Cell cycle and DNA replication

DNA replication:
• Copying genetic information for transmission to the next
generation
• Occurs in S phase of cell cycle
• Process of DNA duplicating itself
• Begins with the unwinding of the double helix to expose the
bases in each strand of DNA
• Each unpaired nucleotide will attract a complementary
nucleotide from the medium
– will form base pairing via hydrogen bonding.

• Enzymes link the aligned nucleotides by phosphodiester

bonds to form a continuous strand.

1
 General Principles for DNA replication

The DNA being replicated must be in a ready state

for the start of replication, and there also has to be a
clear start point from which replication proceeds. As
each piece of DNA must only be copied one, there
also has to be an end point to replication.

DNA replication must be carried out accurately, with

an efficient proof reading and repair mechanism in
place for any mismatches or errors. And finally, the
system of replication must also be able to distinguish
between the original DNA template and then newly
copied DNA.
 Cell cycle
Actively dividing eukaryote cells pass through a
series of stages known collectively as the cell
cycle, two gap phases (G1 and G2); an S (for
synthesis) phase, in which the genetic material is
duplicated; and an M phase, in which mitosis
partitions the genetic material and the cell
divides.
Cell cycle
Replication in terms of the cell:
Must be ready: G1
Must all start at the same time: G1 – S
Must know where to start: Origin of Replication
Must all finish: Complete S
Must ensure that each piece of DNA is only replicated once, so
need to know where to end Replicon
Proof reading and repair: G2
Able to distinguish between original and copy: Epigenetics
4
DNA replication:
– First question asked was whether duplication was
semiconservative or conservative

• Meselson and Stahl expt

• Semiconservative -
– one strand from parent in each new strand

• Conservative-
– both strands from parent and other is all new
strands
2
 DNA replication:
• Complementary base pairing produces semiconservative
replication

– Double helix unwinds

– Each strand acts as template

– Complementary base pairing ensures that T signals

addition of A on new strand, and G signals addition of C

– Two daughter helices produced after replication

3
 Experimental proof of semiconservative replication
– three possible models

• Semiconservative replication –
– Watson and Crick model

• Conservative replication:
– The parental double helix remains intact;
– both strands of the daughter double helix are newly
synthesized

• Dispersive replication:
– At completion, both strands of both double helices contain
both original and newly synthesized material.
5
6
 Meselson-Stahl experiments confirm
semiconservative replication
• Experiment allowed differentiation of parental and
newly formed DNA.

• Bacteria were grown in media containing either

normal isotope of nitrogen (14N) or the heavy
isotope (15N).

• DNA banded after equilibrium density gradient

centrifugation at a position which matched the
density of the DNA:
– heavy DNA was at a higher density than normal DNA.
 Meselson-Stahl experiments confirm
semiconservative replication
• When bacteria grown in 15N were transferred
to normal 14N containing medium,
– the newly synthesized DNA strand had the 14N
while the parental strand had 15N.

• They checked the composition of the resulting

DNA molecules by density gradient
centrifugation,
– found an intermediate band,
– indicating a hybrid molecule
– containing both 14N and 15N DNA.
15N

9
 Protein involved Task Prokaryotes Eukaryotes
Helicase Unwind local segments of DnaB MCM complex
the DNA double
helix (ATP-dependent
reaction) at
the replication fork

Single-stranded DNA-binding Prevents reannealing of SSB (single-strand RPA (replication protein

