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Online Counseling Resource
YCMOU ELearning Drive…
School of Architecture, Science and Technology
Yashwantrao Chavan Maharashtra
Open University, Nashik – 422222, India
OC-SBT044-U01-01
Introduction
Programmes and Courses
 SEP – SBT044 – CP01
 SEP – SBI044 – CP01
 SEP – SGS044 – CP01
School of Science and Technology, Online Counseling Resource…
Credits
 Academic Inputs by
 Mrs. Rasika Bhore
 M.sc (Microbiology)
 [email protected]
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School of Science and Technology, Online Counseling Resource…
How to Use This Resource

Counselor at each study center should use this presentation to deliver
lecture of 40-60 minutes during Face-To-Face counseling.

Discussion about students difficulties or tutorial with assignments should
follow the lecture for about 40-60 minutes.

Handouts (with 6 slides on each A4 size page) of this presentation should
be provided to each student.

Each student should discuss on the discussion forum all the terms which
could not be understood. This will improve his writing skills and enhance
knowledge level about topics, which shall be immensely useful for end
exam.

Appear several times, for all the Self-Tests, available for this course.

Student can use handouts for last minutes preparation just before end
exam.
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Learning Objectives
 After studying this module, you should be able to:
 Discuss the role of all enzymes participated in
Replication.
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School of Science and Technology, Online Counseling Resource…
Introduction
 Like all metabolic processes, DNA replication is
also the combine action of many enzymes.
 Physical, chemical, biochemical and mutation
studies indicate that DNA replication is under
the strict control of enzymes.
 All these replication enzymes & proteins
performs a specific task.
 These entire complex of enzymes works
together for replication & known as ‘DNA
replicase system or replisome’.
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School of Science and Technology, Online Counseling Resource…
Enzymes Of Replication
 Following are the main enzymes of DNA
replication.
1.
2.
3.
4.
5.
Helicases
DNA gyrase or topoisomerase
Primases
DNA polymerases
DNA ligases
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School of Science and Technology, Online Counseling Resource…
Helicases
 Helicases mainly functions in separating the two annealed
nucleic acid strands (DNA, RNA).
 For the replication, it is necessary to separate the double
stranded DNA strand which is done by Helicases.
 For this activity they derived energy from ATP or GTP
hydrolysis which is the output of breaking of hydrogen bonds
between nucleotides during separation.
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School of Science and Technology, Online Counseling Resource…
DNA Gyrase Or Topoisomerase
 DNA gyrase is type II topoisomerase.
 This enzyme relieves the topological stress of helical
DNA structure created on strand separation.
 The stress is removed by introducing the negative
supercoils on other words by relaxing positive
supercoils.
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School of Science and Technology, Online Counseling Resource…
Primases
 For the activity of DNA polymerase primers must be
present on template.
 DNA polymerase started to synthesize new DNA by
adding nucleotides to primer.
 These primers are the short segments necessary for
synthesis of lagging strand in eukaryotes are produced
by the enzyme Primase.
 In bacteria, primase binds to the DNA helicase forming a
complex called the primosome.
 Primase is activated by DNA helicase where it then
synthesizes a short RNA primer approximately 11 to 12
nucleotides long, to which new nucleotides can be added
by DNA polymerase.
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School of Science and Technology, Online Counseling Resource…
DNA Ligases
 Lagging strand synthesis carried out by
formation of okazaki fragments by
polymerase III which adds nucleotides to
3’OH group of RNA primer.
 Polymerase I then replaces the RNA
fragments with DNA & fill the gaps.
 Finally DNA ligase fill the nicks & finishes
the synthesis of DNA.
 It also functions in DNA repair.
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School of Science and Technology, Online Counseling Resource…
Action Of Ligase In DNA Synthesis
 The mechanism of DNA ligase is to form covalent
phosphodiester bonds between 3' hydroxyl ends of
one nucleotide with the 5' phosphate end of
another.
 ATP is required for the ligase reaction.
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School of Science and Technology, Online Counseling Resource…
DNA Polymerase
 DNA polymerase is the enzyme which synthesizes DNA.
 It catalyzes the polymerization of deoxyribonucleotides
along a DNA strand.
 The newly-polymerized molecule is complementary to
the template strand and identical to the template's
partner strand.
 DNA polymerase requires a magnesium ion as a cofactor to function properly.
 No known DNA polymerase is able to begin a new chain
(de novo), it can add a nucleotide onto only a
preexisting 3'-OH group, and, therefore, needs a primer
at which it can add the first nucleotide.
 Thus the elongation of chain takes place.
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School of Science and Technology, Online Counseling Resource…
Prokaryotic DNA Polymerases
 E.coli have 5 known DNA polymerases:
1. Pol I: implicated in DNA repair; has both 5'>3'(Polymerase) activity and 3'->5'
(Proofreading) exonuclease activity.
2. Pol II: involved in replication of damaged DNA;
has 3'->5' exonuclease activity.
3. Pol III: the main polymerase in bacteria
(elongates in DNA replication); has 3'->5'
exonuclease proofreading ability.
4. Pol IV: a Y-family DNA polymerase.
