Download replication of dna

REPLICATION OF DNA
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Restriction enzyme
• Endonuclease – cleave
the nt in the middle of
DNA molecule
• Exonuclease - cleave
the nt from the end of
DNA molecule
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The flow of genetic information
• Duplication of DNA to
produce a new DNA
molecule with the same
base sequence as original
• A necessary process
whenever a cell divides to
produce daughter cells
• Flow of genetic
information
DNA –RNA-Protein
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Replication process
• A complex process
• Ensures fidelity
• 3 important challenge
1. Separating the two strands – the double
helix must be unwound if they are to be
separated. At the same time, the unwound
portions must be protected from the action
of nucleases that prefer to attack ss DNA.
2. Synthesizing DNA from 5’ end to the 3’end.
3. Guarding against error in replication –
ensuring the correct base is added
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Types of replication
• Conservative
The parental strands never completely separate
After on round replication, one daughter duplex contains only
parental strands and the other only daughter strands
• Semi conservative
The process of unwinding, of the double helical daughter
molecules – that is composed of a parental strand and a newly
synthesized strand formed from the complementary strand
• Theta replication – replication in a circular form -prokaryote
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Semiconservative replication
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Steps involved in DNA replication
a) Identification of the origins of replication
b) Unwinding (denaturation) of dsDNA to provide ssDNA
template
c) Formation of the replication fork
d) Initiation of DNA synthesis and elongation
e) Formation of replication bubbles with ligation of the
newly synthesized DNA segments
f) Proof reading process
DNA replication in Prokaryote and eukaryote generally involve
the same steps, except in eukaryote the process is more
complex due to the presence of histone complex
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DNA REPLICATION IN E.COLI-FAMOUS
MODEL
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a) Origin of replication
• Origin of replication (oriC )- spesific sites where
replication starts
• Sequence specific DNA binding proteins (O Protein) will
bind to ori
• Adjacent to ori is A+T region
• Binding of O protein lead to local denaturation and
unwinding of A+T region
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b) Unwinding of DNA to form SS DNA which act as template
• Interaction of O protein provide a short region
of ssDNA essential for initiation of replication
• This region – a template for initiation
• DNA helicases helps in unwinding of DNA
• DNA helices produces nicks in one strand of
double helix
• Single stranded binding proteins (SSB
Proteins) – bind to SS each strand and
stabilize the complex and prevents reannealing
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DNA Replication Process
1. DNA helicase unwinds short segment of parent DNA
2. SSB protein stabilize the unwound parental DNA
3. A primase initiates synthesis of RNA molecule (primer) that is essential for
priming DNA synthesis
4. Initiation of rep-require priming by short length of RNA (primer) (10 to 200nts)
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Template and primer
5’ Primer 3’-OH
AGCTACTGCT…….
3’-OH
Template
5’
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DNA Replication Process
• Priming process-nucleophilic attack by 3’-OH group of the RNA primer on the
α-phoshate of the first entering nucleotide with release of pyrophosphate
• Elongation-3’-OH of the newly attached nt is then free to carry out
nucleophilic attack on the next entering nt
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DNA Replication Process
• DNA polymerase III begin replication by adding new complementary strand nt
to the primer-producing polynucleotide chain
• DNA polymerase III cannot initiate DNA synthesis ‘de novo’
• Leading strand (fwd strand)-the DNA is synthesized continuously in 5’to3’
direction with the same over-all fwd direction
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DNA Replication Process
• Lagging strand – the DNA is synthesized in discontinuous manner-not directly
as leading strand- DNA Pol III only add nt to 3-OH – So the system is reversed
to comply to its function
• As replication fork is produced, a primer is synthesized from 5’ to 3’ and DNA
Pol III begin the polymerization process –producing short DNA strand –
Okazaki fragments
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DNA Replication Process
• Okazaki fragments are 100-250nt long in eukaryotes and 1000-2000bp in
prokaryotes
• RNA primers will be removed by DNA Pol I (using its exonuclease activity)
• Leaving primers leave a gap (at least one nt missing) • The gap will be replaced by nt by DNA Pol I – leaving only a nick
(interruption in the phosphodiester bond with no missing nts)
• These nicks are sealed by DNA ligases producing a long polynucleotide
chains
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Termination of replication (prokaryote)
• The 2 replication forks of
E.Coli meet at terminus
region (Ter)
• Terminal Utilization Protein
(Tus) will bind to Ter –
forming Tus-Tur Complex
and stop the rep forkcompleting 2 interlinked
circular chromosome
(catenated)
• DNA Topoisomerase IV
separated the chromosome
– segregate into daughter
cells
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DNA REPLICATION IN EUKARYOTES
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DNA REPLICATION IN EUKARYOTE
• Mostly studied - yeast
• The understanding is not as much as in
prokaryotes
• More complicated
– Multiple Ori- replicators (around 400 replicators in
yeast)
– Timing must be controlled to that off cell division
– More proteins and enzymes involved
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DNA REPLICATION IN EUKARYOTE
• Cell growth- M, G1, S and
G2
• Gap 1 (G1) Cell prepare for
DNA synthesis
• DNA replication occur in S
phase – synthetic S phase
• Transition from one phase
to another is controlled by
cyclins proteins
• The DNA is replicated once
and only once
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DNA REPLICATION IN EUKARYOTE
• Cancer-causing virus (oncoviruses) and cancer
inducing genes (oncogenes) – capable of
disrupting the entry of mammalian cells from
G1 to S – excessive production of cyclin –
production in an appropriate time – abnormal
cell division
• Once a chromatin has been replicated, it is
marked to further its replication until it again
passes through mitosis
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The DNA Polymerase Complex
E.Coli
Mammalian
I
α
Gap filling and synthesis of lagging strand
II
ξ
DNA Proof reading and repair
β
DNA Repair
γ
Mitochondrial DNA synthesis
δ
Processive, leading strand synthesis
III
Function
In mammalian cell, the polymerase is capable of polymerizing about 100nt
persecond – ten fold slower than in bacterial
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Replication bubble
• In prokaryote- genome size is around 6x106 bp – replication is completed
in 30 mins (replication rate 3x105bp per min)
• In eukaryote – genome size is 3x109
Replicated in the same rate – will take 150 hours to complete!
