DNA

REPLICATION

DESAK MADE WIHANDANI

DEPARTEMEN BIOKIMIA
FK UNUD
/REPLICATION
The Central Dogma of Molecular Biology
Replication

protein
DNA mRNA
A
C
UA A G
CA

Transcription G Translation
A
C
G U
U
CA

Phenotype
 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
 The mechanism of DNA replication

Arthur Kornberg et al
 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
 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
DNA replication
Of the 3
possible
models,
replication
is…

A) Semi-
conservative

Meselson-Stahl
experiments
 B) Starts at origin
Initiator proteins identify specific base
sequences on DNA called sites of origin
(bacteria: 1 ori, human: 100.000 ori)

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

Eukaryotes – multiple sites of origin (replicator)

Prokaryotes Eukaryotes
 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
 C) Uni or bidirectional
◼ Replication forks move in one or opposite directions
 D) Semi-discontinuous replication
Anti parallel strands replicated simultaneously
❑Leading strand synthesis continuously in 5’– 3’
❑Lagging strand synthesis in fragments in 5’-3’
 Semi-discontinuous replication

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

E) Synthesis is ALWAYS in the 5’-3’ direction
Nucleotide recognition
Enzyme catalysed polymerisation (DNA polymerase)
Complementary base pair copied
Substrate used is dNTP
What happens if a base Where does energy for addition
mismatch occurs? of nucleotide come from?

From cleavage of high energy phosphate

DNA polymerase has 3’ → 5’ of incoming triphosphate
exonuclease activity in order to
correct errors
DNA elongation (Fig. 3.3a):
DNA elongation (Fig. 3.3b):
 F) RNA primers required
• DNA polymerase can only join an incoming nucleotide to one that is base-paired

• RNA primase provides a base paired 3’ end as a starting point for DNA pol by
synthesising ~10 nucleotide primers
 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
DNA ligase - - seals nick via
phosphodiester linkage
Supercoiled DNA relaxed by gyrase &
unwound by helicase + proteins:

5’SSB Proteins
Okazaki Fragments
1 ATP

Polymerase III 2
Helicase
3 +
Lagging strand 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’
 Core proteins at the replication fork

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

 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 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’
 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.
 Two types

 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.
 DNA polymerase

• 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
Leading strand

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

Because DNA is more stable

RNA is prone to base-catalysed
hydrolysis.
DNA ligase seals the gaps between Okazaki fragments with a
phosphodiester bond (Fig. 3.7)
 DNA replication in eukaryotes:

• 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.

Fig. 3.13 - Replication forks visible in Drosophila