Faculty of

Medicine
Medical
Education-
Damietta
University

Level 1
Semester 1
Module 1B
Instructor information:

Name : Dr Hazem Moh Abdullah

Official e-mail.: drhazem_abdullah@du.edu.eg

Mobile Number: 01146710982

Office hours: any time

DNA Synthesis
(Replication)
Content of lecture
• Contents:
• Definition, biological importance & general criteria of DNA
replication.
• DNA polymerase enzyme & proteins needed for DNA replication
• Steps of DNA replication
• Post replication modification of DNA
• Eukaryotic & prokaryotic DNA replication
Learning outcomes ILOs:

• List enzymes & proteins needed for DNA replication

• Recognize the steps of DNA replication
• Differentiate eukaryotic & prokaryotic DNA replication.
• Correlate DNA replication to uses of some pharmacological drugs.
Case Scenario or Clinical correlate

• A 23-year-old diabetic woman reports having fevers

and dysuria with the presence of bacteria in her
urine.Her physician suspects a complicated urinary
tract infection and begins a 5-day course of
ciprofloxacin. Such quinolone antibiotics inhibit which
one of the following enzymes?
A. Eukaryotic topoisomerase
B. Helicase
C. Primase
D. Gyrase
E. Poly(A) polymerase
 Learning outcome 1
Identify DNA replication & its general criteria
DNA Synthesis (Replication)

• Replication is the process by which an identical copy of DNA is made.

• Replication occurs every time a cell divides so that information can be
preserved and given to offspring.
• https://www.youtube.com/watch?v=G1AoVF3k9Hg

Just before mitosis

 Principles of DNA SYNTHESIS
(REPLICATION
• Replication is semiconservative
• Both strands serve as template “simultaneously”.
• Replication is bidirectional.
• Direction of replication: “5′  3′ ”.
• Replication is semi-discontinuous.
• Separation of The two strands of the
DNA are.
• Each strand serves as a template for
the replication of a new
complementary strand.
• This produces two daughter DNA
molecules.
• Each of the newly formed DNA
molecules contains one of the original
(parental) strands and a new
complementary strand.
• semiconservative replication.
 2. Bidirectional replication
- Involves two replication forks, which move in
opposite directions.
 3. Semi-discontinuous replication
- The leading strand copies continuously.
- The lagging strand copies in segments (Okazaki
fragments) which must be joined.
 Components of DNA replication
• DNA template.
• Precursors: dATP, dGTP, dCTP, dTTP.
• Cofactors: Mg++, Mn++, ATP.
• Enzyme:DNA-dependent-DNA-polymerases.
Learning outcome 2
• Recognize the steps of DNA replication

PROKARYOTIC DNA
SYNTHESIS
Steps of DNA synthesis

1) RNA primer is synthesized by primosome.

2) Then, the RNA primer is elongated by DNA polymerase III until
another RNA stretch is encountered.
3) Proofreading: (3′→5′ exonuclease).
4) Then, DNA polymerase I excises the RNA primer and fill the
resulting gap.
5) The remaining nick is joined by DNA ligase.
Supercoiled DNA relaxed by topoisomerase & unwound by helicase + proteins:

5’ SSB Proteins
Okazaki Fragments
1 ATP

Polymerase III 2
Helicase
Lagging strand 3 +
Initiator Proteins

3’
primase base pairs

Polymerase III 5’

RNA primer replaced by polymerase I

& gap is sealed by ligase

Leading strand
RNA Primer

3’
2. Proofreading of newly synthesized DNA

• Then, the RNA primer is elongated by DNA polymerase III until

another RNA stretch is encountered.
• Proofreading: (3′→5′ exonuclease).
• Then, DNA polymerase I excises the RNA primer and fill the resulting
gap.
• The remaining nick is joined by DNA ligase.
Prokaryotic DNA-dependent-DNA polymerase

- DNA polymerase III:

- 5→ 3 polymerase activity.
- 3→ 5 exonuclease activity (proofreading).

