REPLICATION

Genetic Information
Transfer

Central Dogma of the Cell

Genetic information stored in DNA in the form

of nucleotide sequence flows from DNA to DNA,
DNA to RNA then from RNA to protein.
 Central dogma

Replication Transcription Translation

DNA RNA protein

Reverse
transcription
• Replication: synthesis of daughter
DNA from parental DNA
• Transcription: synthesis of RNA using
DNA as the template
• Translation: protein synthesis using
mRNA molecules as the template
• Reverse transcription: synthesis of
DNA using RNA as the template
 DNA
Replication
MEDICAL AND BIOLOGICAL IMPORTANCE

1. For the transfer of genetic information from parent

to offspring synthesis of new DNA is essential.
2. New DNA synthesis is essential for cell
multiplication.
3. Inhibitors of DNA synthesis are used in the
treatment of cancer, bacterial and viral infections.
4. For the transfer of genetic information from the
nucleus to cytoplasm new RNA synthesis is
essential.
 MEDICAL AND BIOLOGICAL
IMPORTANCE…………

5. Inhibitors of RNA synthesis are used in the

treatment of bacterial infections.
6. Some toxins work by blocking RNA synthesis.
7. DNA synthesis is essential for the multiplication
of RNA containing viruses (retroviruses).
8. Some RNAs act as enzymes and facilitate their
own formation.
9. Some diseases are due to defective DNA repair.
 Section 1

General Concepts of
DNA Replication
 1. Replication
Synthesis of new DNA or information
copying is known as replication.
 In this process information is transmitted
from parent to daughter.
The new DNA is identical to parent DNA.
 DNA replication

• A reaction in which daughter DNAs are

synthesized using the parental DNAs as
the template.

replication

parental DNA
daughter DNA
 Daughter strand synthesis
• Chemical formulation:

(dNMP)n + dNTP (dNMP)n+1 + PPi

elongated
DNA strand substrate
DNA strand

• The nature of DNA replication is a

series of 3´- 5´phosphodiester bond
formation catalyzed by a group of
enzymes.
Phosphodiester bond formation
 DNA replication system
Template: double stranded DNA
Substrate: dNTP
Primer: short RNA fragment with a
free 3´-OH end
Enzyme: DNA-dependent DNA
polymerase (DDDP),
other enzymes,
protein factor
Characteristics of replication

 Semi-conservative replication
 Bidirectional replication
 Semi-continuous replication
 High fidelity
§1.1 Semi-Conservative Replication
Semiconservative replication
Experiment of DNA semiconservative replication

"Heavy" DNA(15N)

grow in 14N
medium

The first
generation

grow in 14N
medium

The second
generation
 Significance

The genetic information is ensured to be

transferred from one generation to the
next generation with a high fidelity.
§1.2 Bidirectional Replication

• Replication starts from unwinding the

dsDNA at a particular point (called
origin), followed by the synthesis on
each strand.
• The parental dsDNA and two newly
formed dsDNA form a Y-shape
structure called replication fork.
 Replication fork

5'
3'

3'
5'
3'
5'

5'

direction of 3'

replication
 Bidirectional replication

• Once the dsDNA is opened at the

origin, two replication forks are
formed spontaneously.
• These two replication forks move in
opposite directions as the syntheses
continue.
Bidirectional replication
 Replication of eukaryotes

• Chromosomes of eukaryotes have

multiple origins.
• The space between two adjacent
origins is called the replicon, a
functional unit of replication.
origins of DNA replication (every ~150 kb)

5' 3'
3' 5'

bidirectional replication

fusion of bubbles

5' 3'
3' 5'
5' 3'
3' 5'
§1.3 Semi-continuous Replication

The daughter strands on two template

strands are synthesized differently since
the replication process obeys the
principle that DNA is synthesized from
the 5´ end to the 3´end.
 Leading strand
On the template having the 3´- end, the
daughter strand is synthesized
continuously in the 5’-3’ direction. This
strand is referred to as the leading
strand.

