Download and DNA-pol

Genetic Information
Transfer
Central dogma
replication
transcription
DNA
RNA
reverse
transcription
translation
protein
• 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
Lecture 2
DNA
Replication
（DNA Biosynthesis）
Section 1
General Concepts of
DNA Replication
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
material.”
Watson & Crick
Characteristics of replication

Semi-conservative replication

Bidirectional replication

Semi-continuous replication
§1.1 Semi-Conservative Replication
——Meselson and Stahl (1958)
Semiconservative replication
•
Definition: Half of the
parental DNA molecule is
conserved in each new double
helix, paired with a newly
synthesized complementary
strand.
•
Significance: The genetic
information is ensured to be
transferred from one
generation to the next
generation with a high fidelity.
A
T
T
G
C
AT
TA
TA
GC
CG
T
A
A
C
G
Parent
molecule
AT
TA
TA
GC
CG
Daughter
molecule
§1.2 Bidirectional
Replication
• Examination of T7 DNA
replication using electron
microscopy
• 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.
Origin
5'
3'
3'
5'
5'
3'
5'
direction of
replication
3'
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
synthesis continue.
Replication of prokaryotes
•The replication
process starts
from the origin,
and proceeds in
two opposite
directions. It is
named 
replication.
Replication of eukaryotes
• Chromosomes of eukaryotes have multiple
origins.
§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.
解链方向
Semi-continuous replication
• The leading strand :the strand synthesized
continuously;
• The lagging strand :the strand formed from
Okazaki fragments;
• 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.
Section 2
Enzymology
of DNA Replication
Large team of enzymes coordinates
replication
Let’s meet
the team…
DNA replication system
Template:
double stranded DNA
Substrate: dNTP
Primer:
Enzyme:
short RNA fragment with
a free 3´-OH end
DNA-dependent DNA
polymerase (DDDP),
other enzymes
Protein factor
Daughter strand synthesis
• Chemical formulation:
(dNMP)n + dNTP
(dNMP)n+1 + PPi
DNA strand substrate
elongated
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
energy
We come
with our own
energy!
(dNMP)n + dNTP → (dNMP)n+1 + PPi
Where’s the
ENERGY
for the bonding!
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.
Kornberg
liked to refer
to his
scientific
career as a
"love affair
with
enzymes."
•Arthur Kornberg (left) with his son, Roger,
after Roger received the 2006 Nobel Prize
in Chemistry.
• Later, DNA-pol II and DNA-pol III
were identified in experiments using
mutated E.coli cell line.
• DNA-pol I possess the following
biological activity.
1. 53 polymerizing
2. The 3` to 5` exonuclease activity
3. The 5` to 3` exonuclease activity
Why
does a DNA
polymerase also need two
exonuclease activities?
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´
exonuclease
activity
3´→5´
exonuclease
activity
cut primer or
excise mismatched
excise mutated
nuleotides
segment
3'
5'
C T T C A G G A
3'
G A A G T C C G G C G
5'
DNA-pol of E. coli
Klenow fragment
• 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.
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
DNA Polymerase III- does the bulk of copying DNA in Replication
β2
subunit:
sliding
clamp
§2.2 Primase
• Also called DnaG
• Primase (a specific
RNA polymerase) :
synthesize primers
using free NTPs as the
substrate and the
ssDNA as the template.
• Primers: short RNA
fragments (5-50
nucleotides).
§2.3 Helicase
• 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
Dna
C
Dna B
解链方向
§2.4 SSB protein(single strand DNA
binding protein)
• maintains the DNA template in the single
strand form in order to
• prevent the dsDNA formation;
• protect the ssDNA degradation by
nucleases.
§2.5 Topoisomerase
• Opening the
dsDNA will create
supercoil ahead of
replication forks,
the supercoil
constraint needs
to be released by
topoisomerases
(type I and II).
Topoisomerase I
• 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.
Topoisomerase II
• It is named gyrase in
prokaryotes.
• It cuts phosphoester
bonds on both strands
of dsDNA, releases
the supercoil constraint,
and reforms the
phosphoester bonds.
• 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
splicing.
5’
3’
O
3’
O P OO-
HO
5’
ATP（NAD+）
DNA Ligase
AMP
5’
3’
O
O P OO-
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.
• 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
the template
downstream further
to separate enough
space.
• SSB protein binds the
complex to stabilize
ssDNA.
Primer synthesis
• Primase joins and
starts the synthesis of
RNA primers.
• Primasome: protein
5'
complex responsible
3'
for creating RNA
primers on ssDNA
during DNA replication.
• Topoisomerase binds to
the dsDNA region just
before the replication
forks to release the
supercoil constraint.
Dna A
Dna B Dna C
DNA topomerase
primase
3'
5'
3
5
primer
3
5
• The short RNA fragments provide free
3´-OH groups for DNA elongation.
b. 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
phosphodiester bonds.
Lagging strand synthesis
• Primers on Okazaki
fragments are
digested by RNase.
• The gaps are filled by
DNA-pol I in the
5´→3´direction.
3'
5'
5'
3'
RNAase
RNase
3'
OH
5'
• The nick between the
3'
5´end of one
5'
fragment and the
3´end of the next
fragment is sealed by
3'
DNA ligase.
5'
dNTP
P
polymerase
DNA
DNA-pol
I
P
ATP
5'
3'
5'
3'
DNA ligase
5'
3'
flash
• The synthesis direction of
the leading strand is the
same as that of the
replication fork.
• The synthesis direction of
the latest Okazaki fragment
is also the same as that of
the replication fork.
c. Termination
movie
• 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.
ori
82
32