- DNA polymerase I:
- 5→ 3 polymerase activity.
- 3→ 5 exonuclease activity (proofreading).
- 5→ 3 exonuclease activity (excise RNA primer).
G. DNA ligase

• The final phosphodiester

linkage between the 5'-
phosphate group on the
DNA chain synthesized by
DNA polymerase III and the
3'hydroxyl group on the
chain made by DNA
polymerase I is catalyzed by
DNA ligase.
• This reaction requires the
cleavage of ATP to AMP +
PPi.
 Replication fork / Replication bubble
3 5

5 3

DNA polymerase III

leading strand
5
3 3 5
5 5
5 3 3
lagging strand

3 5
5
3 lagging strand leading strand
5 growing
3 replication fork 5
5 growing
replication fork 5
leading strand 3
lagging strand
3
5
5 5
Starting DNA synthesis: RNA primers
Limits of DNA polymerase III
◆ can only build onto 3 end of an
existing DNA strand
5

3 5 3
5
3
3 5

growing 3 primase
replication fork DNA polymerase III
5

RNA 5

RNA primer 3

◆ built by primase
◆ serves as starter sequence for
DNA polymerase III
 Replacing RNA primers with DNA
DNA polymerase I
◆ removes sections of RNA primer and
replaces with DNA nucleotides
DNA polymerase I
5

3

3
5 ligase
growing 3
replication fork
5

RNA 5

3

◆ But DNA polymerase I still can only build

onto 3 end of an existing DNA strand
 Leading strand Lagging strand

1-Direction Towards replication fork Away from replication fork

One only at start multiple
2-Primer Continuous Discontinuous
3-Method of replication
4-Okazaki fragments Absent Present
5-Ligase enzyme
Absent present
Learning outcome 3
Differentiate eukaryotic & prokaryotic DNA replication

EUKARYOTIC DNA SYNTHESIS

• The main steps of DNA synthesis in eukaryotes are nearly similar to prokaryotes
and include the following:
I- Separation of the two strands of DNA at multiple specific
sites
[multiple origins of replication, (ori)].
These sites contain short sequence composed only of (AT)
base pairs
(consensus sequence).
3- Unwinding and separation of the two strands
• Catalyzed by DNA helicases.

• This process starts at each replication fork and

proceeds on both directions.

• The two strands of DNA are kept away and separated

by the single strand binding proteins.
4- Synthesis of the two complementary DNA strands
(leading and lagging strands)

• This is catalyzed by DNA polymerases.

• This step utilizes deoxynucleoside triphosphates (dATP. dGTP.

dCTP and dTTP).

• The different bases are arranged in the complementary

strands according to the sequence present in the parental
strands.

• The presence of a large number of DNA polymerases (more

than 20.000 compared to few dozen in E. coli cell) helps to
decrease the time needed for replication in eukaryotic cell.
Eukaryotic DNA Polymerases
1. Pol α:
• Pol α is a multi-subunit enzyme.
• One subunit has primase activity, which initiates strand synthesis on the leading strand and
at the beginning of each Okazaki fragment on the lagging strand.
• The primase subunit synthesizes a short RNA primer that is extended by the pol α 5' → 3'
polymerase activity, generating a short piece of DNA.

2. Pol ε and pol δ:

• Pol ε complete DNA synthesis on the leading strand.
• Pol δ elongates the Okazaki fragments of the lagging strand.
• Each proofread the newly synthesized DNA using 3' → 5' exonuclease activity.

3. Pol β and pol γ:

• Pol β is involved in "gap filling" in DNA repair.
• Pol γ replicates mitochondrial DNA.
 Synthesis of the Leading and Lagging Strands of DNA

• The DNA polymerases are responsible for synthesis of both strands of DNA.

• They require RNA primers synthesized by primase activity of DNA

polymerase .

