• 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
General Concepts of
Double helix structure of DNA
“It has not escaped our notice that the specific pairing we have postulated
immediately suggests a possible copying mechanism for the genetic
Watson & Crick
Characteristics of replication
§1.1 Semi-Conservative Replication
——Meselson and Stahl (1958)
Definition: Half of the
parental DNA molecule is
conserved in each new double
helix, paired with a newly
Significance: The genetic
information is ensured to be
transferred from one
generation to the next
generation with a high fidelity.
• Examination of T7 DNA
replication using electron
• 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.
• Once the dsDNA is
opened at the origin,
two replication forks
• These two replication
forks move in opposite
directions as the
Replication of prokaryotes
from the origin,
and proceeds in
directions. It is
Replication of eukaryotes
• Chromosomes of eukaryotes have multiple
§1.3 Semi-continuous Replication
• The DNA strands are antiparallel. At a replication
fork, both strands of parental DNA serve as templates
for the synthesis of new DNA;
• All known DNA polymerases synthesize DNA in the 5’
→3’ direction but not in 3’ →5’ direction.
Reiji Okazaki and
his wife Tsuneko Okazaki
• This dilemma was resolved by Reiji Okazaki ( in the
1960s), who found that a significant proportion of
newly synthesized DNA exists as small fragments;
These units of about a thousand nucleotides are
called Okazaki fragments;
– They are 1000 – 2000nt long for prokaryotes and 100150nt long for eukaryotes.
• The leading strand :the strand synthesized
• The lagging strand :the strand formed from
• The 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.
of DNA Replication
Large team of enzymes coordinates
DNA replication system
double stranded DNA
short RNA fragment with
a free 3´-OH end
Daughter strand synthesis
• Chemical formulation:
(dNMP)n + dNTP
(dNMP)n+1 + PPi
DNA strand substrate
• The nature of DNA replication is a
series of 3´,5´phosphodiester bond
formation catalyzed by a group of
Phosphodiester bond formation
with our own
(dNMP)n + dNTP → (dNMP)n+1 + PPi
for the bonding!
Enzymes and protein factors
Dna A protein
Dna B protein
Dna C protein
assist Dna B binding
Elongate the DNA
Dna G protein
synthesize RNA primer
§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
liked to refer
career as a
•Arthur Kornberg (left) with his son, Roger,
after Roger received the 2006 Nobel Prize
• Later, DNA-pol II and DNA-pol III
were identified in experiments using
mutated E.coli cell line.
• DNA-pol I possess the following
1. 53 polymerizing
2. The 3` to 5` exonuclease activity
3. The 5` to 3` exonuclease activity
does a DNA
polymerase also need two
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.
cut primer or
C T T C A G G A
G A A G T C C G G C G
DNA-pol of E. coli
• Klenow fragment: large fragment (604 AA)
of DNA pol I, having DNA polymerization
and 3´→5´exonuclease activities, and is
widely used in molecular biology.
• Temporary functional when DNA-pol
I and DNA-pol III are not
• Still capable for doing synthesis on
the damaged template
• Participating in DNA repairing
• A heterodimer enzyme
composed of ten
• Having the highest
• The true enzyme
responsible for the
DNA Polymerase III- does the bulk of copying DNA in Replication
• Also called DnaG
• Primase (a specific
RNA polymerase) :
using free NTPs as the
substrate and the
ssDNA as the template.
• Primers: short RNA
• Also referred to as DnaB.
• It opens the double strand DNA with
consuming ATP. (Zip opener)
• The opening process with the
assistance of DnaA and DnaC
§2.4 SSB protein(single strand DNA
• maintains the DNA template in the single
strand form in order to
• prevent the dsDNA formation;
• protect the ssDNA degradation by
• Opening the
dsDNA will create
supercoil ahead of
to be released by
(type I and II).
• It cuts a
phosphoester bond on
one DNA strand,
rotates the broken
DNA freely around
the other strand to
relax the constraint,
and reseals the cut.
• It is named gyrase in
• It cuts phosphoester
bonds on both strands
of dsDNA, releases
the supercoil constraint,
and reforms the
• Antibiotics: ciprofloxacin, novobiocin and nalidixic
acid, inhibit the bacterial gyrase.
• Anticancer agents: adriamycin, etoposide, and
doxorubicin, inhibit human topoisomerase.
