single-stranded DNA and causes unwinding of the DNA in
the presence of ATP, SSB protein, and DNA gyrase (a topoisomerase). This results in the
unwinding of DNA duplex and the replication from origin proceeds in both directions
(bidirectional); SSB binding occurs on single-stranded regions and primosomes are loaded
on both strands.
2) ELONGATION:
This step requires the presence of the following enzymes and factors:
1. Helicase (DNA B)

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2. Primase (DNA G)
3. DNA polymerase holo-enzyme (DNA pol III HE)
4. SSB proteins
5. RNAse H – it removes RNA primers
6. DNA polymerase I – it fills the gaps created by RNAse H
7. DNA ligase
The steps of elongation of the DNA chain are as follows:
 As helicase (DNA B) travels in 5´ → 3´ direction, it generates a replication fork by
opening the DNA duplex. The DNA strand having helicase becomes the lagging strand.
 DNA primase associates with helicase, forming the primosome which synthesizes
multiple primers for lagging strand and single RNA primer for the leading strand.
 For the synthesis of the lagging strand, the DNA POL III HE (holo-enzyme) has to work
on the same strand to which helicase is bound, but it travels in the opposite direction
(on the leading strand, it travels in the same direction).
Figure: DNA replication.
Synthesis (elongation) of lagging (the strand that proceeds away from replication fork) and
leading (the strand that proceeds towards the replication fork) strands takes place by
different methods:
(a) Lagging Strand – Discontinuous Synthesis:
1. Primase synthesizes an RNA primer (10 to 20nt or nucleotides long) on the lagging
strand.
2. The RNA primers are recognized by DNA POL III HE (DNA Polymerase III) on the lagging
strand and are utilized for synthesis of precursor or Okazaki fragments (1000-2000
nucleotides in prokaryotes).

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3. On completion of the Okazaki fragments, the RNA primers are excised (cut out) by DNA
Polymerase I, which then fills the resulting gaps with DNA.
4. Afterward, the enzyme DNA ligase forms the phosphodiester bond that links the free 3´
end of the primer replacement (deoxyribonucleotides – that replaced the primers) of
the 5´ end of the Okazaki fragment.
(b) Leading Strand – Continuous Synthesis:
1. In bidirectional DNA replication, the leading strand is primed once (just one RNA
primer).
2. The RNA primer of the leading strand is synthesized by RNA polymerase enzyme (not
primase).
3. DNA POL III HE causes elongation of the leading strand and finally DNA POL I and ligase
enzymes give the final touch to the leading strand as in the case of the lagging strand.
ROLLING CIRCLE MODEL OF DNA REPLICATION
Rolling circle DNA replication is a type of DNA replication that occurs in viruses, and bacteria
(such as E. coli) – during mating. In this model, a nick is made in the circular DNA and this
nick has 3'- OH and 5' –P termini (singular – terminus). Under the influence of a helicase and
SSB protein, a replication fork is generated. Synthesis of a primer is unnecessary, leading-
strand synthesis proceeds by elongation from 3'-OH terminus. The parental template for the
lagging strand is displaced. It is also called as σ replication.
ROLLING CIRCLE DNA REPLICATION IN PROKARYOTES (E. coli):
The steps in the rolling circle model of DNA replication are as follows:
 Rolling circle DNA replication is initiated by an initiator protein, which nicks one strand
of the double-stranded, circular DNA molecule at a site called the double-strand origin
(DSO).
 The initiator protein remains bound to the 5' phosphate end (5’ – P terminus) of the
nicked strand, and the free 3' hydroxyl end (3’-OH terminus) is released to serve as
a primer for DNA synthesis by DNA polymerase III.
 Using the un-nicked strand as a template, replication proceeds around the circular DNA
molecule, displacing the nicked strand as single-stranded DNA.
 Displacement of the nicked strand is carried out by a helicase in the presence of the
plasmid replication initiation protein.

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 Continued DNA synthesis can produce multiple single-stranded linear copies of the
original DNA. These linear copies can be converted to double-stranded circular
molecules through the following process:
1. First, the initiator protein makes another nick in the DNA to terminate the synthesis
of the first (leading) strand.
2. RNA polymerase and DNA polymerase III then replicate the single-stranded origin
(SSO) of DNA to make another double-stranded circle.
3. DNA polymerase I removes the primer, replacing it with DNA, and DNA ligase joins
the ends to make another molecule of double-stranded circular DNA.
Figure: Rolling Circle Model of DNA Replication (SS stands for single-stranded and DS stands
for double-stranded) in prokaryotes/bacteria.