• They are only able to read the parental nucleotide sequence in the 3` to
5`direction.

• This can be completed by two different mechanisms on each strand (similar

to prokaryotes).
• Leading Strand:
• This strand is replicated by the DNA polymerase ε in the direction of the advancing
replication fork.
• It is and synthesized in a continuous manner.

• Lagging Strand:
• This strand is replicated by the DNA polymerase δ in the opposite direction of the advancing
replication fork.
• It is synthesized in a discontinuous manner.
• It is formed in the form of small fragments termed Okazaki fragments (each is formed of
small DNA segment connected to RNA primer).
5- Excision of RNA primers
• Removal of RNA primers by RNase H and FEN1 (Flap Endonulcease 1).
• Once the primers are removed, a free-floating DNA polymerase lands at the 3'
end of the preceding DNA fragment and extends the DNA over the gap.

6. Ligation of different DNA fragments:

• By DNA ligase.
• This needs ATP
 Okazaki
Leading & Lagging strands
Limits of DNA polymerase
◆ can only build onto 3 end of an
existing DNA strand
5

3 5
3
5
3 5 5
 3

Lagging strand
ligase
growing 3
replication fork
5
Leading strand
3
✓ 5

3

Lagging strand DNA polymerase

◆ Okazaki fragments Leading strand

◆ joined by ligase ◆ continuous synthesis
Compare between prokaryotic
and eukaryotic DNA replication
 Replication

Prokaryotic Euokaryotic

DNA Circular Linear

Ori Single Multiple

RNA primer synthesis by Primase (primisome) Primase activity of DNA polymerase α

DNA polymerases

Types III& I Α,β,δ,γ,ξ

Enzymes with proofreading activity III δ,ξ

Removal of RNA primer by Polymerase I RNAase H & Flep endonuclease

Filling the gap left behind the primer Polymerase I Free floating DNA polymerase

Telomerase need No need Mandatory

Case Scenario or clinical correlate (to be
discussed here)
• Comment in lecture:
• Quinolone antibiotics, such as ciprofloxacin, inhibit DNA gyrase and
are used for numerous infections, including complicated urinary tract
infections and lower respiratory tract infections.
 Test yourself
II. True / False
1. Replication process involves replication of certain
genes of DNA.

2. Ligase enzyme plays a major role in the separation of the

two DNA strands for replication.

3. In prokaryotes, replication starts at multiple sites that are

rich in AT sequences.

4. Single Stranded DNA binding proteins bind to the single

stranded DNA keeping the 2 strands separated.

5. DNA polymerases utilizes ribnucleoside triphosphates for

the synthesis of the new strands.
Question 1
• 1. All of the following are true about eukaryotic DNA
replication EXCEPT:
• a. It is semiconservative.
• b. It occurs in the 3→5 direction.
• c. Both strands act as a template simultaneously.
• d. It is bidirectional.
•
Question 2
• 2. Which statement is TRUE about DNA replication:
• a. It occurs on one strand only
• b. It is catalyzed by RNA polymerase.
• c. It occurs in the 3→5 direction.
• d. It is semiconservative
•
Question 3
• 3. DNA replication requires all the following EXCEPT:
• a. RNA polymerase
• b. A short RNA primer.
• c. Deoxynucleoside triphosphates.
• d. DNA template
Question 4
• 4. In replication, separation of the double strands of DNA is
• catalyzed by:
• a. Helicase.
• b. Primase.
• c. DNA polymerase.
• d. RNA [polymerase.
Summary
Discussion & Feedback
References
• Chatterjea’s Textbook of Medical Biochemistry, 8th edition.
• Vasudevan's Textbook of Biochemistry For Medical Students, 6th Edition.
• BRS Biochemistry, Molecular Biology, and Genetics, 5th Edition.
• Marks’ Basic Medical Biochemistry: A Clinical Approach, 2nd Edition