3'
5' 3' 5'
direction of unwinding
3'
5'
Semi-continuous replication
3'

5'
replication fork

3'
5'
3'
replication direction 5'

3'

5'
Okazaki fragment
3'

5'
leading strand
 Okazaki fragments
• Many DNA fragments are synthesized
sequentially on the DNA template
strand having the 5´- end. These DNA
fragments are called Okazaki
fragments. They are 1000 – 2000 nt
long for prokaryotes and 100-150 nt
long for eukaryotes.
• The daughter strand consisting of
Okazaki fragments is called the
lagging strand.
 Semi-continuous replication

Continuous synthesis of the leading

strand and discontinuous synthesis of
the lagging strand represent a unique
feature of DNA replication. It is
referred to as the semi-continuous
replication.
 Section 2

Enzymology
of DNA Replication
 Enzymes and protein factors
protein Mr # function

Dna A protein 50,000 1 recognize origin

Dna B protein 300,000 6 open dsDNA
Dna C protein 29,000 1 assist Dna B binding
DNA pol Elongate the DNA
strands
Dna G protein 60,000 1 synthesize RNA primer
SSB 75,600 4 single-strand binding

DNA topoisomerase 400,000 4 release supercoil

constraint
§2.1 DNA Polymerase
DNA-pol of prokaryotes
• The first DNA-
dependent DNA
polymerase (short for
DNA-pol I) was
discovered in 1958 by
Arthur Kornberg who
received Nobel Prize in
physiology or medicine
in 1959.
• Later, DNA-pol II and DNA-pol III were
identified in experiments using
mutated E.coli cell line.
• All of them possess the following
biological activity.
1. 53 polymerizing
2. exonuclease
DNA-pol of E. coli
 DNA-pol I

• Mainly
responsible for
proofreading
and filling the
gaps, repairing
DNA damage
 Klenow fragment
N end DNA-pol Ⅰ C end

caroid

• small fragment (323 AA): having 5´→3´

exonuclease activity
• large fragment (604 AA): called Klenow
fragment, having DNA polymerization
and 3´→5´exonuclease activity
 DNA-pol II

• Temporary functional when DNA-pol I

and DNA-pol III are not functional
• Still capable for doing synthesis on
the damaged template
• Participating in DNA repairing
 DNA-pol III
• A heterodimer enzyme composed of
ten different subunits
• Having the highest polymerization
activity (105 nt/min)
• The true enzyme responsible for the
elongation process
 Structure of DNA-pol III

α ： has 5´→ 3´
polymerizing activity
ε ： has 3´→ 5´
exonuclease activity
and plays a key role to
ensure the replication
fidelity.
θ: maintain
heterodimer structure
 DNA-pol of eukaryotes
DNA-pol : initiate replication DnaG,
and synthesize primers primase
DNA-pol : replication with repairing
low fidelity

DNA-pol : polymerization in
mitochondria
DNA-pol : elongation DNA-pol III
DNA-pol : proofreading and DNA-pol I
filling gap
§2.2 Primase
• Also called DnaG
• Primase is able to synthesize primers
using free NTPs as the substrate and
the ssDNA as the template.
• Primers are short RNA fragments of a
several decades of nucleotides long.
• Primers provide free 3´-OH groups to
react with the -P atom of dNTP to
form phosphoester bonds.
• Primase, DnaB, DnaC and an origin
form a primosome complex at the
initiation phase.
§2.3 Helicase

• Also referred to as DnaB.

• It opens the double strand DNA with
consuming ATP.
• The opening process with the
assistance of DnaA and DnaC
§2.4 SSB protein
• Stand for single strand DNA binding
protein
• SSB protein maintains the DNA
template in the single strand form in
order to
• prevent the dsDNA formation;
• protect the vulnerable ssDNA from
nucleases.
§2.5 Topoisomerase

• Opening the dsDNA will create

supercoil ahead of replication forks.
• The supercoil constraint needs to be
released by topoisomerases.
• The interconversion of topoisomers
of dsDNA is catalyzed by a
topoisomerase in a three-step
process:
• Cleavage of one or both strands
of DNA
• Passage of a segment of DNA
through this break
• Resealing of the DNA break
§2.6 DNA Ligase

3' 5'
5' 3'
RNAase
3' 5'
5' OH P 3'

dNTP DNA polymerase

3' 5'
5' P 3'
ATP DNA ligase
3' 5'
5' 3'
• Connect two adjacent ssDNA strands
by joining the 3´-OH of one DNA
strand to the 5´-P of another DNA
strand.
• Sealing the nick in the process of
replication, repairing, recombination,
and splicing.
§2.7 Replication Fidelity
• Replication based on the principle of
base pairing is crucial to the high
accuracy of the genetic information
transfer.
• Enzymes use two mechanisms to
ensure the replication fidelity.
– Proofreading and real-time correction
– Base selection
 Proofreading and correction
• DNA-pol I has the function to correct
the mismatched nucleotides.
• It identifies the mismatched
nucleotide, removes it using the 3´- 5´
exonuclease activity, add a correct
base, and continues the replication.
 Exonuclease functions
5´→3´ 3´→5´
exonuclease exonuclease
activity activity
cut primer or excise mismatched
excise mutated nuleotides
segment
5' 3'
C T T C A G G A

