Reference Books:

The ABC of Gene Cloning by Dominic W.S. Wong (2nd Edition)

Gene Cloning and DNA Analysis by T.A. Brown (6th Edition)
Essential Cell Biology by Alberts et al
Biochemistry by Voet & Voet

1
Basic Molecular Biology: The Genetic Process

Central dogma of molecular

biology

Biochemistry (3e) by D Voet & J Voet

Molecular biology of the cell (4th ed) by Alberts et al
Essential Cell Biology by Alberts et al
Life: The Science of Biology by Sadava et al (8th ed )
2
Central dogma of molecular biology

3
Basic Molecular Biology: The Genetic
Process

Replication

4
 Learning objectives
Understand the basic rules governing DNA replication
Understand the function of key proteins involved in a
generalised replication model

5
 `It has not escaped our notice that the specific
pairing we have postulated immediately
suggests a possible copying mechanism for
the genetic material’

Watson & Crick

Nature (1953)

Original drawing by Francis Crick 6

Why has DNA
evolved as the
genetic material but
not RNA?

Because DNA is more stable

RNA is prone to base-catalysed
hydrolysis.

7
 Four requirements for DNA to be
genetic material
Must carry information
– Cracking the genetic code
Must replicate
– DNA replication
Must allow for information to change
– Mutation
Must govern the expression of the phenotype
– Gene function

8
 DNA stores information in the sequence of its bases

Much of DNA’s sequence-specific information is accessible only

when the double helix is unwound

Proteins read the DNA sequence of nucleotides as the DNA helix

unwinds.

Proteins can either bind to a DNA sequence, or initiate the copying

of it.

Some proteins recognize the base sequence of DNA without

unwinding it (e.g. a restriction enzyme).

restriction
enzyme EcoR V 9
 DNA Replication
Process of duplication of the entire genome prior to cell division

Biological significance
• extreme accuracy of DNA replication is necessary in order to
preserve the integrity of the genome in successive generations
• In eukaryotes , replication only occurs during the S phase of
the cell cycle.
• Replication rate in eukaryotes is slower resulting in a higher
fidelity/accuracy of replication in eukaryotes

10
 THE EUKARYOTIC CELL CYCLE

S phase
DNA synthesis
G1
Growth phase 1 Main checkpoints

G2
Go M
Growth phase 2
Quiescent Mitosis
11
Three main features of the DNA synthesis reaction:

1. DNA polymerase 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 of the dNTP.

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

12
 The mechanism of DNA replication

– Initiation
• Proteins bind to DNA and open up double helix
• Prepare DNA for complementary base pairing
– Elongation
• Proteins connect the correct sequences of nucleotides into a
continuous new strand of DNA
– Termination
• Proteins release the replication complex

13
 Basic rules of replication

A. Semi-conservative
B. Starts at the ‘origin’
C. Can be uni or bidirectional
D. Semi-discontinuous
E. Synthesis always in the 5-3’ direction
F. RNA primers required

14
 DNA replication
Of the 3
possible
models,
replication is…

A) Semi-
conservative

15
 B) Starts at origin
Initiator proteins identify specific base sequences on DNA
called sites of origin

Prokaryotes – single origin site E.g E.coli - oriC

Eukaryotes – multiple sites of origin (replicator)
E.g. yeast - ARS (autonomously replicating sequences)

Prokaryotes Eukaryotes
16
 C) Uni or bidirectional
 Replication forks move in one or opposite directions

17
 D) Semi-discontinuous replication
Anti parallel strands replicated simultaneously
 Leading strand synthesis continuously in 5’– 3’
 Lagging strand synthesis in fragments in 5’-3’

18
 D) Semi-discontinuous replication

New strand synthesis always in the 5’-3’ direction

19
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

20
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. 21
Model of replication in E. coli

22
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 is

synthesized in short fragments. 23
E) Synthesis is ALWAYS in the 5’-3’ direction
Nucleotide recognition
Enzyme catalysed polymerisation (DNA polymerase)
Complementary base pair copied
Substrate used is dNTP

24
(SSB)

25
 Replication Enzymes

• DNA Polymerase - Matches the correct

nucleotides then joins adjacent
nucleotides to each other
• Primase - Provides an RNA primer to
start polymerization
• Ligase - Joins adjacent DNA strands
together (fixes “nicks”)
26
• Helicase - Unwinds the DNA and melts it
• Single Strand Binding Proteins - Keep the
DNA single stranded after it has been
melted by helicase
• Gyrase - A topoisomerase that Relieves
torsional strain in the DNA molecule
• Telomerase - Finishes off the ends of
DNA strands
27
 DNA polymerase

OH 3’
5’
3’ 5’

OH 3’
5’
3’ 5’

OH 3’
5’
3’ 5’

DNA synthesis always occurs by adding

nucleotides to the 3’-OH of the growing strand.

Synthesis is always in the 5’- 3’ direction

28
29
SSB

30
DnaB

31
In prokaryotes, there are three main types of DNA polymerase

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

I Yes Yes Yes

II Yes Yes No

III Yes Yes No

•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.

32
Eukaryotic enzymes:
Five common 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 the nucleus and mtDNA

• Some polymerases proofread; others do not.

• Some polymerases used for replication; others for repair.

• Polymerases vary by species.

33
 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’
34
DNA ligase seals the gaps between Okazaki fragments with a
phosphodiester bond

35
Model of DNA Replication

36
Model of DNA replication

37
Model of DNA replication

38
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).

39
• 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!

40
What about the ends (or telomeres) of linear chromosomes?

DNA polymerase/ligase cannot fill gap at end of chromosome after RNA primer is
removed. 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
new repeats.

3. Compensates by lengthening the chromosome.

4. Absence or mutation of telomerase activity results in chromosome shortening

41
and limited cell division.
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.

42
 Core proteins at the replication fork

Topoisomerases - Prevents torsion by DNA breaks

Helicases - separates 2 strands
Primase - RNA primer synthesis
Single strand - prevent reannealing
binding proteins of single strands
DNA polymerase - synthesis of new strand
Tethering protein - stabilises polymerase
DNA ligase - seals nick via phosphodiester linkage

43
 Core proteins at the replication fork

Nature (2003) vol 421,pp431-435 Figure in ‘Big’ Alberts too

44
45