DNA

replicatio
n
Topics covered:
Definition, Significance,
Replication Machinery,
Process, Challenges

BOT-601: Molecular Week 2: Lecture

Biology
BS Botany 7th (M+E)
3
Credit hour: 3(2-1)
 Definition
DNA replication is the process by which a
double-stranded DNA molecule is copied to
produce two identical DNA molecules.

The process by which DNA makes a copy of

itself during cell division.

It is the process by which DNA is essentially

doubled.
The Significance of DNA
Replication
Ensures genetic
01 continuity

02 Regulates reproduction of
cells & organisms

03 Required for regenertaion

04 Source of variation
 Replication Machinery
The cellular
machinery
involoved in DNA
replication is
comprised of 2
ENZYME main components PROTEIN
S S
 ENDONUCLEASE
An endonuclease produces an internal cut
(single- or double-stranded) in a DNA
molecule. But a restriction endonuclease
produces cuts only at those sites that have a
specific base sequence.

During DNA replication, an endonuclease may

induce a nick to initiate DNA replication, or it
may induce nicks to generate a swivel (twist)
for DNA unwinding.
 DNA GYRASE
(DNA TOPOISOMERASE)
DNA gyrase, or simply gyrase, is an enzyme
within the class of topoisomerase (Type II
topoisomerase).

It relieves (removes) strain (stress/ pressure)

while double-stranded DNA is being unwound
by helicase.
 DNA HELICASE
Helicase effects strand separation at the
forks and use one ATP molecule for each base
that is separated.

In E. coli, DNA functions as helicase; this

protein is a hexamer and it moves with the
replication fork.

Helicase breaks the hydrogen bonding

between bases.
 SINGLE STRAND BINDING
PROTEINS (SSBP)
SSB protein binds to single-stranded DNA,
and prevents it from forming duplex DNA or
secondary structures.

SSB binds as a monomer, but it facilitates the

binding of one SSB molecule facilitates
binding of more SSB monomers to the same
DNA strand.

E. coli SSB is a tetramer.

 PRIMASE
This enzyme activity catalyzes the synthesis
of RNA primers to initiate DNA replication.

In E. coli, DnaG functions as primase.

But in eukaryotes, DNA polymerase α

provides this function. There are, however,
several other ways in which primers are
produced, e.g., the 3′-OH generated by a nick
in the template DNA molecule.
 DNA POLYMERASE
(The Chief of All Replication
Enzymes)
 DNA POLYMERASE
DNA polymerase is the chief enzyme of DNA
replication. DNA polymerase activity was
discovered by
Arthur Kornberg in 1956.

E. coli has 4 more enzymes, DNA polymerase

II, III, IV and V;
DNA polymerase III (Pol III) is concerned with
DNA replication, while the remaining four
enzymes are involved in DNA repair.
 DNA POLYMERASE
All DNA polymerases require the following:

(1) A template DNA strand,

(2) A short primer (either RNA or DNA), and
(3) A free 3′ -OH in the primer.

They add one nucleotide at a time to the free

3′ -OH of the primer, and extend the primer
chain in 5′ → 3′ direction.
 DNA POLYMERASE
(Types)
The best‐studied bacterium, E. coli, has three
DNA polymerase types.

DNA polymerase I is simplest and best

understood of the DNA polymerases, not
responsible for most chromosomal DNA
replication in E. coli,
but
functions in vivo both in DNA replication and
DNA repair.
 DNA POLYMERASE
(Types)
DNA polymerase I has to perform 3 activities

1. The 5′ → 3′ polymerase activity is responsible for

primer extension or DNA synthesis.

2. The 5′ → 3′ exonuclease activity is involved in excision

of DNA strands during DNA repair; it removes ~ 10
bases at a time.
An exonuclease digests nucleic acids (here DNA)
from one end, and it does not cut DNA internally.

3. The 3′ → 5′ exonuclease activity is responsible for proof-

reading.
 DNA POLYMERASE
(Types)
DNA polymerase II has to perform 2 activities

1. DNA polymerase II enzyme functions in DNA-

repair.

2. It has 5′ → 3′ polymerase and 3′ → 5′ exonuclease

activities, and uses as template only such DNA
duplexes that have short gaps.
 DNA POLYMERASE
(Types)
DNA polymerase III has to perform 2
activities
1. DNA polymerase III is involved in replication alongwith
DNA polymerase I.

2. DNA polymerase I and are single polypeptides but DNA

polymerase III is a 10 subunits long protein with a
molecular mass of 900KD).

3. It is responsible for DNA replication in vivo.

4. It has 5′ → 3′ polymerase and 3′ → 5′ exonuclease activities.
5. It catalyses polymeraization at very higher rates, 15,000
bases/ minute at 37⁰C.
 LIGASE
DNA ligase or polynucleotide ligase catalyzes
the formation of phosphodiester linkage
between two immediate neighbour
nucleotides of a DNA strand.

Thus it seals the nicks remaining in a DNA

strand either following DNA replication or
DNA repair.

However, this enzyme cannot fill the gaps in

DNA strands.
QUICK RECAP
 Terminology
Replication Origin of
fork
Y-shaped area of replication
A particular sequence in a
highest activity during genome at which replication is
replication. initiated.