ter
Replication of prokaryotes
•The replication
process starts
from the origin,
and proceeds in
two opposite
directions. It is
named 
replication.
§ Replication Fidelity
• Replication based on the principle of
base pairing is crucial to the high
accuracy of the genetic information
transfer.
• Enzymes use three mechanisms to
ensure the replication fidelity.
1×10-5
1×10-2
1×10-2
1×10-9
§3.2 Replication of Eukaryotes
• DNA replication is
closely related
with cell cycle: Sphase.
• Multiple origins on
one chromosome.
• Cell cycle
DNA-pol of eukaryotes
DNA-pol : initiate replication
and synthesize primers
DnaG,
primase
DNA-pol : replication with
low fidelity
repairing
DNA-pol : polymerization in
mitochondria
DNA-pol : elongation
DNA-pol III
DNA-pol : proofreading and
filling gap
DNA-pol I
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.
3
5
5
3
Leading strand
3
5
Lagging strand
primer
nucleosome
3
5
c. Termination
3'
5'
5'
3'
3'
5'
5'
3'
3'
5'
connection of discontinuous
segment
5'
3'
3'
5'
5'
3'
The End Replication Problem:
Telomeres shorten with each S phase
5'
3'
3'
5'
3'
5'
3'
5'
5'
Ori
Telomere
• Telomere: the terminal structure
of eukaryotic DNA of
chromosomes.
• composed of terminal DNA
sequence and protein.
• Function: keep the termini of
chromosomes in the cell from
becoming entangled and sticking to
each other.
Repetitive DNA sequence
(TTAGGG in vertebrates)
Form a 'capped' end structure
shoelace
The Nobel Prize in Physiology or
Medicine 2009
Elizabeth Blackburn
Carol Greider
Jack Szostak
"for the discovery of how chromosomes are
protected by telomeres and the enzyme
telomerase"
Telomerase
• Telomerase: the enzyme that essentially
builds new telomeres, 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.
inchworm
Inchworm model
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
cellular clock
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
the telomerase.
•Telomerase is an attractive target for cancer
chemotherapy.
SUMMARY
Telomeres are essential for chromosome stability
Telomere shortening occurs owing to the biochemistry of
DNA replication
Short telomeres cause replicative senescence
Telomerase prevents telomere shortening and
replicative senescence
Section 4
Reverse Transcription
Reverse Transcription
• 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
reverse transcription.
Viral infection of RNA virus
Reverse transcription
•Reverse transcription is
a process in which
ssRNA is used as the
template to synthesize
dsDNA.
•Synthesis of ssDNA
complementary to ssRNA,
cDNA, forming a RNA-DNA
hybrid.
•Hydrolysis of ssRNA: RNase
activity of reverse
transcriptase, leaving ssDNA.
•Synthesis of the second
ssDNA, forming a DNA-DNA
duplex.
David Baltimore
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
molecular biology
•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
dogma.
Section 5
DNA Damage and Repair
§5.1 Mutation
•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
process.
• 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
UV radiation
Physical
factors
Chemical
modification
carcinogens
DNA
damage
infection
spontaneous
mutation
T
G
viruses
evolution
Physical damage
O
O
N
P
N
O
UV
O
N
R
R
CH3
N
N
O
CH3
P
Physical
factors
CH3
O
R
N
O
N
CH3
O
)
R
N
(TT)
viruses
Mutation caused by
chemicals
• Carcinogens can cause mutation.
• Carcinogens include:
• Food additives and food
preservatives; spoiled food
• Pollutants: automobile emission;
chemical wastes
• 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
pyrimidine.
• Transversion: the base alternation
between purine and pyrimidine, and vise
versa.
• 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
serine.
• 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
codon.
Hb mutation causing anemia
•Single base mutation leads to one AA
change, causing disease.
HbS
HbA
 chains
CAC
CTC
 mRNA
GUG
GAG
AA residue 6 in  chain
Val
Glu
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.
Frame-shift mutation
Normal
5´… …GCA GUA CAU GUC … …
Ala Val His Val
Deletion C
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
DNA replication.
Photoreactivation repair
(or lignt repair)
O
O
N
P
N
O
UV
O
N
R
R
CH3
O
CH3
P
CH3
O 300~600nm
N
N
R
N
O
N
CH3
O
)
R
N
(TT)
Excision repairing
• One of the most
important and effective
repairing approach.
• UvrA and UvrB:
recognize and bind the
damaged region of
DNA.
• UvrC: excise the
damaged segment.
• DNA-pol Ⅰ: synthesize
the DNA segment to
fill the gap.
• DNA ligase: seal the
nick.
UvrC
UvrA
UvrB
OH
P
DNA-pol Ⅰ
OH P
DNA ligase NAD+
Xeroderma
pigmentosum (XP)
• 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,
and often
important part of
treatment is
avoiding exposure
to sunlight.
Recombination repairing
• It is used for repairing when a large
segment of DNA is damaged.
• Recombination protein RecA, RecB and RecC
participate in this repairing.
SOS 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
network.
Points
I. General characteristics
•
Semi-conservative; Specific origins; Bidirectional; Semidiscontinuous
replication
II. Bacterial Replication
A. Polymerization
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,
helicase, topoisomerase
B. Semidiscontinuous replication: leading strand and lagging
strand synthesis
1. RNA primer synthesized by the primases
2. polymerization by Pol III
3. completion by Pol I and ligase
4. Okazaki fragment
Points (continue)
Ⅲ. Eukaryotic Replication
• S phase; Telomere and Telomerase
Ⅳ. Reverse transcription
•
Definition; Significance
Ⅴ. Mutation, DNA damage and repair
•
Point mutation; insertion and deletion, Frameshift
mutations
•
Physical and chemical damage;
•
photoreactivation repair; excision repair
•
Xeroderma pigmentosum (XP)