Replication 13

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DNA STRUCTUREDNA STRUCTURE
ANDAND
REPLICATIONREPLICATION
DR.K.S.SODHI
PROFESSOR
MMIMS&R

2
Central Dogma
RNA
DNA
Protein
轉錄
轉譯
Transcription
Translation
Replication
複製
逆轉錄
Reverse
Transcription
Juang RH (2004) BCbasics

33
ARTHUR KORNBERG1958ARTHUR KORNBERG1958

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23 paternal
directories
23 maternal
equivalents
Total 35,000 files
Replication
Nucleus
23 x 2
In 46 chromosomes
Homologous chromosomes
Before cell
division
3,000 MB
Cell
Nucleus

5
Site-directed mutagenesis
CAG
GTC
CAG
GCC
CAG
GCC
CAG
+ polymerase
+ primer
replication
GCC
CGG
Mutant
Thr
translation
Wild type
GTC
CAG
Val
translation
Only one amino acid changed
Wild type protein
Mutant protein
primer
(1)
(2)
(3)
(5)
(4)
(6)
Val → ThrSmith (1993)
JuangRH(2004)BCbasics

6
AbbreviationsAbbreviations
 dsDNA.dsDNA.
 ssDNA.ssDNA.
 Ori.Ori.
 SSBsSSBs
 DnaK,dnaJ,dnaEDnaK,dnaJ,dnaE
 DNA Helicase.DNA Helicase.
 dRibosedRibose
 ApoptosisApoptosis
 Leading & Laging strands.Leading & Laging strands.
 Gyrase.Gyrase.
 Speed:100nts/sec. total 9 hours toSpeed:100nts/sec. total 9 hours to
complete in a typical cell.complete in a typical cell.
 Double stranded DNADouble stranded DNA
 Single stranded DNASingle stranded DNA
 Origin of replication.Origin of replication.
 Single strand Binding.Single strand Binding.
 Heat shock proteins EHeat shock proteins E
 Unwind short segmenUnwind short segmen
 De-OxyriboseDe-Oxyribose
 Programmed cell deathProgrammed cell death
 One strech, multiple streches.One strech, multiple streches.
 Negative supercoiling using ATPNegative supercoiling using ATP

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 cDNA asingle stranded DNA molecule thatcDNA asingle stranded DNA molecule that
is complementary to mRNA and isis complementary to mRNA and is
synthesised from it by the action ofsynthesised from it by the action of
reverse transcriptase.reverse transcriptase.
 miRNAS micro RNAs 21-25 nucleotidemiRNAS micro RNAs 21-25 nucleotide
long.long.
 Sines Short interspread repeatSines Short interspread repeat
sequences.sequences.
 Si RNA silencing RNA 21-25 nt length canSi RNA silencing RNA 21-25 nt length can
cause gene knockdown.cause gene knockdown.

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PROTEINS & FUNCTIONSPROTEINS & FUNCTIONS
 DNA polymerasesDNA polymerases
 Helicases.Helicases.
 Topoisomerases.Topoisomerases.
 DNA primaseDNA primase
 SSB proteinsSSB proteins
 DNA LigaseDNA Ligase
 Polymerisation.Polymerisation.
 Unwinding of DNA.Unwinding of DNA.
 Remove supercoiling.Remove supercoiling.
 Initiates synth. of RNAInitiates synth. of RNA
primer.primer.
 Prevent reanealing ofPrevent reanealing of
dsDNA.dsDNA.
 Seals the nick inSeals the nick in
okazaki fragments.okazaki fragments.

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REQUIREMENTSREQUIREMENTS
 Four activated precursors ofFour activated precursors of
dATP,dGTP,dCTP andTTP MgdATP,dGTP,dCTP andTTP Mg++++
..
 Template Strand.Template Strand.
 Primer with free3’-OH group(10-200)Primer with free3’-OH group(10-200)
 Elongation proceeds 5’—3’ direction.Elongation proceeds 5’—3’ direction.
 Removal of mismatched nucleotides.Removal of mismatched nucleotides.
 Error rate is less than 10Error rate is less than 10 -8-8 per
bp.
 3’3’
hydroxyl group attack(nucleophillic) on
po4 of recently attached nucleotide.

