Section E
DNA Replication
E1 DNA Replication: An Overview
E2 Bacterial DNA Replication
E3 Eukaryotic DNA Replication
E4 Other types of DNA Replication
Section D: Chromosome Structure
Section E: DNA replication
Yang Xu, College of Life Sciences
E1 DNA Replication:
An Overview
•
•
•
•
Semi-conservative mechanism
Replicons, origins and termini
Semi-discontinuous replication
RNA priming
Section D: Chromosome Structure
Section E: DNA replication
Yang Xu, College of Life Sciences
Semi-conservative mechanism
• This semi-conservative mechanism was demonstrated
experimentally in 1958 by Meselson and Stahl
N15
DNA
0
N14
1.0
2.0
3.0
4.0
0 + 2.0
0 + 4.0
S
Section D: Chromosome Structure
Section E: DNA replication
Yang Xu, College of Life Sciences
Replicons, origins and termini
• Replication fork: The point at which 
separation of the double strands and 
synthesis of new DNA takes place.
• Replcon: Any piece of DNA which replicates
as a single unit is called a replicon.
• Origin: The initiation of DNA replication
within a replicon always occurs at a fixed
point known as origin.
• Terminus: Usually, two replication forks
proceed bi-directionally away from the origin
and the strands are copied as they separate
until the terminus.
Section D: Chromosome Structure
Section E: DNA replication
Yang Xu, College of Life Sciences
Semi-discontinuous replication-I
3‘
5‘
3‘
5‘
• Definitions: At each replication fork:
– leading strand is synthesized as one continuous piece
– lagging strand is synthesized discontinuously as short
fragments in the reverse direction. They are joined by DNA ligase.
• These lagging strand fragments (Okazaki fragments) are:
– 1000-2000 nt long in prokaryotes;
– and 100-200 nt long in eukaryotes.
Section D: Chromosome Structure
Section E: DNA replication
Yang Xu, College of Life Sciences
RNA priming
• Finding fact: Close examination on Okazaki fragments
has shown that the first few nucleotides at their 5‘-ends
are ribo-nucleotides, as the first few nucleotides of the
leading strand are ribonucleotides.
• Conclusion: Hence, DNA synthesis is primed by RNA.
These primers are removed and the resulting gaps filled
with DNA before the fragments are joined.
• Reason: The reason for initiating each piece of DNA
with RNA appears to relate to the need for DNA
replication to be of high fidelity (see F4).
Section D: Chromosome Structure
Section E: DNA replication
Yang Xu, College of Life Sciences
Synthesis of progeny strand in lagging DNA
ppp
primosome
DNA polymerase III
RNA as primer
ppp
DNA polymerase I
DNA
ligase
Section D: Chromosome Structure
Section E: DNA replication
Yang Xu, College of Life Sciences
E2 Bacterial DNA Replication
•
•
•
•
Initiation
Unwinding
Elongation
Termination and segregation
Section D: Chromosome Structure
Section E: DNA replication
Yang Xu, College of Life Sciences
Initiation-I
Initiation model: the oriC-minichromosome:
• Making: E. coli origin gene has been cloned into plasmids
called oriC-minichromosome.
• Function: It behaves like the E. coli chromosome. oriC contains
four 9 bp binding sites for the initiator protein DnaA. Synthesis
of DnaA is coupled to growth rate so that the initiation of
replication is also coupled to growth rate.
Prokaryotic chromosomes:
• Initiation Feature: at high cellular
growth rates the replication of the
DNA can re-initiate a second round
at the two new origins before the
first round is completed.
Section D: Chromosome Structure
Section E: DNA replication
Q
Yang Xu, College of Life Sciences
Initiation-II
• DnaA protein : initiator
– Once DnaA attain a critical level:
– the DnaA proteins form a complex of 30-40 molecules,
– each protein bounds to an ATP molecule,
– around which the oriC DNA becomes wrapped in
negatively supercoiling way (Fig. 2).
