DNA replication

DNA replication is necessary for the reproduction of a species and for any dividing cell
Objectives
1. Define replication and its importance to living organisms.
2. Learn about semiconservative and conservative DNA replication and Meselson-
Stahl experiment.
3. List various enzymes and proteins that participate in DNA replication with their
functions.
4. Describe briefly the sequential process of DNA replication
a. Initiation: Define ori.
b. Unwinding of DNA helix.
c. Clearly understand how ‘nick’ is formed, the enzymes that form and reseal it.
d. Learn Polymerisation process.
e. Understand how replication fork and replication bubbles are formed and the
enzymatic machinery associated with it.
5. Learn how the synthesis of primer takes place and its importance.
6. Learn how the strands are joined together.
7. Understand the relationship between cell cycle and replication.
8. Make a tabular form to differentiate prokaryotic and eukaryotic DNA replication

STRAND SEPARATIONAND ORIGINS OF REPLICATION

1. The human genome has about 3 billion base pairs packed into multiple
chromosomes.
2. The replisome or replication complex is a set of specialized proteins that assist
the DNA polymerases. To begin the process of replication, DNA unwinds at
points called origins of replication.
3. The model of DNA as proposed by Watson and Crick suggests that because one
strand of DNA is the complement of the other, upon unwinding of the double helix
(dsDNA), each strand (ssDNA) acts as a template for the formation of a new
strand. This process is called as DNA replication
4. The generation of new DNA proceeds in both directions, creating replication forks
on both sides of the origin
Semiconservative replication: The process of unwinding of the double-helical
daughter molecules, each of which is composed of a parental strand and a newly
synthesized strand formed from the complementary strand. This is called
semiconservative replication.
Meselson and Stahl demonstrated experimentally that the process of replication in E.
coli was semiconservative.
They carried out the following experiment:
Bacteria were grown in a medium containing the heavy isotope of nitrogen 15N, when
the entire DNA was labeled with heavy nitrogen. These cells were allowed to divide in a
medium containing normal nitrogen, 14N. In the first generation, all DNA molecules

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were half-labelled. In the second generation, half-labeled and completely unlabeled
molecules were present in equal numbers. From this Pioneer experiment, it was
proved that DNA replication in vivo is semiconservative.

Fig. 1. (a) Semi-conservative replication of DNA at the replication fork. (b)

Proof of semi-conservative mechanism. The centrifuge tubes
on the left show the positions of fully 15N-labeled (HH), hybrid density (LH) and
fully 14N-labeled (LL) DNA in CsCl density gradients after each generation and the
diagram on the right shows the corresponding DNA molecules. The original
parental 15N molecules are shown as thick lines and all 14N daughter DNA as thin
lines.

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MAJOR ENZYMES AND OTHER PROTEINS INVOLVED IN DNA
REPLICATION
1. DNA polymerases: Chief enzyme mainly involved in repair and deoxynucleotide
polymerisation.
In prokaryotes: Three types of DNA polymerases found. They are DNA
polymerase I, DNA polymerase II and DNA polymerase III.
In eukaryotes: There are five types of DNA polymerases and they are called
as:α, ε, β, γ, δ
2. DNA helicases: Required for unwinding of dsDNA.
3. DNA Primase: Required for synthesis of RNA primer.
4. Nick sealing enzymes:
Two enzymes:
a. Topoisomerases

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 b. DNA ligase.
5. Single strand binding proteins (SSB proteins).