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DNADNA
 DNA stands for deoxyribose nucleic acid
 This chemical substance is present in the
nucleus of all cells in all living organisms
 DNA controls all the chemical changes
which take place in cells
 The kind of cell which is formed, (muscle,
blood, nerve etc) is controlled by DNA
 The kind of organism which is produced
(buttercup, giraffe, herring, human etc) is
controlled by DNA The kind of organism
which is produced (buttercup, giraffe,
herring, human etc) is controlled by DNA

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Ribose is a sugar, like glucose, but with only five
carbon atoms in its molecule
Deoxyribose is almost the same but lacks one
oxygen atom
Both molecules may be represented by the
symbol
Ribose & deoxyriboseRibose & deoxyribose

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The most common organic bases are
Adenine (A)
Thymine (T)
Cytosine (C)
Guanine (G)
The basesThe bases

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The deoxyribose, the phosphate and one of the bases
adenine
deoxyribose
PO4
Combine to form a nucleotide
NucleotidesNucleotides

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A molecule of
DNA is formed by
millions of
nucleotides joined
together in a long
chain
PO4
PO4
PO4
PO4
sugar-phosphate
backbone + bases
Joined nucleotidesJoined nucleotides

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PO4
PO4
PO4
PO4
PO4
PO4
PO4
PO4
PO4
PO4
PO4
PO4
PO4
PO4
PO4
PO4
2-stranded DNA2-stranded DNA

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REPLICATION STEPSREPLICATION STEPS
 A. INITIATIONA. INITIATION
 B. ELONGATION.B. ELONGATION.
 C. TERMINATION.C. TERMINATION.

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DNA ReplicationDNA Replication
 Priming:Priming:
1.1. RNA primersRNA primers: before new DNA strands can: before new DNA strands can
form, there must be small pre-existingform, there must be small pre-existing
primers (RNA)primers (RNA) present to start the addition ofpresent to start the addition of
new nucleotidesnew nucleotides (DNA Polymerase)(DNA Polymerase)..
2.2. PrimasePrimase: enzyme that polymerizes: enzyme that polymerizes
(synthesizes) the(synthesizes) the RNA PrimerRNA Primer..

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This DNA polymerase replaces the RNA primer
with DNA.
This is a different type of DNA polymerase
from the main DNA polymerase which
synthesises DNA on a DNA template.
Another DNA polymerase:

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In E. coli the main enzyme is DNA
polymerase III .
And the enzyme that replaces the RNA
primer with DNA is DNA polymerase I.
When the RNA primer has been
replaced with DNA, there is a gap
between the two Okazaki fragments
and this is sealed by DNA ligase.

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DNA ligase seals the gap left between Okazaki
fragments after the primer is removed. As the
Okazaki fragments are joined, the new lagging
strand becomes longer and longer.
DNA ligase:
Location: At the replication fork.
Function: Unwinds the DNA double helix.
Helicase:

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Location: On the template strands.
Function: Synthesizes new DNA in the
5' to 3' direction using the base
information on the template strand to
specify the nucleotide to insert on the new
chain. Also does some proofreading; that
is, it checks that the new nucleotide being
added to the chain carries the correct base
as specified by the template DNA.
DNA polymerase:

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The new DNA strand made discontinuously
in the direction opposite to the direction in
which the replication fork is moving.
The new DNA strand made continuously in
the same direction as movement of the
replication fork.
Lagging Strand:
Leading strand:

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If an incorrect base pair is formed,
DNA polymerase can delete the new
nucleotide and try again. In E. coli
the enzyme used for all new DNA
synthesis except for the replacement
of the RNA primers is DNA
polymerase III. DNA polymerase I
replaces the primers.

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Location: On the template strand which
dictates new DNA synthesis away from the
direction of replication fork movement.
Okazaki fragment:

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Function: A building block for DNA
synthesis of the lagging strand. On one
template strand, DNA polymerase
synthesizes new DNA in a direction away
from the replication fork movement.
Because of this, the new DNA synthesized
on that template is made in a
discontinuous fashion; each segment is
called an Okazaki fragment.

