SBS 2023

GENETICS

LECTURE 2
DNA REPLICATION

DR. MOHD NAZMI BIN ABD MANAP

 OBJECTIVES
 To describe the three possible models of DNA
replication
 To show how the Meselson-Stahl Experiment proves
the semiconservative DNA replication
 To describe the semiconservative DNA replication in
eukaryotes
 To describe the enzymes involved in DNA replication
 To describe semi discontinuous DNA replication
 To describe the replication of circular DNA and the
supercoiling problem
 To describe DNA replication in eukaryotes
 To describe initiation of Replication
 To describe replicating the ends of chromosomes
Lecture 2: DNA Replication

1. Alternative models of DNA replication

2. Semiconservative model: Meselson-Stahl

Experiment

3. DNA synthesis and elongation

4. DNA polymerases

5. Origin and initiation of DNA replication

6. Prokaryote/eukaryote models (circular/linear

chromosomes)

7. Telomeres (replicating the ends of chromosomes)

Alternative models of DNA replication
Equilibrium density gradient centrifugation

The Meselson-Stahl Experiment:

Meselson and Stahl (1958) grew E. coli in a heavy (not radioactive) isotope of
nitrogen, 15N in the form of 15NH4Cl (Ammonium chloride). Because it is
heavier, DNA containing 15N is more dense than DNA with normal 14N,
and so can be separated by CsCl density gradient centrifugation.
1958: Matthew Meselson & Frank Stahl’s Experiment

Semiconservative model of DNA replication

 DNA REPLICATION

Basic Steps
Unwinding the DNA molecule
Separating the two strands
Making a complementary copy for each strand
1955: Arthur Kornberg

Worked with E. coli.

Discovered the mechanisms of DNA synthesis.

Five components are required:

1. dNTPs: dATP, dTTP, dGTP, dCTP

(deoxyribonucleoside 5’-triphosphates)
(sugar-base + 3 phosphates)

2. DNA template

3. DNA polymerase I (formerly the Kornberg enzyme)

(DNA polymerase II & III discovered soon after)

4. Mg 2+ (optimizes DNA polymerase activity)

5. DNA Primers (Short DNA chain)

Three main features of the DNA synthesis reaction:

1. DNA polymerase I catalyzes formation of phosphodiester

bond
between 3’-OH of the deoxyribose (on the last
nucleotide) and
the 5’-phosphate of the dNTP.

• Energy for this reaction is derived from the release of

two of the three phosphates.

2. DNA polymerase I “finds” the correct complementary

dNTP at each step in the lengthening process
(elongation).

• rate ≤ 800 dNTPs/second

• low error rate

3. Direction of synthesis is 5’ to 3’
DNA elongation
DNA elongation
Not all polymerases are the same

Polymerase Polymerization (5’-3’) Exonuclease (3’-5’) Exonuclease (5’-3’)

I Yes Yes Yes

II Yes Yes No

III Yes Yes No

•Polymerase I & III replicate 5’ to 3’

•Polymerase II’s role is not well understood

•3’ to 5’ exonuclease activity = ability to remove nucleotides from the

3’ end of the chain

Important proofreading ability

Without proofreading error rate (mutation rate) is 1 x 10-6

With proofreading error rate is 1 x 10-9 (1000-fold effect)

•5’ to 3’ exonuclease activity functions in DNA replication & repair.

Origin of replication (e.g., the prokaryote):

 Begins with double-helix denaturing into single-strands thus

exposing the bases.

 Exposes a replication bubble from which replication proceeds in both

directions.

~245 bp in E. coli
Initiation of replication: The major elements:

 Segments of single-stranded DNA are called template

strands.

 Gyrase (a type of topoisomerase) relaxes the

supercoiled DNA.

 Special sequence (Ori C) in DNA will direct initiation of

replication ( Cluster of 13bp AT rich seq. and four copies
of 9bp repeats)

 DnaA initiator protein will binds to OriC and stimulates

denaturation – (denature AT reach region)

 DNA Helicases will untwist the DNA in both direction

from the origin of replication – This is done by breaking
the hydrogen bond

 DNA helicases will recruit DNA primase (forming a

complex called the primosome)
Initiation of replication in E. coli

The DnaA initiator protein binds to oriC (the replicator) and

stimulates denaturation of the DNA. DNA helicases are recruited
and begin to untwist the DNA to form two head-to-head replication
forks.
Initiation of replication: The major elements

 DNA primase is important as to synthesis short RNA

primer (5-10 nucleotides)- DNA polymerase cannot start
synthesizing without this short primer sequence. This
primer sequence will be removed and replace later.

 Polymerase III adds nucleotides 5’ to 3’ on both strands

beginning at the RNA primer.

 The RNA primer is later removed and replaced with DNA

by polymerase I, and the gap is sealed with DNA ligase.

 Single-stranded DNA-binding (SSB) proteins stabilize

the single-stranded template DNA during the process.
Model of replication in E. coli
Nature of DNA replication :

DNA replication is continuous on the leading strand and

semidiscontinuous on the lagging strand:

Unwinding of any single DNA replication fork proceeds in one

direction.

The two DNA strands are of opposite polarity, and DNA polymerases
only synthesize DNA 5’ to 3’.

Solution: DNA is made in opposite directions on each template.

