DNA REPLICATION IN

PROKARYOTES

Salman Ahmed
Lecturer MLT

05/15/2025 1
 Contents
 Cell Cycle

 Replication fact

 DNA Replication in Prokaryotes

 Initiation

 Elongation

 Termination

05/15/2025 2
Cell cycle or Cell division cycle

The cell cycle is the series of events that take place in a cell leading to
its division and duplication of its DNA (DNA replication) to produce two
daughter cells.

or

The cell cycle is a cycle of stages that cells pass through to allow them
to divide and produce new cells.

05/15/2025 3
 Continue…..
Identical Daughter Cells

Two identical
daughter
cells

Parent Cell

05/15/2025 4
 Cell division

All cells are derived from pre-existing cells

New cells are produced for growth and to replace damaged or old cells

Differs in prokaryotes (bacteria) and eukaryotes (protists, fungi, plants, &

animals)

05/15/2025 5
 Keeping the cells identical

The instructions for making cell parts are

encoded in the DNA, so each new cell
must get a complete set of the DNA
molecules

05/15/2025 6
 Types of cell reproduction

Asexual reproduction involves a single cell dividing to make 2 new, identical

daughter cells

example: Mitosis & binary fission are examples of asexual reproduction

Sexual reproduction involves two cells (egg & sperm) joining to make a new
cell (zygote) that is NOT identical to the original cells

example: Meiosis is an example

05/15/2025 7
 Replication facts

• DNA must be copied before a cell divides

• DNA is copied during the S or synthesis phase of interphase
• New cells will need identical DNA strands
• Interphase  G1, S and G2 Phase
• Mitosis  Prophase, Metaphase, Anaphase, Telophase

05/15/2025 8
 Continue…..
• S phase during interphase of the cell cycle
• Nucleus of eukaryotes
S
DNA replication phase
takes
place in the S phase.
G1 interphase G2

Mitosis
-prophase
-metaphase
-anaphase
-telophase

05/15/2025 9
 DNA Replication
In molecular biology DNA replication is the biological process of
producing two identical replicas of DNA from one original DNA
molecule .
• DNA replication was postulated by Watson and Crick after they
discovered the structure of DNA.

• DNA replication is semiconservative, each strand in the double

helix acts as a template for synthesis of a new,
complementary.

• The process of DNA replication is vital for cell growth, repair

and reproduction in organisms.`
05/15/2025 10
 DNA Replication
• DNA replication is the process by which DNA makes a
copy of itself during cell division.

• This process takes us from one starting molecule to two

daughter molecules, with each newly formed double
helix containing one new and one old strand.

• DNA found within the nucleus, must be replicated in

order to ensure that each new cell receives the correct
number of chromosomes. The process of duplication is
call DNA Replication.
05/15/2025 11
 DNA Replication

05/15/2025 12
 DNA Replication

Matthew Meselson & Franklin Stahl, 1958

investigated the process of DNA replication
considered 3 possible mechanisms:
– conservative model
– semiconservative model
– dispersive model

05/15/2025 13
05/15/2025 14
 Steps in Prokaryotic DNA
Synthesis
1. Separation of the two complementary DNA strands
2. Formation of the replication fork
3. Direction of DNA replication
4. RNA primer
5. Chain elongation
6. Excision of RNA primers and their replacement by DNA
7. DNA ligase
8. Termination

05/15/2025 15
 Separation of the two
complementary strand
• DNA replication begins at a single, unique
nucleotide sequence, a site called the origin of
replication, or ori

• The ori includes short, AT-rich segments that

facilitate melting

• In Prokaryotes  Single ori

• In eukaryotes  Multiple ori

05/15/2025 16
05/15/2025 17
 Formation of the replication
fork
• As the two strands unwind and separate, synthesis occurs at two
replication forks that move away from the origin in opposite directions
(bidirectionally) generating a replication bubble

• A group of proteins are required to initiate the replication and to

recognize the ori which are responsible for:

i. maintaining the separation of the parental strands

ii. unwinding the double helix ahead of the advancing replication fork
05/15/2025 18
 Proteins required to form pre-
priming complex
I. DnaA Protein
binds to specific nucleotide sequences within the ori
Melt AT-rich regions in the ori
Melting is adenosine triphosphate (ATP) dependent
II. DNA Helicase (dnaB protein)
• Actually an enzyme  unwind the DNA double helix
• Helicases require energy provided by ATP
• Unwinding at the replication fork causes supercoiling in other
regions of the DNA molecule
05/15/2025 19
 Proteins required to form pre-
priming complex
III. Single strand binding proteins

• This protein binds to the ssDNA generated by helicases

• Binding is cooperative

• The SSB proteins are not enzymes

• These proteins keep the two strands of DNA separated in the area of the
replication origin,

• providing the single-stranded template required by polymerases,

• protect
05/15/2025 the DNA from nucleases that degrade ssDNA. 20
05/15/2025 21
 DNA Topoisomerases

• DNA strands separation

leads to formation of
Positive supercoils in the
region of DNA ahead of the
replication fork as a result of
over-winding

05/15/2025 22
 DNA Topoisomerases

• DNA Topoisomerases are

responsible for removing
supercoils in the helix by
transiently cleaving one
or both DNA strands.