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Location: Wherever the synthesis of a new
DNA fragment is to commence.
Function: DNA polymerase cannot start the
synthesis of a new DNA chain, it can only
extend a nucleotide chain primer. Primase
synthesizes a short RNA chain that is used as
the primer for DNA synthesis by DNA
polymerase.
Primase:

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Location: On single-stranded DNA near the
replication fork. Function: Binds to single-
stranded DNA to make it stable.
Single-strand binding (SSB)
proteins

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Alternative models of DNA replication

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DNA Replication 1
 Models of DNA replication: -Meselson-Stahl
Experiment
 DNA synthesis and elongation
 DNA polymerases
 Origin and initiation of DNA replication
 Prokaryote/eukaryote models
 Telomere replication

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Let us animate theLet us animate the
process of replicationprocess of replication

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H bonds break Two strands seperate

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Sugar phosphate backbone is made by joining the adjcent
nucleotides ( DNA polymarase enzyme( ) )
Nucleotides with Complementary bases are assembled
alongside each strands

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Two identical DNA molecules are formed

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1958: Matthew Meselson & Frank Stahl’s Experiment
Semiconservative model of DNA replication (Fig. 3.2)

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1955: Arthur Kornberg
Worked with E. coli.
Discovered the mechanisms of DNA synthesis.
Four components are required:
1. dNTPs: dATP, dTTP, dGTP, dCTP
(deoxyribonucleoside 5’-triphosphates)
(sugar-base + 3 phosphates)
2. DNA template
3. DNA polymerase (Kornberg enzyme)
4. Mg 2+
(optimizes DNA polymerase activity)
1959: Arthur Kornberg (Stanford University) & Severo Ochoa (NYU)

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Three main features of the DNA synthesis
reaction:
1. DNA polymerase I catalyzes formation of phosphodiester bond
between 3’-OH of the deoxyribose (on the last nucleotide) and
the 5’-phosphate of the dNTP.
• Energy for this reaction is derived from the release of two of the
three phosphates.
2. DNA polymerase “finds” the correct complementary dNTP at each
step in the lengthening process.
• rate ≤ 800 dNTPs/second
• low error rate
3. Direction of synthesis is 5’ to 3’

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DNA elongation (Fig. 3.3a):

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There are many different types of DNA polymerase
Polymerase
Polymerization
(5’-3’)
Exonucleas
e (3’-5’)
Exonuclease
(5’-3’)
#Copies
I YES YES YES 400
II YES NO YES?
III YES YES YES20-40

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 3’ to 5’ exonuclease activity = ability to remove
nucleotides from the 3’ end of the chain
 Important proofreading ability
– Without proofreading error rate (mutation rate) is 1
x 10-6
– With proofreading error rate is 1 x 10-9 (1000-fold
decrease)
 5’ to 3’ exonuclease activity functions in DNA
replication & repair.

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Eukaryotic enzymes:
 Five DNA polymerases from mammals.
 Polymerase α (alpha): nuclear, DNA replication,
no proofreading
 Polymerase β (beta): nuclear, DNA repair, no
proofreading
 Polymerase γ (gamma): mitochondria, DNA
repl., proofreading
 Polymerase δ (delta): nuclear, DNA replication,
proofreading

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 Polymerase ε (epsilon): nuclear, DNA
repair (?), proofreading
 Different polymerases for nucleus and
mtDNA
 Some polymerases proofread; others do not.
 Some polymerases used for replication;
others for repair

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Origin of replication (e.g., the prokaryote example):
 Begins with double-helix denaturing into single-strands thus exposing the
bases.
 Exposes a replication bubble from which replication proceeds in both
directions.

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Initiation of replication, major elements:
 Segments of single-stranded DNA are called template
strands.
 Gyrase (a type of topoisomerase) relaxes the supercoiled
DNA.
 Initiator proteins and DNA helicase binds to the DNA at
the replication fork and untwist the DNA using energy
derived from ATP (adenosine triphosphate).
(Hydrolysis of ATP causes a shape change in DNA
helicase)
 DNA primase next binds to helicase producing a
complex called a primosome (primase is required for
synthesis),

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 Primase synthesizes a short RNA primer of 10-12
nucleotides, to which DNA polymerase III adds
nucleotides.
 Polymerase III adds nucleotides 5’ to 3’ on both strands
beginning at the RNA primer.
 The RNA primer is removed and replaced with DNA by
polymerase I, and the gap is sealed with DNA ligase.
 Single-stranded DNA-binding (SSB) proteins (>200)
stabilize the single-stranded template DNA during the
process.