proteins (SSBs) separated strands binding protein) A)
Topoisomerase Type I Cleaves only one of Topoisomerase I (type Topoisomerase I (type
•Relieve supercoils that topoisomera both DNA strands IA topoisomerases) IB topoisomerases)
develop se •Has a nuclease (cuts the
during DNAelongation as strand) and
parent DNA is separated a ligase (reseals the
by helicase. strand)
•Does not require ATP
Type II Temporarily cleaves Topoisomerase II (DNA Topoisomerase II
topoisomera both DNA strands for gyrase) •Inhibited by the
se larger structural and topoisomerase IV anticancer
alterations of the DNA •Both inhibited drug etoposide
•Requires ATP by gyrase
inhibitors (e.g., fluoroq
uinolonessuch
as ciprofloxacin; also
the quinolone nalidixic
acid)
 Protein
Proteininvolved
involved Task Prokaryotes Eukaryotes
Primase
Primase(DNA-dependent
(DNA- RNA Synthesis of Primase (DnaG) Primase activity as a component
polymerase)
dependent RNA polymerase) short RNA sequences (RNA of DNA polymerase α
primers)
DNA-dependent
DNA-dependentDNADNA Extends RNA primers by ∅ Polymerase α
polymerase
polymerase adding 50–100 nucleotides
Replicates the lagging DNA polymerase III Polymerase δ
strand
Extend the leading strand Polymerase ε
Removal of primers RNase H and DNA RNase H and FEN-1(flap
polymerase I (5'→3' endonuclease-1)
exonuclease activity)
Gaps between fragments DNA polymerase I Polymerase δ
are filled after primer
removal
DNA
DNAclamp
clamp A subunit of DNA β-subunit of DNA PCNA (proliferating cell nuclear
polymerase that anchors polymerase III antigen)
polymerase to the DNA
Ligase
Ligase Links newly DNA ligase DNA ligase
synthesized DNA fragment
+
s (ATP or NAD+-
dependentreaction) by
catalyzing the formation of
phosphodiester bonds
 Protein involved Task Prokaryotes Eukaryotes
Replisome The complex Replisome Replisome
of proteins (i.e., DNA
polymerase, helicase, etc.)
that builds to participate
in DNA replication

Telomerase Ensures complete ∅ Telomerase

replication of the ends of
linear chromosomes
 CONDITIONS FOR DNA POLYMERASE ACTION:
a) All four dNTPs (dATP, dGTP, dTTP, dCTP) along with Mg2+ ions.

b) A primer chain with a free 3'-OH group. Invitro conditions require DNA primer strand
while invivo condition require RNA primer strand. Generally DNA Pol – I utilizes DNA
primer whereas DNA Pol – III utilizes RNA Primer.
c) A DNA template, which can be single stranded or double stranded DNA. Double
stranded DNA is an effective template only if its sugar phosphate backbone is broken at
one or more sites.
ii) 3'-->5' exonuclease activity:
This is the proofreading or editing activity of DNA polymerase, which gets activated
when a wrong base pair is incorporated into the chain. DNA pol-I removes deoxy
nucleotide at 3'-end of the progeny DNA. Because of this, the activity is referred as 3'-->5'
exonuclease activity. Due to this activity, it repairs DNA during replication.
5'-->3' exonuclease activity:
With the help of this activity, enzyme removes nucleotides from 5'-end. Two functions
can be carried out by this activity. They are removal of RNA primer and DNA repair.
 DNA directed DNA Polymerase-III

Holoenzyme of DNA pol-III consists of ten subunits. They are aeqbgdd'cjt . Core enzyme
of DNA poly-III contain three subunit aeq. Of three subunits alpha subunit posses
polymerizing activity and epsilon posses proof reading activity. These are the two
function of the enzyme. It does not have 5'-->3' exonuclease activity.
Kornberg enzyme
I. DNA directed DNA polymerase - I (Kornberg
enzyme):
Its Molecular weight is 109kd and the concentration is
around 400 molecules per cell. When treated with
proteases like Trypsin, it gets cleaved into two fragments
possessing different function. The large fragment called
klenow fragment posses both polymerase and 3'-->5'
exonuclease activity. The small fragment possesses only
5'-->3' exonuclease activity. Overall, three activities are
possessed by DNA Pol-I.
i) DNA polymerase activity
ii) 3'--> 5' exonuclease activity
iii) 5'-->3' exonuclease activity
 [Link] SUBUNITS FUNCTION
1. a Polymerization
2. e Repair and Proof reading function
3. q Assembly of DNA pol-III
4. b Processivity - It holds the template
5. gdd' Lagging strand synthesis - They aid the formation of loop.
6. cj Normal function of DNA pol-III
7. t Dimerization

gdd'c and j subunits called as a gamma complex, which mediates transfer of the beta subunit
to the duplex DNA primer strand. DNA pol-III possesses high processivity than other DNA
polymerases. This is because of beta subunit. It form a donut shaped dimers around duplex DNA
and then associate with and hold the catalytic core aeq polymerase at the primer template
terminus.
Once tightly associated with DNA, the beta subunit dimers functions like a "clamp" which can
move freely along the DNA, like a ring on a string carrying the associated core polymerase with
it. In this way, the active sites remain near the growing fork and the processivity of the core
polymerase maximized.
Processivity: Number of nucleotides added by the enzyme before it gets released from the
template strand after its attachment.
What Really Happens….