Problem is overcome by having multiple origin in chromosome and
replication occur in both direction along all of the chromosome
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Termination of replication in eukaryotes
• Require a special mechanism because the DNA is linear
• Leading strand -5’ end template is not a problem,
because the DNA is synthesized from 5’to3’ (continuous
process and require only one primer in the beginning.
• Lagging strand- problem
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Termination of replication in eukaryotes
The last RNA Primer need to
be removed, the gap need to
be filled with DNA, but
without RNA, the DNA cannot
be synthesized
The DNA can become
shorter and shorter
every time replication
occur
Lagging strand
3’
5’
3’
Lagging strand
3’
Telomerase added telomere
repeats to the 3’end of the new
strand-serve as primer
5’
5’ ????????
5’
3’
5’
3’
TTAGGG
3’
The last segment of DNA can be
synthesized- complete the
replication
5’
3’
3’
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REPLICATION ERRORS AND THEIR
REPAIR
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Causes of DNA Damage
DNA damage during DNA
replication can occur through:
• Mis-incorporation of dntp
during replication
• By spontaneous
deamination of bases during
normal genetic functions
• From X- radiation that cause
nicks in the DNA
• From various chemicals that
interact with DNA
The rapid repair is needed
as they may be lethal to
the cell
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Effect of replication errors
• Mutations – permanent
changes- not fx
• Prevent it to be used as
the template for
replication and
transcription
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Types of damages to DNA
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•
•
•
Single base alteration
Two base alteration
Chain breaks
Cross-linkage
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Cell cycle checkpoint
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Proof reading mechanism
• Error can occur once in 104 to 105 base pairs
• Proof reading -Removal of incorrect nt immediately
after they are added to the growing DNA during the
replication process
• Performed by DNA Pol I
• DNA Pol I have two major components –
– Klenow fragment (for polymerase & proofreading
activity)
– 5’ to 3’ repair activity
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Proof reading mechanism
• Occur at the last stage of replication, after removing of
primers -Through cut and patch process
– Cut – removal the DNA mistakes as it moves along the
DNA
– Patch – Fills in the right nt
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Some replication errors escapes
proofreading
• Proof reading activity- increase the fidelity
• However, there are some errors escape
• These errors need to be minimized before it
become permanent damaged in the DNA
sequence
• Performed through genome scanning by the
protein MutS
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1. The newly synthesized
DNA has a mismatched
Mismatched repair
2. MutH, Mut S, Mut L
complex will bring the
mismatch with the nearest
methylation site – to
identify the parent strand.
The methylated strand is
the parent strand
3. An exonuclease removes
DNA from the red strand
between proteins
(damaged dna)
4. DNA Polymerase replace
the removed DNA with the
correct sequence
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Base excision repair
• To repair base that is
damaged by oxidation or
chemical modification
• The damaged base is
removed by DNA
glycosylase –leaving AP
site
• AP endonuclease then
removes s the sugar and
PO4 from the group
• Excision nuclease removes
several more bases
• DNA Pol I fill the gap
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Nucleotide excision repair
• For DNA damaged due to
the UV or chemical effect
– lead to deformed DNA
structure
• ABC excinuclease remove
the large section of
damaged DNA
• DNA Pol I add new nt
• DNA ligases seal the gap
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Prokaryotes
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Initiation point specific (ori)
DNA Polymerase- 3 types – I, II, III
DNA Pol I has diverse function
Not applicable
No repair function
Replication with few replication
forks
Theta structure observed
Accessory proteins few with limited
functions
Only unwinding takes place in
prokaryotes
Not at all or few replication
bubbles
RNA as primer
Eukaryotes
• Initiation point specific but different
in prokaryotes
• DNA Pol – 5 types –αξβϒδ
• Functional variety of DNA
polymerase is specific
• γ DNA polymerase In mitochondria
• Β- Polymerase functions as repair
enzyme
• Many replication forks
• Theta structure not observed
• Many accessory proteins with
diverse functions
• Histone separation from DNA as
well as unwinding takes place
• Many replication bubbles
• RNA/DNA as primer
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Summary
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Identification of sites of the origin of replication (ori)
Unwinding of parental DNA (dsDNA ssDNA)
Formation of replication fork
Synthesis of RNA primer, complementary to DNA template,
the enzyme required is primase
Leading strand is synthesized in the 5’to 3’ direction by the
enzyme DNA polymerase
Lagging strand is synthesized as Okazaki fragments
RNA pieces are removed when polymerization is complete
The gaps are filled by nt and the pieces are joined by DNA
ligases
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