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DNA replication is continuous on the leading strand and semidiscontinuous on the
lagging strand:
Unwinding of any single DNA replication fork proceeds in one direction.
The two DNA strands are of opposite polarity, and DNA polymerases only
synthesize DNA 5’ to 3’.
Solution: DNA is made in opposite directions on each template.
•Leading strand synthesized 5’ to 3’ in the direction of
the replication fork movement.
continuous
requires a single RNA primer
•Lagging strand synthesized 5’ to 3’ in the opposite
direction.
semidiscontinuous (i.e., not continuous)
requires many RNA primers

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3
Polymerase III
5’ →
3’
Leading strand
base pairs
5’
5’
3’
3’
Supercoiled DNA relaxed by gyrase & unwound by helicase + pr
Helicase
+
Initiator Proteins
ATP
SSB Proteins
RNA Primer
primase
2Polymerase III
Lagging strand
Okazaki Fragments
1
RNA primer replaced by polymerase I
& gap is sealed by ligase

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Two Libraries ： cDNA Library vs Genomic Library
mRNA
cDNA
Reverse transcription
Chromosomal DNA
Restriction digestion
Genes in expression Total Gene
Complete
gene Gene fragments
Smaller
Library
Larger
Library
Vector:
Plasmid or phage
Vector: Plasmid

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Restriction Mapping of DNA
A B 10 kb
8 kb
2 kb
A
7 kb
3 kb
B
5 kb
3 kb
2 kb
A
+
B
CK A B A+B M
Restriction
enzymes

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The Specific Cutting and Ligation of DNA
GAATTC
CTTAAG
GAATTC
CTTAAG
G
CTTAA
AATTC
G
AATTC
G
G
CTTAA
G
CTTAA
AATTC
G
G
CTTAA
AATTC
G
G
CTTAA
AATTC
G
EcoRI
DNA Ligase
EcoRI sticky end EcoRI sticky end

56
DNA ligase seals the gaps between Okazaki fragments with a
phosphodiester bond (Fig. 3.7)
TIME: E.coli 30 minutes,Humans: 24 Hours.

57Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
Fig. 3.5 - Model of DNA replication

Fig. 3.5 - Model of DNA replication

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Concepts and terms to understand:
Why are gyrase and helicase required?
The difference between a template and a primer?
The difference between primase and polymerase?
What is a replication fork and how many are there?
Why are single-stranded binding (SSB) proteins required?
How does synthesis differ on leading strand and lagging strand?
Which is continuous and semi-discontinuous?
What are Okazaki fragments?

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Replication of
circular DNA in
E. coli (3.10):
1. Two replication forks result in
a theta-like (θ) structure.
2. As strands separate, positive
supercoils form elsewhere in
the molecule.
3. Topoisomerases relieve
tensions in the supercoils,
allowing the DNA to continue
to separate.

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Rolling circle model of DNA
replication (3.11):
1. Common in several
bacteriophages including
λ.
2. Begins with a nick at the
origin of replication.
3. 5’ end of the molecule is
displaced and acts as
primer for DNA synthesis.
4. Can result in a DNA
molecule many multiples
of the genome length (and
make multiple copies
quickly).
5. During viral assembly the
DNA is cut into individual
viral chromosomes.

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DNA replication in eukaryotes:
Copying each eukaryotic chromosome during the S phase of the cell cycle
presents some challenges:
Major checkpoints in the system
1. Cells must be large enough, and the environment favorable.
2. Cell will not enter the mitotic phase unless all the DNA has replicated.
3. Chromosomes also must be attached to the mitotic spindle for mitosis
to complete.
4. Checkpoints in the system include proteins call cyclins and enzymes
called cyclin-dependent kinases (Cdks).