§2.6 DNA Ligase
• 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
O P OO-
O P OO-
• 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
• The replication
starts at a
• Genome of E. coli
• The structure of the origin
is 248 bp long and AT-rich.
DNA sequences at the Bacterial origin of Replication
Formation of replication fork
• DnaA recognizes origin.
• DnaB(helicase) and
DnaC join the DNADnaA complex, open
the local AT-rich
region, and move on
to separate enough
• SSB protein binds the
complex to stabilize
• Primase joins and
starts the synthesis of
• Primasome: protein
for creating RNA
primers on ssDNA
during DNA replication.
• Topoisomerase binds to
the dsDNA region just
before the replication
forks to release the
Dna B Dna C
• The short RNA fragments provide free
3´-OH groups for DNA elongation.
• dNTPs are continuously connected to
the primer or the nascent DNA
chain by DNA-pol III.
• The nature of the chain elongation
is the series formation of the
Lagging strand synthesis
• Primers on Okazaki
digested by RNase.
• The gaps are filled by
DNA-pol I in the
• The nick between the
5´end of one
fragment and the
3´end of the next
fragment is sealed by
• The synthesis direction of
the leading strand is the
same as that of the
• The synthesis direction of
the latest Okazaki fragment
is also the same as that of
the replication fork.
• The replication of E. coli is bidirectional
from one origin, and the two replication
forks must meet at one point called ter
• All the primers will be removed, and all
the fragments will be connected by
DNA-pol I and ligase.
Replication of prokaryotes
from the origin,
and proceeds in
directions. It is
§ Replication Fidelity
• Replication based on the principle of
base pairing is crucial to the high
accuracy of the genetic information
• Enzymes use three mechanisms to
ensure the replication fidelity.
§3.2 Replication of Eukaryotes
• DNA replication is
with cell cycle: Sphase.
• Multiple origins on
• Cell cycle
DNA-pol of eukaryotes
DNA-pol : initiate replication
and synthesize primers
DNA-pol : replication with
DNA-pol : polymerization in
DNA-pol : elongation
DNA-pol : proofreading and
• 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.
• DNA replication and nucleosome
assembling occur simultaneously.
• Overall replication speed is
compatible with that of prokaryotes.
connection of discontinuous
The End Replication Problem:
Telomeres shorten with each S phase
• Telomere: the terminal structure
of eukaryotic DNA of
• composed of terminal DNA
sequence and protein.
• Function: keep the termini of
chromosomes in the cell from
becoming entangled and sticking to
Repetitive DNA sequence
(TTAGGG in vertebrates)
Form a 'capped' end structure
The Nobel Prize in Physiology or
"for the discovery of how chromosomes are
protected by telomeres and the enzyme
• Telomerase: the enzyme that essentially
builds new telomeres, maintain the integrity
of DNA telomere.
• The telomerase is composed of
telomerase association protein
telomerase reverse transcriptase
• It is able to synthesize DNA using RNA as
Significance of Telomerase
• Telomerase is highly active in the
embryo, and after birth it is active
in the reproductive and stem cells.
• Telomerase may play important roles
in cell aging and cancer cell biology.
Telomerase and Senescence
In most somatic tissues, telomerase is expressed at very
low levels or not at all -- as cells divide, telomeres shorten
Short telomeres signal cells to senesce (stop dividing)
Telomerase and Cancer
•Strong evidence to suggest that the absence of
senesence in cancer cells is linked to the activation of
•Telomerase is an attractive target for cancer
Telomeres are essential for chromosome stability
Telomere shortening occurs owing to the biochemistry of
Short telomeres cause replicative senescence
Telomerase prevents telomere shortening and
• The genetic information carrier of some
biological systems is ssRNA instead of
dsDNA (such as ssRNA viruses).
• The information flow is from RNA to DNA,
opposite to the normal process.
• This special replication mode is called
Viral infection of RNA virus
•Reverse transcription is
a process in which
ssRNA is used as the
template to synthesize
•Synthesis of ssDNA
complementary to ssRNA,
cDNA, forming a RNA-DNA
•Hydrolysis of ssRNA: RNase
activity of reverse
transcriptase, leaving ssDNA.