•Leading strand synthesized 5’ to 3’ in the direction of

the replication fork movement.

continuous

requires a single RNA primer

•Lagging strand synthesized 5’ to 3’ in the opposite

direction.

semidiscontinuous (i.e., not continuous)

requires many RNA primers

DNA REPLICATION
DNA REPLICATION
Supercoiled DNA relaxed by gyrase & 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’
DNA ligase seals the gaps between Okazaki fragments with a
phosphodiester bond
Model for the replisome, the complex of key replication proteins,
with the DNA at the replication fork. The DNA polymerase III on the
lagging-strand template (top of figure) is just finishing the synthesis
of an Okazaki fragment.
Model for the events occurring around a single replication fork of the
E. coli chromosome

Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
Model for the events occurring around a single replication fork of the
E. coli chromosome

Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
 Summary

Process of Replication : Synthesis of Leading and

Lagging strands of DNA
 Summary
Enzymes involved in DNA replication

Type Function
1. DNA gyrase : catalyzes formation of -ve supercoil
2. DNA helicase : catalyzes unwinding
3. DNA ligase : catalyzes covalent joining of
Okazaki fragments; joins sections of
nucleotide chain to one another

4. DNA single-stranded : maintains an extended single-

binding protein stranded template

5. DNA primase : catalyzes RNA primer synthesis

6. Topoisomerase : prevents DNA from tangling up by

introducing –ve supercoil as the
replication fork migrates
 Summary
Concepts and terms to understand:

Why are gyrase and helicase required?

The difference between a template and a primer?

Primase and polymerase?

Replication fork?

Why are single-stranded binding (SSB) proteins required?

Leading strand and lagging strand?

Continuous and semi-discontinuous?

Okazaki fragments?
Replication of circular DNA in
E. coli

Bidirectional replication of
circular DNA molecules –
the two replisomes are
mirror images moving away
from each other

1. Two replication forks result

in a theta-like () structure.

2. As strands separate,
positive supercoils form
elsewhere in the molecule.

3. Topoisomerases relieve
tensions in the supercoils,
allowing the DNA to
continue to separate.
Rolling circle model of DNA
replication in phage  :

The replication process of ds circular

DNA molecules through the
rolling circle mechanism.

1. Common in several
bacteriophages including .

2. Begins with a nick at the origin

of replication.

3. 5’ end of the molecule is

displaced and acts as primer for
DNA synthesis.

4. Can result in a DNA molecule

many multiples of the genome
length (and make multiple copies
quickly).

5. During viral assembly the DNA is

cut into individual viral
chromosomes.
 DNA Replication in Eukaryotes
DNA replication is very similar in both prokaryotes and eukaryotes,
except that eukaryotes have more than one chromosome.

• Eukaryotic chromosomes generally contain much more DNA than

those of prokaryotes, and their replication forks move much more
slowly.

• Eukaryotic chromosomes contain multiple origins, at which DNA

denatures and replication then proceeds bidirectionally until an
adjacent replication fork is encountered. The DNA replicated from a
single origin is called a replicon, or replication unit.

• In eukaryotes, replicon size is smaller than it is in prokaryotes,

replication is slower, and each chromosome contains many
replicons. Number and size of replicons vary with cell type.

• Not all origins within a genome initiate DNA synthesis

simultaneously.
DNA replication in eukaryotes:

Copying each eukaryotic chromosome during the S phase of

the cell cycle presents some challenges:

Major checkpoints in the system

1. Cells must be large enough, and the environment

favorable.

2. Cell will not enter the mitotic phase unless all the DNA has
replicated

3. Chromosomes also must be attached to the mitotic spindle

for mitosis to complete.

4. Checkpoints in the system include proteins call cyclins and

enzymes called cyclin-dependent kinases (Cdks).
Eukaryotic enzymes:

Five DNA polymerases from mammals.

1. Polymerase  (alpha): nuclear, DNA replication, no

proofreading

2. Polymerase  (beta): nuclear, DNA repair, no proofreading

3. Polymerase  (gamma): mitochondria, DNA repl.,

proofreading

4. Polymerase  (delta): nuclear, DNA replication,

proofreading

5. Polymerase  (epsilon): nuclear, DNA repair (?),

proofreading

• Different polymerases for nucleus and mtDNA

• Some proofread; others do not.

• Some used for replication; others for repair.

• Each eukaryotic chromosome is one linear DNA double helix

• Average ~108 base pairs long

• With a replication rate of 2 kb/minute, replicating one human

chromosome would require ~35 days.

• Solution ---> DNA replication initiates at many different sites

simultaneously.

Rates are cell

specific!
Origin of replication in eukaryotic chromosomes
What about the ends (or telomeres) of linear chromosomes?

DNA polymerase/ligase cannot fill gap at end of chromosome after RNA

primer is removed. Big problem---If this gap is not filled,
chromosomes would become shorter each round of replication!

Solution:

1. Eukaryotes have tandemly repeated sequences at the ends of their

chromosomes.

2. Telomerase (composed of protein and RNA complementary to the

telomere repeat) binds to the terminal telomere repeat and catalyzes
the addition of of new repeats.

3. Compensates by lengthening the chromosome.

4. Absence or mutation of telomerase activity results in chromosome

shortening and limited cell division.
Synthesis of telomeric DNA by telomerase

Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.