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• Each eukaryotic chromosome is one linear DNA
double helix
• Average ~108
base pairs long
• With a replication rate of 2 kb/minute, replicating
one human chromosome would require ~35 days.
• Solution ---> DNA replication initiates at many
different sites simultaneously.
Fig. 3.14

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Fig. 3.13 - Replication forks visible in Drosophila

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(or telomeres What about the ends ) of linear
chromosomes?
DNA polymerase/ligase cannot fill gap at end of chromosome after
RNA primer is removed. this gap is not filled, chromosomes
would become shorter each round of replication!
Solution:
1. Eukaryotes have tandemly repeated sequences at the ends of
their chromosomes.
2. Telomerase (composed of protein and RNA complementary to
the telomere repeat) binds to the terminal telomere repeat and
catalyzes the addition of of new repeats.
3. Compensates by lengthening the chromosome.
4. Absence or mutation of telomerase activity results in
chromosome shortening and limited cell division.

Fig. 3.16 Synthesis of telomeric DNA by telomerase

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Final Step - Assembly into Nucleosomes:
• As DNA unwinds, nucleosomes must disassemble.
• Histones and the associated chromatin proteins must
be duplicated by new protein synthesis.
• Newly replicated DNA is assembled into nucleosomes
almost immediately.
• Histone chaperone proteins control the assembly.
Fig. 3.17

6868
DNA DAMAGEDNA DAMAGE
AND REPAIRAND REPAIR

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DNA DAMAGE
1. SPONTANEOUS
2. ENVIRONMENTAL
AGENTS
3. REPLICATION
PHYSICAL
CHEMICAL
BIOLOGICAL

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Mis-
incorporation
of
bases
Chemicals UV-radiation
X-radiation
Spontaneous
De-amination
of bases
CAUSES
OF
DAMAGE

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Single
Base
alterations
Cross
Linkage
Chain
breaks
Two
Base
alterations
TYPES
OF
DAMAGE

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1.1. SINGLE BASE ALTERATIONSSINGLE BASE ALTERATIONS
 DEPURINATIONDEPURINATION
 DEAMINATION OF CYTOSINE TO URACILDEAMINATION OF CYTOSINE TO URACIL
 DEAMINATION OF ADENINE TODEAMINATION OF ADENINE TO
HYPOXANTHINEHYPOXANTHINE
 ALKYLATION OF BASESALKYLATION OF BASES
 INSERTION OR DELETION OF NUCLEOTIDESINSERTION OR DELETION OF NUCLEOTIDES
 BASE ANALOG INCORPORATIONBASE ANALOG INCORPORATION

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2.2. TWO BASE ALTERATIONSTWO BASE ALTERATIONS
 UV INDUCED THYMINE-THYMINEUV INDUCED THYMINE-THYMINE
DIMERSDIMERS
 BIFUNCTIONAL ALKYLATING AGENTSBIFUNCTIONAL ALKYLATING AGENTS

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3.3. CHAIN BREAKSCHAIN BREAKS
 IONIZING RADIATION INDUCEDIONIZING RADIATION INDUCED
 RADIOACTIVE DISINTEGRATION OFRADIOACTIVE DISINTEGRATION OF
BACKBONE ELEMENTBACKBONE ELEMENT
 FREE-RADICAL INDUCEDFREE-RADICAL INDUCED

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4. CROSS LINKAGE4. CROSS LINKAGE
 BETWEEN BASES IN SAME ANDBETWEEN BASES IN SAME AND
OPPOSITE STRANDOPPOSITE STRAND
 BETWEEN DNA AND PROTEINBETWEEN DNA AND PROTEIN
MOLECULESMOLECULES

76
1. Proof
reading
and editing
2. Mismatch
Repair
system
3. Base
Excision
repair
4. Nucleotide
Excision
Repair
5. Photo-
Reactivation
Or
Direct
repair
6. Double
Strand
Break
Repair
7.Transcription-
Coupled
repair

77
1. PROOF READING AND1. PROOF READING AND
EDITINGEDITING Despite doubleDespite double
monitoring duringmonitoring during
replication, first at time ofreplication, first at time of
incorporation of bases andincorporation of bases and
second by later follow upsecond by later follow up
energy requiringenergy requiring
processes, some mispairedprocesses, some mispaired
bases persist which havebases persist which have
to be removed by otherto be removed by other
enzyme systems.enzyme systems.
 The proof reading activityThe proof reading activity
is carried out by 3’-5’is carried out by 3’-5’
exonuclease activities ofexonuclease activities of
DNA polymerase III andDNA polymerase III and
I.I.