05/15/2025 23
 DNA Topoisomerases

 Type I DNA Topoisomerases

• Cut one strand of DNA

• Does not require ATP

 Type II DNA Topoisomerases
• Cut both strands
• Require ATP

05/15/2025 24
 • DNA gyrase, a type II topoisomerase found in
bacteria and plants, has the unusual property of
being able to introduce negative supercoils into
circular DNA
• Negative supercoils neutralize the positive
supercoils introduced during opening of the
double helix
• Anticancer agents, such as the camptothecins,
target human type I topoisomerases,
• Etoposide targets human type II
topoisomerases.
• fluoroquinolones targets Bacterial DNA gyrase
and act as antimicrobial agents
05/15/2025 25
05/15/2025 26
 DNA Polymerases

• DNA polymerases: Chief enzyme mainly involved in

repair and deoxynucleotide polymerization.
• In prokaryotes: Three types of DNA polymerases found.
• DNA polymerase I,
• DNA polymerase II and
• DNA polymerase III.
• In eukaryotes: There are five types of DNA polymerases
• α, ε, β, γ and δ.

05/15/2025 27
 DNA Polymerases

05/15/2025 28
 Direction of DNA replication

• The DNA polymerases for DNA Replication

• Read” the parental nucleotide sequences in the 3ʹ→5ʹ direction,

• Synthesize the new DNA strands only in the 5ʹ→3ʹ (antiparallel)

direction.

• two newly synthesized DNA strands must grow in opposite directions,

• One in the 5ʹ→3ʹ direction toward the replication fork and one in the
5ʹ→3ʹ direction away from the replication fork
05/15/2025 29
 Leading & Lagging strands
1. Leading strand:
• The DNA is synthesized continuously in 5’ → 3’ direction with same
over-all forward direction.
• The strand that is being copied in the direction of the advancing replication
fork is called the leading strand and is synthesized continuously.
2. Lagging strand (Retrograde strand):
• The DNA is synthesized in a discontinuous manner.
• The strand that is being copied in the direction away from the replication
fork
• Okazaki fragments
• Okazaki fragments, are eventually joined (ligated) to become a single,
continuous
05/15/2025 strand 30
05/15/2025 31
 RNA Primer
• DNA polymerases cannot initiate synthesis of a complementary
strand of DNA on a totally single-stranded template.

• They require an RNA primer, which is a short, double-stranded

region consisting of RNA base paired to the DNA template, with a
free hydroxyl group on the 3ʹ-end of the RNA strand

• This hydroxyl group serves as the first acceptor of a deoxynucleotide

by action of a DNA polymerase
05/15/2025 32
 Primer DnaG

• A specific RNA polymerase, called primase (DnaG), synthesizes

the short stretches of RNA (approximately ten nucleotides long)

• Short RNA sequences are constantly being synthesized at the

replication fork on the lagging strand,

• Only one RNA sequence at the origin of replication is required on

the leading strand

05/15/2025 33
 Chain Elongation
• DNA polymerases (DNA pol) elongate a new DNA strand by
adding deoxyribonucleotides, one at a time, to the 3ʹ-end of the
growing chain
• DNA polymerase III:
• Multisubunit enzyme,
• Adds new nucleotides to the 3ʹ- hydroxyl group of the RNA primer
• DNA pol III is a highly “processive” enzyme (that is, it remains
bound to the template strand as it moves along and does not
diffuse away and then rebind before adding each new nucleotide

05/15/2025 34
 Chain Elongation Continue…….
• All four substrates
• Deoxyadenosine triphosphate [dATP],
• Deoxythymidine triphosphate [dTTP],
• Deoxycytidine triphosphate [dCTP], and
• Deoxyguanosine triphosphate [dGTP])
• Must be present for DNA elongation to occur
• If one of the four is in short supply, DNA synthesis stops when that
nucleotide is depleted

05/15/2025 35
 Proofreading of newly
synthesized DNA
• All four substrates
• Deoxyadenosine triphosphate [dATP],
• Deoxythymidine triphosphate [dTTP],
• Deoxycytidine triphosphate [dCTP], and
• Deoxyguanosine triphosphate [dGTP])
• Must be present for DNA elongation to occur
• If one of the four is in short supply, DNA synthesis stops when that
nucleotide is depleted

05/15/2025 36
 DNA Ligase
• The final phosphodiester linkage between the 5ʹ-phosphate group
on the DNA chain synthesized by DNA pol III and the 3ʹ-hydroxyl
group on the chain made by DNA pol I is catalyzed by DNA ligase
• ATP is required

05/15/2025 37
 Termination
• Replication termination in E. coli is mediated by sequence-specific
binding of the protein,

• Tus (terminus utilization substance) to replication termination sites

(ter sites) on the DNA, stopping the movement of DNA polymerase

References

• Lippincott’s Illustrated Reviews:BiochemistryFifth Edition

05/15/2025 38
 for attending….

Be Aware – Be Healthy – Be Safe

05/15/2025 39