5. Pol V: a Y-family DNA polymerase; participates
in bypassing DNA damage.
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School of Science and Technology, Online Counseling Resource…
Proofreading Or Exonuclease Activity
 DNA polymerases shows the 3’->5’ exonuclease activity.
 Exonuclease activity double checks each nucleotide after
it added.
 This activity permits the enzyme to remove a newly
added nucleotide & is highly specific for mismatched
base pairs.
 If the polymerase has added the wrong nucleotide,
translocation of the enzyme to the position where the
next nucleotide is to be added is halted.
 This pause provides the opportunity for a correction.
 Thus the 3’->5’ exonuclease activity removes the
mispaired nucleotide, & he polymerase moves ahead.
 This activity is known as ‘proofreading’.
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School of Science and Technology, Online Counseling Resource…
DNA Polymerase I
 Polymerase I is not a primary enzyme of replication,
instead it performs a clean-up functions during
replication, repair & recombination.
 It is a single-chain protein with a mass of about
109,000 Da.
 Each of its three enzymatic activities are encapsulated
into distinct domains of the holoenzyme .
 The proteolytic deletions can separate the 3’->5’
(proofreading) exonuclease domain.
 The remaining fragment is large fragment or called
Klenow fragment retains the polymerization &
proofreading activities & widely used in recombinant
DNA work.
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School of Science and Technology, Online Counseling Resource…
DNA Polymerase III
 Polymerase III is the primary enzyme complex having 10
types of subunits.
 It was discovered by Thomas Kornberg and Malcolm Gefter in
1970.
 The complex has high processivity and, specifically referring
to the replication of the E.coli genome, works in conjunction
with four other DNA polymerases (Pol I, Pol II, Pol IV, and Pol
V).
 DNA Pol III holoenzyme also has proofreading capabilities that
correct replication mistakes by means of exonuclease activity
working 3'->5'.
 DNA Pol III is a component of the replisome, which is located
at the replication fork.
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School of Science and Technology, Online Counseling Resource…
Subunits Of DNA Pol III
SUBUNIT NO.OF SUBUNIT PER
HOLOENZYME
FUNCTION OF SUBUNIT
α
2
POLYMERIZATION ACTIVITY
ε
2
3’->5’ PROOFREADING
EXONUCLEASE
θ
2
τ
2
STABLE TEMPLATE BINDING
√
1
CLAMP LOADER
♪
1
CLAMP OPENER
♪’
1
CLAMP LOADER
ҳ
1
INTERACTION WITH SSB
Ψ
1
INTERACTION WITH √ & ҳ
β
4
DNA CLAMP REQUIRED FOR OPTIMAL
PROCESSIVITY
_
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School of Science and Technology, Online Counseling Resource…
Structure Of DNA Pol III
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Eukaryotic DNA Polymerases -1
 Eukaryotes have at least 15 DNA Polymerases:
1. Pol α : acts as a primase (synthesizing a RNA
primer).
2. Pol β: Implicated in repairing DNA, in base excision
repair and gap-filling synthesis.
3. Pol γ: Replicates mitochondrial DNA.
4. Pol δ: Highly processive and has proofreading 3'->5'
exonuclease activity. Thought to be the main
polymerase involved in lagging strand synthesis.
5. Pol ε: Also highly processive and has proofreading
3'->5' exonuclease activity. Highly related to pol δ,
and thought to be the main polymerase involved in
leading strand synthesis.
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School of Science and Technology, Online Counseling Resource…
Eukaryotic DNA Polymerases -2
6. η, ι, κ, and Rev1 are Y-family DNA polymerases
and Pol ζ is a B-family DNA polymerase. These
polymerases are involved in the bypass of DNA
damage.
7. There are also other eukaryotic polymerases
known, which are not as well characterized: θ, λ,
φ, σ, and μ.
8. None of the eukaryotic polymerases have 5'->3'
exonuclease activity; that function is carried out by
other enzymes.
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School of Science and Technology, Online Counseling Resource…
What We Learn………….
 Helicase enzyme initiate the replication by separating
the DNA strands.
 The topological stress produced on separation is relieved
by gyrase by introducing negative supercoils in DNA.
 Primase synthesizes RNA primer without which DNA
polymerase can’t elongate the chain of nucleotides.
 There are 5 DNA polymerases in prokaryotes while 15
in eukaryotes.
 Prokaryotic pol III have high processivity & have 3’->5’
exonuclease activity.
 Prokaryotic pol I is primary enzyme of replication.
School of Science and Technology, Online Counseling Resource…
Critical Thinking Questions
 What will be the consequences if 3’->5’
proofreading activity has lost from DNA
polymerase?
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School of Science and Technology, Online Counseling Resource…
Tips For Critical Thinking Questions
 Proofreading activity replaces the
mismatched nucleotide with correct ones,
thus, helps in accurately copying DNA.
© 2007, YCMOU. All Rights Reserved.
School of Science and Technology, Online Counseling Resource…
Study Tips
 Article
 Title: Enzymes & reactions at eukaryotic DNA replication
fork.
 Author: Robert Bambara, R. Murante & L. Henricksen
 Publication: Vol. 272, No.8, Issue of Feb 21, 1997
 Book
 Title: DNA Replication
 Author: Adam, Roger L. P
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School of Science and Technology, Online Counseling Resource…
Study Tips
www.en.wikipedia.org
Polymerase, primase, Helicase etc.
www.biochemistry.org
DNA replication.
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School of Science and Technology, Online Counseling Resource…
Acknowledgement




www.vivo.colostate
www.nature.com
www.medpham.yc.ac
www.library.thinkquest.org
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End of the Presentation
Thank You !