G A A G T C C G G C G
3' 5'
 Section 3

DNA Replication
Process
 Sequential actions

• Initiation: recognize the starting point,

separate dsDNA, primer synthesis, …
• Elongation: add dNTPs to the existing
strand, form phosphoester bonds,
correct the mismatch bases, extending
the DNA strand, …
• Termination: stop the replication
§3.1 Replication of prokaryotes
a. Initiation

• The replication starts at a particular

point called origin.
• The origin of E. coli, ori C, is at the
location of 82.
• The structure of the origin is 248 bp
long and AT-rich.
Genome of E. coli
 Structure of ori C
• Three 13 bp consensus sequences
• Two pairs of anti-consensus repeats
Formation of preprimosome
 Formation of replication fork
• DnaA recognizes ori C.
• DnaB and DnaC join the DNA-DnaA
complex, open the local AT-rich
region, and move on the template
downstream further to separate
enough space.
• DnaA is replaced gradually.
• SSB protein binds the complex to
stabilize ssDNA.
 Primer synthesis

• Primase joins and forms a complex

called primosome.
• Primase starts the synthesis of
primers on the ssDNA template using
NTP as the substrates in the 5´- 3´
direction at the expense of ATP.
• The short RNA fragments provide free
3´-OH groups for DNA elongation.
 Primosome complex

Dna B Dna C
Dna A primase 3'

5'
3'

5'
DNA topomerase
 Releasing supercoil constraint
• The supercoil constraints are
generated ahead of the replication
forks.
• Topoisomerase binds to the dsDNA
region just before the replication
forks to release the supercoil
constraint.
• The negatively supercoiled DNA
serves as a better template than the
positively supercoiled DNA.
b. Elongation
• dNTPs are continuously connected to
the primer or the nascent DNA chain
by DNA-pol III.
• The core enzymes ( 、、 and  )
catalyze the synthesis of leading and
lagging strands, respectively.
• The nature of the chain elongation is
the series formation of the
phosphodiester bonds.
• The synthesis
direction of the
leading strand is
the same as that of
the replication fork.

• The synthesis
direction of the
Okazaki fragment is
also the same as
that of the
replication fork.
 Lagging strand synthesis
• Primers on Okazaki fragments are
digested by RNase.
• The gaps are filled by DNA-pol I in the
5´→3´direction.
• The nick between the 5´end of one
fragment and the 3´end of the next
fragment is sealed by ligase.
Formation of phosphodiester bond
by DNA Ligase
c. Termination

• The replication of E. coli is

bidirectional from one origin, and the
two replication forks must meet at
one point called ter at 32.
• All the primers will be removed, and
all the fragments will be connected
by DNA-pol I and ligase.
§3.2 Replication of Eukaryotes
• DNA replication is closely related
with cell cycle.
• Multiple origins on one chromosome,
and replications are activated in a
sequential order rather than
simultaneously.
Cell cycle
 Initiation
• The eukaryotic origins are shorter
than that of E. coli.
• Requires DNA-pol  (primase
activity) and DNA-pol  (polymerase
activity and helicase activity).
• Needs topoisomerase and replication
factors (RF) to assist.
b. Elongation

• DNA replication and nucleosome

assembling occur simultaneously.
• Overall replication speed is
compatible with that of prokaryotes.
c. Termination
3' 5'
5' 3'
3' 5'
5' 3'

connection of discontinuous
segment
3' 5'
5' 3'
3' 5'
5' 3'
• Telomerase
• Eukaryotic cells face a special problem in
replicating the ends of their linear DNA
molecules.
• Following removal of the RNA primer from the
extreme 5'-end of the lagging strand, there is no
way to fill in the remaining gap with DNA.
• To solve this problem, and to protect the ends of
the chromosomes from attack by nucleases,
noncoding sequences of DNA complexed with
protein found at these ends. Called Telomeres.
 Telomere
• The terminal structure of eukaryotic
DNA of chromosomes is called
telomere.
• Telomere is composed of terminal
DNA sequence and protein.
• The sequence of typical telomeres is
rich in T and G.
• The telomere structure is crucial to
keep the termini of chromosomes in
the cell from becoming entangled and
sticking to each other.
 Telomerase
• The eukaryotic cells use telomerase to
maintain the integrity of DNA telomere.
• The telomerase is composed of
telomerase RNA
telomerase association protein
telomerase reverse transcriptase

• It is able to synthesize DNA using RNA

as the template.
 Significance of Telomerase

• Telomerase may play important

roles in cancer cell biology and in
cell aging.