•Synthesis of the second
ssDNA, forming a DNA-DNA
Howard M. Temin
• In 1970
• Discover RNA-dependant DNA polymerase
which later known as reverse transcriptase.
• 1975 Nobel Prize in Physiology or Medicine
Significance of RT
• An important discovery in life science and
•RNA plays a key role just like DNA in
the genetic information transfer and
gene expression process.
•RNA could be the molecule developed
earlier than DNA in evolution.
•RT is the supplementary to the central
DNA Damage and Repair
•Definition: mutation is a change of nucleic
acids in genomic DNA of an organism.
•The mutation could occur in the replication
process as well as in other steps of life
• Consequences of mutation
•To create a diversity of the biological world; a
natural evolution of biological systems
•To lead to the functional alternation of
biomolecules, death of cells or tissues, and
some diseases as well
§5.2 Causes of Mutation
Mutation caused by
• Carcinogens can cause mutation.
• Carcinogens include:
• Food additives and food
preservatives; spoiled food
• Pollutants: automobile emission;
• Chemicals: pesticides; alkyl
derivatives; nitrous acid(HNO2)
§5.3 Types of Mutation
a. Point mutation (mismatch)
Point mutation is referred to as the
single nucleotide alternation.
• Transition: the base alternation from
purine to purine, or from pyrimidine to
• Transversion: the base alternation
between purine and pyrimidine, and vise
• Nitrous acid (HNO2): react with base that
contain amino groups, deaminates C to produce
U, resulting in G·C A·U
• Nitrous acid formed by digestion of
nitrites (preservatives) in foods.
Consequences of point mutations
• Silent mutation: The code containing the
changed base may code for the same
amino acid. UCA, UCU, all code for
• Missense mutation: the changed base
may code for a different amino acid.
UCA for serine, ACA for threonine.
• Nonsense mutation: the codon with the
altered base may become a termination
codon. UCA for serine, UAA for stop
Hb mutation causing anemia
•Single base mutation leads to one AA
change, causing disease.
AA residue 6 in chain
b. Deletion and insertion
• Deletion: one or more nucleotides
are deleted from the DNA sequence.
• Insertion: one or more nucleotides
are inserted into the DNA sequence.
Deletion and insertion can cause the
reading frame shifted.
5´… …GCA GUA CAU GUC … …
Ala Val His Val
5´… …GAG UAC AUG UC … …
Glu Tyr Met Ser
§5.4 DNA Repairing
• DNA repairing is a kind response made
by cells after DNA damage occurs,
which may resume their natural
structures and normal biological functions.
• DNA repairing is a supplementary to the
proofreading-correction mechanism in
(or lignt repair)
• One of the most
important and effective
• UvrA and UvrB:
recognize and bind the
damaged region of
• UvrC: excise the
• DNA-pol Ⅰ: synthesize
the DNA segment to
fill the gap.
• DNA ligase: seal the
DNA ligase NAD+
• XP is an genetic disease.
• Patients will be suffered
with hyper-sensitivity to
UV which results in
multiple skin cancers.
• The cause is due to the
low enzymatic activity for
the nucleotide excisionrepairing process,
particular thymine dimer.
• The most obvious,
important part of
• It is used for repairing when a large
segment of DNA is damaged.
• Recombination protein RecA, RecB and RecC
participate in this repairing.
• It is responsible for the situation that
DNA is severely damaged and the
replication is hard to continue.
• If workable, the cell could be survived,
but may leave many errors.
• In E. coli, uvr gene and rec gene as well
as Lex A protein constitute a regulatory
I. General characteristics
Semi-conservative; Specific origins; Bidirectional; Semidiscontinuous
II. Bacterial Replication
1. template, primer, dNTP, proceed in 5` to 3` direction
2. Pol I, Pol II, Pol III
3. other replication proteins at the replication fork – SSB,
B. Semidiscontinuous replication: leading strand and lagging
1. RNA primer synthesized by the primases
2. polymerization by Pol III
3. completion by Pol I and ligase
4. Okazaki fragment
Ⅲ. Eukaryotic Replication
• S phase; Telomere and Telomerase
Ⅳ. Reverse transcription
Ⅴ. Mutation, DNA damage and repair
Point mutation; insertion and deletion, Frameshift
Physical and chemical damage;
photoreactivation repair; excision repair
Xeroderma pigmentosum (XP)