– N-supercoiled  facilitates melting of three 13 bp AT-rich
sequences;  they open to allow binding of DnaB.
orC DNA
DnaA
AT-rich
sequence
Section D: Chromosome Structure
Section E: DNA replication
DnaB
Yang Xu, College of Life Sciences
Unwinding
Factors related to unwinding
• DnaB: the DNA helicases must travel along the
template strands to open the double helix for
copying;
• A second DNA helicase: may bind to the other
strand to assist unwinding besides DnaB.
• Ssb: (Single Strand Binding protein) Binding of
Ssb protein further promotes unwinding.
• DNA gyrase, a type II topoisomerase:
In a closed-circular DNA molecule, however,
removal of helical turns at the replication fork
leads to the positive supercoiling (see Topic C4).
This positive supercoiling must be relaxed by the
introduction of further negative supercoils by
called DNA gyrase (see Topic C4).
Section D: Chromosome Structure
Section E: DNA replication
Yang Xu, College of Life Sciences
Elongation-I
Proteins related to elongation:
1. Primosome: It is a mobile complex, which includes:
 DnaB helicase and  Primase,
synthesizes RNA primers every 1000-2000 nt on lagging strand.
2. DNA polymerase III holoenzyme:
• Both leading and lagging strand primers are elongated by DNA
polymerase III holoenzyme. This complex is a dimer,
– One half synthesizes the leading strand;
– The other synthesizes the lagging strand;
– The two polymerases in a single complex ensures that both strands
are synthesized at the same rate.
• Same subunits in the both halves of the dimer contain：
– an  subunit, the actual polymerase;
– an  subunit, is a 3’5’ proofreading exonuclease;
– a  subunits clamp the polymerase to the DNA.
• Different subunits: to synthesize  short and  long stretches of DNA
on the  lagging and  leading strands, respectively.
Section D: Chromosome Structure
Section E: DNA replication
Yang Xu, College of Life Sciences
Elongation-II
3. DNA polymerase I: Once the lagging strand primers
have been elongated by DNA polymerase III, they are
removed and the gaps filled by DNA polymerase I,
which has:
– 5'3' exonuclease: removes the primers (Fig. 3);
– 5'3' polymerase: fills the gaps with DNA by
elongating the 3'-end of the adjacent Okazaki
fragment;
– 3'5' proofreading exonuclease:
4. DNA ligase: The final phosphodiester bond between
the fragments is made by DNA ligase. The enzyme
from E. coli uses the co-factor NAD+ as an unusual
energy source.
• Replisome: In vivo,  the DNA helicases,  the
primosome and  the DNA polymerase III
holoenzyme dimer, are physically associated in a
complex called replisome.
Section D: Chromosome Structure
Section E: DNA replication
Yang Xu, College of Life Sciences
Replisome
Section D: Chromosome Structure
Section E: DNA replication
Yang Xu, College of Life Sciences
Replisome model
Section D: Chromosome Structure
Section E: DNA replication
Yang Xu, College of Life Sciences
Termination and segregation
• Termination:
The two replication forks meet about 180 opposite oriC.
– Terminator: in this region there are several terminator
sites which arrest the movement of the forks by binding
the tus gene product, which is an inhibitor of the DnaB
helicase;
– Hence, if one fork is delayed for some reason, they will
still meet within the terminus.
• Segregation:
– Topoisomerase IV: Once replication is completed, the
two daughter circles remain interlinked. They are
unlinked by topoisomerase IV (a type II DNA
topoisomerase);
– They can be segregated into the two daughter cells by
movement apart of their membrane attachment sites.
Section D: Chromosome Structure
Section E: DNA replication
Yang Xu, College of Life Sciences
Segregation
Section D: Chromosome Structure
Section E: DNA replication
Yang Xu, College of Life Sciences
E3 Eukaryotic DNA Replication
• Origins and initiation
• Replication forks
• Telomere replication
Section D: Chromosome Structure
Section E: DNA replication
Yang Xu, College of Life Sciences
Origins and initiation-I
• Individual origin structure:
– Size: only 11 bp
– Structure: the sequence is [A/T]TTTAT[A/G]TTT[A/T],
• The initiation system for eukaryotic replication includes:
– multiple copies of this origin are required for efficiency;
– the origin recognition complex (ORC) which permits
opening of the origins for copying;
– ORC is activated by CDKs.