DNA pol works as a dimer

Lagging strand must
loop around to
accommodate
dimerization

Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc.,

publishing as Benjamin Cummings.

21
Origin of Rep
 Origins of
Replication
• Replication proceeds in both directions
(bidirectionally) from a single origin of
replication on the prokaryotic circular
chromosome

• Replication proceeds in both directions

(bidirectionally) from hundreds or thousands
of origins of replication on each of the linear
eukaryotic chromosomes.
14
Origins of Replication

• Bacteria have 1 origin of

replication per one
chromosome

• They only have one

chromosome = 1 origin!

15
Molecular Biology of the Cell, 4th Edition.
Eukaryotic Origins of
Replication

16
 The process of DNA replication

1. Initiation
Specific proteins (comprising a prepriming complex) recognize and bind to
the origin of replication
◦ Origin of replication (ori): a specific DNA sequence in
the genome where DNA replication starts
◦ Eukaryotes have multiple oris (30,000–50,000 in humans): ensures a timely
replication .
◦ Prokaryotes with circular genomes only have a single ori
At the ori, helicase separates and begins unwinding dsDNA into single strands,
forming 2 replication forks
◦ Replication fork: Y-shaped region in the chromosome where both leading
and lagging strands are replicated from the DNA template
◦ Both forks moves bidirectionally (in opposite directions) along the DNA as
synthesis occurs
SSBs prevent the single strands from reannealing and protects ssDNA from
cleavage
Supercoil relaxation: DNA topoisomerases relieve overwinding
(positive supercoils) or underwinding (negative supercoils) that develop
during DNA separation and elongation.
Replication initiation in bacteria (a) and eukaryotes (b). In bacteria (a), the binding of the initiator (DnaA) to the replication origin leads
to DNA melting (top). The initiator then recruits the replicative DNA helicase (DnaB) in an active form around single-stranded DNA
(ssDNA). This is followed by replisome assembly and the start of DNA synthesis. Since DnaB translocates in the 5’ to 3’ direction
along the DNA, it encircles the lagging-strand template. In eukaryotes (b), the initiator (Orc1-6) loads the replicative DNA helicase
(Mcm2-7) in an inactive, double-hexameric form around double-stranded DNA (dsDNA) during the G1-phase. Activation of the
helicase is temporally separated from helicase loading and only occurs during S-phase by recruitment of Cdc45 and GINS to form
the active Cdc45/Mcm2-7/GINS (CMG) holo-helicase. Since CMG translocates in the 3’ to 5’ direction along the DNA, it encircles the
leading strand template
 Elongation
Primer synthesis : Primase synthesizes a short, 5–10 nucleotide long RNA
primer that is complementary to the template strand
[Link] synthesis: For simultaneous replication of both parent
strands, DNA replication occurs continuously on the leading strand and
discontinuously on the lagging strand in a 5'→3' direction as
complementary deoxynucleotides are added to the free 3'OH group of
the daughter strand
2. Reaction catalyzed by DNA polymerase (polymerase
δ in eukaryotes, DNA polymerase III in prokaryotes)
2.5'→3' DNA polymerase activity catalyzes the nucleophilic attack of
the 3'OH group to the α-phosphate of the
incoming deoxynucleotide triphosphate, forming an ester bond
[Link] strands are elongated by a single nucleotide.
[Link] (PPi) is released
[Link] is cleaved into two phosphates by
pyrophosphatase.
[Link] strand: A complementary daughter
strand of DNA whose replication is continuous due to its
free 3'OH end (initially on the primer)
[Link] one primer is required.
[Link] strand: A complementary daughter
strand of DNA whose replication is discontinuous as
new RNA primers are constantly being synthesized at the
moving replication fork to ensure a free 3'OH end for
elongation.
[Link] fragments: Discontinuous segments of 1000–
2000 nucleotides that arise during lagging
strand creation.
[Link] DNA segment requires its own RNA primer.
[Link] primer is synthesized at the
moving replication fork.
Proofreading: Some polymerases (e.g., DNA polymerase I and III)
have 3'→5' exonuclease activity and remove incorrectly
paired nucleotides.
Primer removal: RNA primers are excised in the opposite direction of
synthesis (so 5'→3')
◦ In prokaryotes, by RNase H and 5'→3' exonuclease of the DNA polymerase I
◦ In eukaryotes, by enzyme FEN-1 (flap endonuclease-1)