Leading 5’ and 3’
strand Each strand of DNA or
That template strand at RNA has a 5' end and a
which replication occurs in 3' end, named due to the
a continuous manner. carbon position on the
deoxyribose (or ribose)
ring.
Lagging Klenow fragment
strand
That template strand at which A large protein fragment
replication occurs in a dis- produced after cleavage of
continuous manner. DNA polymerase I by
protease subtilisin.
Retains replication and
repair activities but in
 Terminology
Template / Parent RNA primer
Strand used by DNA A primer is a short single-
strand stranded nucleotide stretch
polymerase to add new
nucleotide bases used by all living organisms
complementary to the in the initiation of DNA
existing / old DNA strand. replication.
New / Daughter Upstream
strand
Newly synthesized strand of DNA When considering double-
that is copied via the addition of
stranded DNA, upstream is
complementary nucleotides from
toward the 5' end of the
one strand of pre-existing DNA
coding strand
during DNA replication.

Okazaki fragments
Downstream
Short stretches of newly When considering double-
synthesized DNA found on the stranded DNA, downstream
lagging strand during DNA is towards the 3' end.
replication.
Functional domains in the Klenow Fragment
(left) and DNA Polymerase I (PDB).
Steps of DNA
Replication
Process
 There are 4 main steps in replication
process

Initiatio Primer Elongatio Terminati

1 2 Synthesis of 3 4
n Annealing n
Addition of on
Daughter DNA
Unwinding of primer at DNA
double helix polynucleotides strand is
template on the
DNA template in daughter released while
site of origin of
starnd by strand by DNA template strand
replication by
helicase polymerase re-winds,
primase
 DNA Replication in Prokaryotes
1. The two strands of DNA unwind at the origin of replication.
2. Helicase opens the DNA and replication forks are formed.
3. Helicase opens the DNA and replication forks are formed.

4. The DNA is coated by the single-strand binding proteins

around the replication fork to prevent rewinding of DNA.
5. Topoisomerase prevents the supercoiling of DNA.
6. RNA primers are synthesised by primase. These primers are
complementary to the DNA strand.

7. DNA polymerase III starts adding nucleotides at the end of the

primers.
8. The leading and lagging strands continue to elongate.
9. The primers are removed and the gaps are filled with DNA
Polymerase I and sealed by ligase.
 Replisome
&
Replication fork

What is the
difference?
 The replisome is a large protein complex that
carries out DNA replication, starting at
the replication origin.

It contains several enzymatic activities, such as

helicase, primase and DNA polymerase.

Replisome creates a replication fork to

duplicate both the leading and lagging strand.
Replication
fork
Replication
Fork
 Challenges of Replication
Many of the structural and chemical problems
Efficient
associated with DNA replication are managed by
ng
replisome molecular machinery that is highly conserved acrossEnsuri sivity
s
assembly is organisms. ro cse
p
required

Separating
gh
duplex into
nt ain ing hi ut
Mai t hrougho +
leading & y
fidelit ion process
lagging at
replic
strands

Protecting
separated tion
Error correc
strands from ’
through 3’-5
damage activity
exonuclease
se
of polymera

Priming the Sync

hro
leading & lagging Gap filling at polym nized
Primer eriza
stran tion a
strands Okazaki ds d e t bot
removal antip spite h
fragments aralle
l natu
re
 Useful animation links
DNA DNA
replication replication
3D
https://youtu.be/T https://youtu.be/
NKWgcFPHqw Qqe4thU-os8

(Source: McGraw (Source: The

Hill) Amoeba Sisters)

Replication Nucleotide
fork 3D polymerizati
https://youtu.be/u on
wZsnDFeL7o https://youtu.be/9
QtQrVyiEK0
(Source: McGraw
Hill) (Source: McGraw
Hill)
 SUMMARY DNA Clamp is a
protein that
NA duplication is
called replication prevents
elongating DNA
DNA polymerase polymerase from
cannot initiate dissociating from
The assembly of replication, always growing strand
molecular depends on primer
structures Termination of
(enzymes and replication occurs
proteins) at origin when progress
of replication is stops at
called replisome Polymerases are
replication fork
highly accurate : 1
mistake /107
Replisome nucleotides added
creates Eukaryotes have
replication linear
fork chromosomes

Replication occurs
DNA polymerase is Prokaryotes have
in 3 coordinated
a unidirectional circular
steps: initiation,
enzyme chromosome
elongation and
termination
 References
BOOKS
1. Philip W. Kuchel, Ph.D; Simon Easterbrook-Smith; Vanessa Gysbers; J. Mitchell Guss. Schaum’s Outline
of Biochemistry, Third Edition. DNA Replication and Repair, Chapter (McGraw-Hill
Companies, Inc., 2009, 1998, 1988).
https://www.accessengineeringlibrary.com/content/book/9780071472272/chapter/chapter8.
2. Victor W. Rodwell, David A. Bender, Kathleen M. Botham, Peter J. Kennelly, P. Anthony Weil. Harper's
Illustrated Biochemistry, 30edition, Chapter 35: DNA Organization, Replication, & Repair.
(McGraw-Hill Companies, Inc., 2009)

WEB LINKS
I. https://slidesgo.com
II. https://socratic.org/questions/why-is-dna-replication-necessar
III. https://teachmephysiology.com/biochemistry/cell-growth-death/dna-replication/y
IV. https://www.yourgenome.org/facts/what-is-dna-replication
V. https://www.sparknotes.com/biology/molecular/dnareplicationandrepair/terms/
VI. https://www.sciencedirect.com/topics/neuroscience/primer-rna
VII. https://en.wikipedia.org/wiki/Replisome#Challenges_of_DNA_replication
THANK YOU