• Licensing factor:
– It is a protein which is absolutely required for initiation and
inactivated after use,
– It can only enter into the nucleus when the nuclear envelope
dissolves at mitosis, thus preventing premature re-initiation.
Section D: Chromosome Structure
Section E: DNA replication
Yang Xu, College of Life Sciences
Origins and initiation-II
• Features of eukaryotic initiation:  at defined times in S-phase 
each replicon can only initiate once per cell cycle  tandem arrays
of about 20-50 replicons after.
• Initiation Order in S-phase comprise:
– the first part is in euchromatin (which includes
transcriptionally active DNA);
– the second parts are within heterochromatin
– the last are for centromeric and telomeric.
• ARSs (Autonomously replicating sequences):
– Individual yeast replication origins have been cloned into
prokaryotic plasmids.
– Since the origins allow these plasmids to replicate in yeast (a
eukaryote) they are termed ARSs.
– Eukaryotic origins + prokaryotic plasmids  eukaryote
Section D: Chromosome Structure
Section E: DNA replication
Yang Xu, College of Life Sciences
Replication forks-I
135bp
• DNA between the forks and nucleosomes
– Free state: Before copying, the DNA must be unwound
from the nucleosomes at the replication forks.
– Bending state: After the fork has passed, new
nucleosomes are assembled.
• Proteins related to replication:
– DNA helicases are required to separate strands;
– RP-A (Replication protein A) is for binding ssDNA;
– DNA polymerases (three types) are used for elongation.
Section D: Chromosome Structure
Section E: DNA replication
Yang Xu, College of Life Sciences
Replication forks-II
The three polymerases
• DNA polymerase : Function:  synthesizes RNA primers
by its primase domain;  initiates and elongates the leading
strands and each lagging strand fragment by its DNA
polymerase domain;
• DNA polymerase : Function:  replaces DNA polymerase
 on the leading strand;  proofreads the leading strand; 
synthesizes long DNA.
• DNA polymerase : Function:  replaces DNA polymerase
 on the lagging strand;  proofreads the lagging strand.
Section D: Chromosome Structure
Section E: DNA replication
Yang Xu, College of Life Sciences
Telomere Structure
• The problem of dsDNA ends: The ends of
linear chromosomes cannot be fully replicated
by semi-discontinuous. Thus, genetic
information could be lost from the DNA.
• Telomere structure: To overcome this, the ends
of eukaryotic chromosomes (telomeres) consist
of hundreds of copies of a simple, noninformational repeat sequence (e.g. TTAGGG
in humans) with the 3‘-end overhanging (突出
于) the 5'-end.
3'-AATCCCAATCCC-5'
5'-TTAGGGTTAGGG (TTAGGG)n TTAGGG-3'
Fig. 3. Human telomeric DNA (n=several hundred)
Section D: Chromosome Structure
Section E: DNA replication
Yang Xu, College of Life Sciences
Telomere replication
• Telomerase structure: Telomerase contains a short RNA
molecule: one part is a 150 bp RNA and the other part
is a complementary sequence to this repeat.
• Telomere replication:
1. Elongation : This RNA acts as a template for the addition
polymerization;
2. Translocation: Then telomerase moves and leaves a 3'overhang, which is the template of next polymerization.
• Special function (biological clock):
Telomerase activity is repressed in somatic cells,
resulting in a gradual shortening of the telomere with
each cell generation. As this shortening reaches
informational DNA, the cells die.
Section D: Chromosome Structure
Section E: DNA replication
Yang Xu, College of Life Sciences
G –T2G4---- TTGGGGT TGGG TTGGGGTTG
C – A2C4---aaaAACCCCAACuuac--aaaAACCCCAACuuac--5’
5’
telomerasetelomerase
3’
3’
Ploymerization
Section D: Chromosome Structure
Section E: DNA replication
Translocation
Ploymerization
Translocation
Yang Xu, College of Life Sciences
That’s all for Section E
Section D: Chromosome Structure
Section E: DNA replication
Yang Xu, College of Life Sciences