Filling the gaps: During primer removal, DNA polymerase fills the gaps
with deoxynucleotides complementary to the parent strand until the free
ends meet.
◦ In prokaryotes, DNA polymerase I adds deoxynucleotides one at a time and
“proofreads”
◦ In eukaryotes, by Polymerase δ
•Joining of the ends DNA ligase joins (ligates) the free
ends of the newly synthesized daughter strand.
•Ligase reaction
• DNA ligase transfers an AMP residue to the 5' phosphate end of one of
the DNA fragments to be bound.
• AMP is cleaved and the 5' phosphate end is bound to the 3'OH end of the
other fragment.

Termination
•Termination of replication is initiated by binding
termination proteins to termination sequences.
•There are various termination mechanisms for
circular and linear DNA molecules.
 Telomeres
Structure: a non-coding DNA fragment of several thousand bp (composed of tandem
repeats of TTAGGG) at the 3' ends of chromosomes
Function
• Prevent the loss of structural genes during replication of linear DNA double-strands
◦ The lagging strand becomes shorter with each process of DNA replication.
• DNA polymerase cannot synthesize the last part of the 5' end of the lagging
strand because there is no 3'OH group present following removal of the RNA
primer to fill the gaps.
• A section of the telomere is lost instead of the coding segment of the DNA.
• Telomeres extend the cell's lifespan by slowing the cell's aging process
Maintenance: by telomerase
• Telomerase
◦ A special reverse transcriptase that carries its
own RNA template (ribonucleoprotein) and can elongate telomeres
◦ Has a strong presence in frequently dividing cells (to prevent loss of function).
Not present in all cells!
◦ Activity is enhanced in cancer cells++
 Replication Termination
The ends of chromosomes (telomeres) cannot be replicated on
the lagging strand because there is no primer available.
Telomerases
◦ enzymes that contain RNA primers which extend the ends of
chromosomes (not normally expressed in significant levels)
◦ Telomeres form a sort of single stranded cap around the chromosome
ends to protect them from being degraded
◦ chromosome ends are progressively shortened with each round of
replication.
◦ “old” cells with shortened telomeres undergo apoptosis -
◦ Protective for normal cells
◦ Kill the old and possibly mutated

◦ Telomerase is over expressed in cancer cells

◦ Hypothesis is that cancer cells do not undergo apoptosis because
their telomeres do not shorten over time. 22
 The problem of
replicating
completely a linear
chromosome in
eukaryotes

23
Peter J. Russell, iGenetics: Copyright © Pearson Education
, Inc., publishing as Benjamin Cummings.
 Replicating the Ends of Chromosomes

• Telomerase adds an RNA primer complementary to telomere

sequences
– chromosomal replication proceeds by adding to the 3’ end
of the primer
• Fills the gap left behind by replication
• Telomerase enzyme can also add DNA basepairs to the
TEMPLATE DNA
– complementary to the RNA primer basepairs

– Using an RNA template to make DNA, telomerase

functions as a reverse transcriptase called TERT
(telomerase reverse transcriptase).
• This goes against the Central Dogma….
• Evolutionarily thought to be derived from a Retrovirus
24
Synthesis of telomeric DNA by
Telomerase
New template DNA!

25
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
 Replication at the chromosomal level
• Replication is bidirectional.
• For circular DNA (and linear chromosomes)
– the unwinding at the replication forks causes supercoiling.

• DNA topoisomerases
– enzymes that help relax the DNA by nicking the strands
– releasing the twists
– then rejoining the DNA ends.
– Example is DNA gyrase

26
 The bidirectional
replication of a circular
chromosome
(Prokaryotic)

.18
 Assembling Newly Replicated DNA
into Nucleosomes
• When eukaryotic DNA is replicated, it complexes
with histones.
– This requires synthesis of histone proteins and assembly of
new nucleosomes.

• Transcription of histone genes is initiated near the

end of G1 phase, and translation of histone proteins
occurs throughout S phase.

• Assembly of newly replicated DNA into nucleosomes

28
The Assembly of Nucleosomes after
Replication

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Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.