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REPLICATION OF DNA
By
Sumit sharma
Assistant Professor
HMV jalandhar
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• To impart the basic concepts of DNA replication process
• To demonstrate the functions of various enzymes involved
in the replication process
• To highlight on the different stages and mechanism of the
replication process
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DNA RNA protein
transcription translation replication
reverse
transcription
Central dogma
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• 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
www.cuchd.in Campus: Gharuan, Mohali
CENTRAL DOGMA
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DNA
Replication
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General Concepts of
DNA Replication
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• A reaction in which daughter DNAs are
synthesized using the parental DNAs as
the template.
• Transferring the genetic information to the
descendant generation with a high fidelity
replication
parental DNA
daughter DNA
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• Chemical formulation:
(dNMP)n + dNTP (dNMP)n+1 + PPi
DNA strand substrateelongated
DNA strand
• The nature of DNA replication is a
series of 3´- 5´phosphodiester bond
formation catalyzed by a group of
enzymes.
www.cuchd.in
DAUGHTER STRAND SYNTHESIS
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• Putting the DNA backbone together
– refer to the 3 and 5 ends of the DNA
OH
O
PO4
base
CH2
O
base
O
P
O
C
O–O
CH2
1
2
4
5
1
2
3
3
4
5
www.cuchd.in Campus: Gharuan, Mohali
THE DNA BACKBONE
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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
www.cuchd.in
DNA REPLICATION SYSTEM
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Characteristics of replication
Semi-conservative replication
Bidirectional replication
Semi-continuous replication
High fidelity
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Half of the parental DNA molecule is
conserved in each new double helix,
paired with a newly synthesized
complementary strand. This is called
semiconservative replication
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Semiconservative replication
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replication
"Heavy" DNA(15N)
grow in 14N
medium
The first generation
grow in 14N
medium
The second generation
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Significance
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The genetic information is ensured to be
transferred from one generation to the
next generation with a high fidelity.
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• 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.
Bidirectional replication
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3'
5'
5'
3'
5'
3'
5'3'
direction of
replication
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Replication Fork
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• 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.
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The replication
process starts
from the origin,
and proceeds
in two opposite
directions. It is
named
replication.
www.cuchd.in
REPLICATION OF PROKARYOTES
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• Chromosomes of eukaryotes have
multiple origins.
• The space between two adjacent
origins is called the replicon, a
functional unit of replication.
Replication in eukaryotes
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5'
3'
3'
5'
bidirectional replication
fusion of bubbles
3'5'3'5'
5'3'5'3'
origins of DNA replication (every ~150 kb)
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§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.
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5'
3'
3'
5'
5'
direction of unwinding3'
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.
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Leading strand
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3'
5'
5'3'
replication direction
Okazaki fragment
3'
5'
leading strand
3'
5'
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3'
5'
replication fork
Semi continuous replication
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• 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.
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Okkazaki fragments
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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.
Semi continuous replication
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Enzymology
of DNA Replication
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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
Enzyme and Protein factors
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• 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.
DNA-pol of prokaryotes
DNA Polymerase
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• 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. 53 polymerizing
2. exonuclease
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DNA-pol I
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• Mainly
responsible for
proofreading
and filling the
gaps, repairing
DNA damage
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Klewno fragment
• small fragment (323 AA): having 5´→3´exonuclease activity
• large fragment (604 AA): called Klenow
fragment, having DNA polymerization
and 3´→5´exonuclease activity
N end C end
caroid
DNA-pol Ⅰ
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• 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
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• 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-ppl lll
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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
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DNA-pol : elongation DNA-pol III
DNA-pol : initiate replication and synthesize primers
DnaG, primase
DNA-pol : replication with
low fidelity
DNA-pol : polymerization in
mitochondria
DNA-pol : proofreading and
filling gap
DNA-pol I
repairing
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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.
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• 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.
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§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
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§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.
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§2.5 Topoisomerase• Opening the dsDNA will create
supercoil ahead of replication forks.
• The supercoil constraint needs to be
released by topoisomerases.
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• 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
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• Also called -protein in prokaryotes.
• 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 I (topo I)
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• It is named gyrase in prokaryotes.
• It cuts phosphoester bonds on both
strands of dsDNA, releases the
supercoil constraint, and reforms the
phosphoester bonds.
• It can change dsDNA into the
negative supercoil state with
consumption of ATP.
Topoisomerase II (topo II)
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§2.6 DNA Ligase3'
5'
5'
3'
RNAase
POH
3'
5'
5'
3'
DNA polymerase
P
3'
5'
5'
3'
dNTP
DNA ligase
3'
5'
5'
3'
ATP
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• 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.
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§2.7 Replication Fidelity
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• 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
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• 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.
Proofreading and correction
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3´→5´exonuclease
activityexcise mismatched
nuleotides
5´→3´exonuclease
activitycut primer or
excise mutated
segment
C T T C A G G A
G A A G T C C G G C G
5' 3'
3' 5'
Exonuclease functions
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References:
• Lehninger, Principle of Biochemistry, 5th edition
• Kumar,P. and Mina,U.2013.Life Sciences Fundamentals and
Practice.Vol(II).3rd ed.Pathfinder Publication,New Delhi
• Maloy, Stanley R. , Cronan, John E. and
Freifelder,David,Microbial Genetics.2nd edition.Narosa
Publication,New Delhi
• http://themedicalbiochemistrypage.org/dna.php#intro
• Satyanarayan,U and Chakrapani,U.2010.Biochemistry.3rd
ed.Book and Allied(P) Ltd.Kolkata