78
2. MISMATCH REPAIR SYSTEM2. MISMATCH REPAIR SYSTEM
 This mechanism operates immediately afterThis mechanism operates immediately after
DNA replication.DNA replication.
 Sometimes the replication errors escape theSometimes the replication errors escape the
DNA proofreading function. This mechanismDNA proofreading function. This mechanism
checks for the correction of escaped bases.checks for the correction of escaped bases.
 Specific proteins scan the newly synthesizedSpecific proteins scan the newly synthesized
DNA by the following mechanisms:DNA by the following mechanisms:
1.1. Identification of mismatched strandIdentification of mismatched strand
2.2. Repair of mispaired base.Repair of mispaired base.

79
 In error detection,In error detection,
parent strand isparent strand is
identified first with theidentified first with the
help of GATC-help of GATC-
sequences, that occursequences, that occur
approx. once after everyapprox. once after every
thousand nucleotides.thousand nucleotides.
 It is methylated atIt is methylated at
adenine residue.adenine residue.
 The methylation doesThe methylation does
not occur immediatelynot occur immediately
after replication. So, theafter replication. So, the
new strand is notnew strand is not
methylated and is easilymethylated and is easily
identified.identified.

80
 Secondly, on the new strand, GATC-endonucleaseSecondly, on the new strand, GATC-endonuclease
‘nicks’ the mismatched strand.‘nicks’ the mismatched strand.
 This faulty strand is digested by exonuclease.This faulty strand is digested by exonuclease.
 An extensive region, from the mismatched area tillAn extensive region, from the mismatched area till
the next GATC-sequence is removed.the next GATC-sequence is removed.
 This gap is filled by the DNA polymerase I, in 5’-3’This gap is filled by the DNA polymerase I, in 5’-3’
direction.direction.
 Clinical significance-Clinical significance- A defect in mismatch repairA defect in mismatch repair
in humans has been known to cause hereditary non-in humans has been known to cause hereditary non-
polyposis colon cancer (HNPCC).polyposis colon cancer (HNPCC).

81
3. BASE EXCISION REPAIR3. BASE EXCISION REPAIR
 This mechanism operates all the time in the cells.This mechanism operates all the time in the cells.
 The bases of DNA can be altered:The bases of DNA can be altered:
a)a) SpontaneouslySpontaneously -- cytosine uracil.cytosine uracil.
b)b) Deaminating compounds -Deaminating compounds - like NO, which is formedlike NO, which is formed
from nitrosamines,nitrites, and nitrates.from nitrosamines,nitrites, and nitrates.
- NO is a potent de-aminating compound, that converts:- NO is a potent de-aminating compound, that converts:
i.i. Ctytosine uracilCtytosine uracil
ii.ii. Adenine hypoxanthineAdenine hypoxanthine
iii.iii. Guanine xanthineGuanine xanthine
c)c) Bases can also be lost spontaneously -Bases can also be lost spontaneously - approximately,approximately,
10,000 purine bases are lost spontaneously per cell per10,000 purine bases are lost spontaneously per cell per
day.day.

82
 Following mechanismsFollowing mechanisms
operate to correct such baseoperate to correct such base
alterations or losses:alterations or losses:
1.1. Removal of abnormalRemoval of abnormal
bases-bases- Abnormal bases areAbnormal bases are
recognized by specificrecognized by specific
glycosylasesglycosylases..
-they hydrolytically cleave-they hydrolytically cleave
them from deoxy-ribose-them from deoxy-ribose-
phosphate backbone of thephosphate backbone of the
strand.strand.
-this results in either A-this results in either Apurinicpurinic
or Apyrimidinicor Apyrimidinic site,site,
referred to asreferred to as AP- siteAP- site..

