DNA Replication

Prokaryotes
Eukaryotes
 DNA Replication

1. Models of DNA replication: Meselson-Stahl Experiment.

2. Unique origin and bidirectional replication (Cairns). A-T rich physical
markers by Schnos and Inman.

1. DNA synthesis and elongation

2. DNA polymerases

3. Origin and initiation of DNA replication

4. Prokaryote/eukaryote models (circular/linear chromosomes)

5. Telomere replication (eukaryotes)

1955: Arthur Kornberg

Worked with E. coli.

Discovered the mechanisms of DNA synthesis.

Four components are required:

1. dNTPs: dATP, dTTP, dGTP, dCTP

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

2. DNA template

3. DNA polymerase (Kornberg enzyme)

4. Mg 2+
(optimizes DNA polymerase activity)

1959: Arthur Kornberg (Stanford University) & Severo Ochoa (NYU)

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 “finds” the correct complementary dNTP at

each step in the lengthening process.

• rate ≤ 800 dNTPs/second

• low error rate

3. Direction of synthesis is 5’ to 3’

DNA polymerase
Image credit:
Protein Data Bank
DNA elongation (Fig. 3.3b):
DNA elongation (Fig. 3.3a):
There are many different types of DNA polymerase

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

I Yes Yes Yes 400

II Yes Yes No ?

III Yes Yes No 10-20

•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 decrease)

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

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 polymerases proofread; others do not.

• Some polymerases used for replication; others for repair.

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

 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, major elements:

 Segments of single-stranded DNA are called template strands.

 Gyrase (a type of topoisomerase) relaxes the supercoiled DNA.

 Initiator proteins and DNA helicase binds to the DNA at the

replication fork and untwist the DNA using energy derived from
ATP (adenosine triphosphate).
(Hydrolysis of ATP causes a shape change in DNA helicase)

 DNA primase next binds to helicase producing a complex called

a primosome (primase is required for synthesis),

 Primase synthesizes a short RNA primer of 10-12 nucleotides,

to which DNA polymerase III adds nucleotides.

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

beginning at the RNA primer.

 The RNA primer is removed and replaced with DNA by

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

 Single-stranded DNA-binding (SSB) proteins (>200) stabilize

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

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
3’

5’
Leading strand
RNA Primer

3’
Fig. 3.8 Model of DNA Replication
DNA ligase seals the gaps between Okazaki fragments with a
phosphodiester bond (Fig. 3.7)
 Fig. 3.5 - Model of DNA replication

Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
 Fig. 3.5 - Model of DNA replication

Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
Replication of circular DNA in
E. coli (3.10):

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 (3.11):

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 in Eukaryotes:

Replication of DNA as well as histones as
well as non-histones proteins.

Packaging of DNA and histone proteins .

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 polymerases proofread; others do not.

• Some polymerases used for replication; others for repair.

 Multiple Origins in Eukaryotes
• Eukaryotes replicate their DNA only in S-
phase
• Eukaryotes have larger chromosomes
• Replication speed 2,600 npm.
• Largest Drosophila chromosome is 6.5 x 107
nucl., but it can replicate in 3-4 min. From a
single origin, bidirectional replication would
take 8.5 days. ==> The chromosome must have
some 7,000 origins of replication.
A replicating Drosophila chromosome
Origins initiate
replication at
different times.
 Two DNA polymerases are involved in
eukaryotic replication

• DNA polymerase  has no primase activity

and is thought to be the polymerase that
synthesizes the leading strand.
• DNA polymerase  has associated primase
activity and is thought to be the polymerase
that synthesizes the lagging strand.
 DNA Synthesis at the Origin

• Additional factors:
• PCNA (proliferating
cell nuclear antigen)
• DNA helicase
• Replication factor C
• OTHERS
 Replication of Nucleosomes

• Eukaryotic DNA is packaged with histones in

structures called nucleosomes.
• What happens to the nucleosome when the
replication fork and the replication machinery pass
by and open up the DNA double strand?
• Nucleosomes are found properly spaced on both
postreplicative DNA strands immediately after
passage of replication fork.
A model for nucleosome replication
Fig. 3.16 Synthesis of telomeric DNA by telomerase

Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
Final Step - Assembly into Nucleosomes:

• As DNA unwinds, nucleosomes must disassemble.

• Histones and the associated chromatin proteins must be

duplicated by new protein synthesis.

• Newly replicated DNA is assembled into nucleosomes almost

immediately.

• Histone chaperone proteins control the assembly.

Fig. 3.17
The lagging strand of telomeres cannot be
replicated by the usual mechanism
 Telomere and Telomerase
Solution:
• special telomere sequence: tandem repeats of
TTAGGG (human)
• telomerase, a specific enzyme with integrated
RNA template.
Telomere replication
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).
• 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!

Fig. 3.14
Fig. 3.13 - Replication forks visible in Drosophila
What about the ends (or telomeres) of linear chromosomes?

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

RNA primer is removed. 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.