83
2.2. Repair of AP-site -Repair of AP-site - AP-endonucleaseAP-endonuclease recognizes therecognizes the
empty site and starts excision by making a cut at 5’-end ofempty site and starts excision by making a cut at 5’-end of
AP-site.AP-site.
-- deoxy-ribose-phosphate lyasedeoxy-ribose-phosphate lyase removes the single, empty,removes the single, empty,
sugar-phosphate residue and gap is finally filled bysugar-phosphate residue and gap is finally filled by DNADNA
polymerase Ipolymerase I and nick is sealed byand nick is sealed by DNA ligaseDNA ligase..
NOTE..NOTE..
 By the similar series of steps involving initially theBy the similar series of steps involving initially the
recognition of the defect, the alkylated bases and baserecognition of the defect, the alkylated bases and base
analogs can be removed from DNA. And thus, DNAanalogs can be removed from DNA. And thus, DNA
returns to its original information content.returns to its original information content.
 This mechanism is efficient only for replacement of aThis mechanism is efficient only for replacement of a
single base but is not efficient for replacing regions ofsingle base but is not efficient for replacing regions of
damaged DNA.damaged DNA.

84
Recognition and excision ofRecognition and excision of
defectdefect
(eg:-UV induced dimers)(eg:-UV induced dimers)
 First,a UV-specificFirst,a UV-specific
endonuclease recognizes theendonuclease recognizes the
dimer and cleaves thedimer and cleaves the
damaged strand atdamaged strand at
phosphodiester bonds on bothphosphodiester bonds on both
5’ side and 3’ side of the5’ side and 3’ side of the
dimer.dimer.
 The damaged oligonucleotideThe damaged oligonucleotide
is released, leaving a gap inis released, leaving a gap in
the DNA strand that formerlythe DNA strand that formerly
contained the dimer.contained the dimer.
 This gap is filled byThis gap is filled by DNADNA
polymerase Ipolymerase I and nick isand nick is
sealed bysealed by DNA ligaseDNA ligase..

85
5. PHOTO-REACTIVATION OR5. PHOTO-REACTIVATION OR
DIRECT REPAIRDIRECT REPAIR
 This is also called ‘This is also called ‘lightlight
induced repair’.induced repair’.
 The enzyme photo-The enzyme photo-
reactivating enzyme (PRreactivating enzyme (PR
enzyme) brings about anenzyme) brings about an
enzymatic cleavage ofenzymatic cleavage of
thymine dimers activated bythymine dimers activated by
the visible lightthe visible light
 It leads to restoration ofIt leads to restoration of
monomeric condition.monomeric condition.
 Co-enzymes required for theCo-enzymes required for the
reaction are FADH2 andreaction are FADH2 and
THF.THF.

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6. DOUBLE STRAND BREAK6. DOUBLE STRAND BREAK
REPAIRREPAIR High energy radiation, oxidative free radicals or someHigh energy radiation, oxidative free radicals or some
chemotherapeutic agents bring about double-strandedchemotherapeutic agents bring about double-stranded
breaks in DNA, or may also occur naturally duringbreaks in DNA, or may also occur naturally during
naturally during gene rearrangements.naturally during gene rearrangements.
 They are potentially lethal to the cell.They are potentially lethal to the cell.
 They cannot be repaired by excising single strand andThey cannot be repaired by excising single strand and
using the other strand as template to replace missingusing the other strand as template to replace missing
nucleotides.nucleotides.
 It is repaired by 2 ways:It is repaired by 2 ways:
1.1. Non-homologous end-joining repair.Non-homologous end-joining repair.
2.2. Homologous recombination repair.Homologous recombination repair.

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1.1. Non-homologous end joining repair-Non-homologous end joining repair-
 In this system the two ends of DNA areIn this system the two ends of DNA are
brought together by a group of proteins andbrought together by a group of proteins and
thereby the ends are re-ligated.thereby the ends are re-ligated.
 This system does not require that the 2 DNAThis system does not require that the 2 DNA
sequences have any homology.sequences have any homology.
2.2. Homologous recombination repair-Homologous recombination repair-
 This system uses the enzymes that normallyThis system uses the enzymes that normally
perform genetic recombination betweenperform genetic recombination between
homologous chromosomes during meiosis.homologous chromosomes during meiosis.
 This is called sister-strand exchange.This is called sister-strand exchange.

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7. TRANSCRIPTION COUPLED7. TRANSCRIPTION COUPLED
REPAIRREPAIR
When RNA polymerase transcribes aWhen RNA polymerase transcribes a
gene, as it encounters a damagedgene, as it encounters a damaged
region, the transcription stops.region, the transcription stops.
The excision repair enzymes repairThe excision repair enzymes repair
the area and then transcriptionthe area and then transcription
resumes.resumes.

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CLINICALCLINICAL
DISORDERSDISORDERS

91
1.1. XERODERMAXERODERMA
PIGMENTOSAPIGMENTOSA
 Autosomal recessive in nature.Autosomal recessive in nature.
 UV- specific exonuclease is deficient.UV- specific exonuclease is deficient.
 Cutaneous hypersensitivity to UV-rays.Cutaneous hypersensitivity to UV-rays.
 Blisters on skin.Blisters on skin.
 Hyperpigmentation.Hyperpigmentation.
 Corneal ulcer.Corneal ulcer.
 Death occurs due to formation of cancers ofDeath occurs due to formation of cancers of
skin.skin.

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2. ATAXIA TELANGIECTASIA2. ATAXIA TELANGIECTASIA
 Autosomal recessive inAutosomal recessive in
nature.nature.
 Increased sensitivity to X-Increased sensitivity to X-
rays and UV-rays.rays and UV-rays.
 Progressive cerebellarProgressive cerebellar
ataxia.ataxia.
 Oculo-cutaneousOculo-cutaneous
telangiectasia.telangiectasia.
 Frequent sino-pulmonaryFrequent sino-pulmonary
infections.infections.
 Lympho-reticular neoplasm.Lympho-reticular neoplasm.

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3. BLOOM’S SYNDROME3. BLOOM’S SYNDROME
 Chromosomal breaks orChromosomal breaks or
rearrangements are seen.rearrangements are seen.
 Defect lies in DNA helicase orDefect lies in DNA helicase or
ligase.ligase.
 Facial erythmia.Facial erythmia.
 Photosensitivity.Photosensitivity.
 Lympho-reticularLympho-reticular
malignancies.malignancies.

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4.FANCONI SYNDROME4.FANCONI SYNDROME
 Lethal aplastic anaemia,due to defectiveLethal aplastic anaemia,due to defective
DNA repair.DNA repair.
 Cells can not repair interstrand cross-links,Cells can not repair interstrand cross-links,
or damage induced by X-Rays.or damage induced by X-Rays.

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5.Cockayne’sSyndrome5.Cockayne’sSyndrome
&Retinoblastoma&Retinoblastoma
 Defects in DNA repair.Defects in DNA repair.
 Cells from patients with someCells from patients with some
chromosomal abnormalities eg Downchromosomal abnormalities eg Down
Syndrome may also show aberrant DNASyndrome may also show aberrant DNA
repair.repair.

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INHIBITOR OF DNAINHIBITOR OF DNA
REPLICATIONREPLICATION
 Anthracyclines cause chainAnthracyclines cause chain
breakageSu.bstances that act directly onbreakageSu.bstances that act directly on
DNA Polymerases eg. Acyclovir inhibitsDNA Polymerases eg. Acyclovir inhibits
the DNA polymerase of herpes simplex.the DNA polymerase of herpes simplex.
 2’-dideoxyazidocytidine is a inhibitor of2’-dideoxyazidocytidine is a inhibitor of
bacterial primase,andbacterial primase,and
cournermycin,novobiocin,oxolinic acid andcournermycin,novobiocin,oxolinic acid and
nalidixic acid are effective inhibitor of DNAnalidixic acid are effective inhibitor of DNA
gyrase in bacteria.gyrase in bacteria.

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TOPOISOMERASE ITOPOISOMERASE I
INHIBITORINHIBITOR
 Topoisomerase is essential for DNA replicationTopoisomerase is essential for DNA replication
and cell growth.and cell growth.
 Certain drugs produces double strand breaks inCertain drugs produces double strand breaks in
DNA that are irreversible and can lead to cellDNA that are irreversible and can lead to cell
death.death.
 Eg. Quilnolne antibiotics,anthracyclines activeEg. Quilnolne antibiotics,anthracyclines active
for treatment of lung, ovarian and colorectalfor treatment of lung, ovarian and colorectal
cancer.The Camptothecins were discoveredcancer.The Camptothecins were discovered
from extract of tree Camptotheca acuminata.from extract of tree Camptotheca acuminata.

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THE END
kingkul@yahoo.co.in

Replication 13

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