SEQUENTIAL EVENTS OF DNA REPLICATION

The steps involved in DNA replication in Eukaryotes can be arbitrarily divided into
five steps for better understanding. They are:
1. Identification of sites of origin of replication (ori)
2. Unwinding of dsDNA to provide ssDNA which can act as template
3. Formation of the replication fork
4. Initiation and chain elongation
5. Formation of replication bubbles and ligation of the newly synthesised
DNA segments.
Types of polymerases function
Prokaryotes Eukaryotes
I α Gap filling and synthesis of lagging strand
II ε Proofreading of DNA and repair
β DNA repair
γ Required for mitochondrial DNA synthesis
III δ Helps in leading strand synthesis

A. Identification of Site of the Origin of Replication

There are specific sites, called origin of replication (ori); where replication starts. At
the origin of replication, there is an association of sequence-specific DNA binding
proteins with a series of direct repeat DNA sequences. Adjacent to ‘ori‘ is A + T rich
region.
• A primase initiates synthesis of RNA molecule (Primer) that is essential for priming
DNA synthesis.
• The DNA polymerase initiates nascent, daughter strand synthesis (ssDNA) and,
• SSB proteins bind to ssDNA and prevent premature reannealing of ssDNA to dsDNA.
DNA polymerases only synthesise DNA in the 5’→ 3’ direction and only one of the
several different types of polymerases is involved in formation of replication fork.
An enzyme capable of polymerising DNA in 3’ → 5’ direction does not exist in any
organism, so that both of the newly replicated DNA strands cannot grow in the same
direction simultaneously. Again the same enzyme does not replicate both strands at the
same time. As the DNA strands are antiparallel, the polymerase functions
asymmetrically

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Note: everything in molecular biology is 5′ to 3′. DNA polymerase reads 3′ to 5′, but the
following processes occur 5′ to 3′:
DNA synthesis
DNA repair
RNA transcription
RNA translation (reading of codons)
• Leading strand (forward strand): The DNA is synthesized continuously in 5’ → 3’
direction with same over-all forward direction.
• Lagging strand (Retrograde strand): The DNA is synthesised in a discontinuous
manner. In this strand, the DNA is synthesized in short spurts, 1-5 kb fragments, the
so-called Okazaki fragments. Several Okazaki fragments, approximately 250 must be
synthesized, in sequence, for each replication forks
The DNA helicase associates with primase and a mobile complex is formed which is
called as Primosome.
This association of helicase with primase allows the primase to get access to template;
which makes the RNA primer and in turn allows polymerase to begin the DNA
replication. This is an important step as DNA polymerases cannot initiate DNA
synthesis de novo.
When synthesis of one Okazaki fragment is completed, the polymerase is released and
a new primer is synthesized. The same polymerase molecule remains associated with
the replication fork and forms a new Okazaki fragments. This process is repeated to
form several Okazaki fragments, on the Lagging strands.

The RNA must eventually be removed. This is accomplished by the enzyme DNA
polymerase I (prokaryotes) or RNase H (eukaryotes). Then, DNA polymerase I
(prokaryotes) or DNA polymeraseδ(eukaryotes) adds DNA nucleotides where the RNA
primer had been. DNA ligase seals the ends of the DNA molecules together, creating
one continuous strand of DNA.

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Telomere replication: Telomeric DNA consists of multiple copies of a simple
repeat sequence. The 3’-ends overhang the 5’-ends. Telomeric DNA is copied by
telomerase, which carries the template for the repeat as a small RNA molecule.
Telomerase is repressed in somatic cells but reactivated in many cancer cells.

DNA polymerase control the fidelity of DNA replication

Proofreading refers to any mechanism for correcting errors in protein or nucleic
acid synthesis that involves scrutiny of individual units after they have been
added to the chain Processive DNA polymerases have 3’→5’ exonuclease
activity

DNA Synthesis and Cell Cycle

DNA synthesis occurs during the S phase of the cell cycle. This period is referred to as
the synthetic S phase. This is usually temporarily separated from the mitotic phase by
nonsynthetic period referred to as gap I (G1) and gap-2 (G2) occurring before and after
the S phase respectively.
The cell regulates its DNA synthesis grossly by allowing it to occur only at specific
times.
During S phase, the nuclear DNA is completely replicated once and only once.
Methylation of DNA is suggested to be covalent marker for further cycle of replication.

Role of Cyclins in DNA Synthesis

The cyclins are a family of proteins whose concentration increases and decreases
throughout the cell cycle.
The cyclins, at the appropriate time, turn on different cyclin-dependent protein kinases
(CDKs) that phosphorylate substrates which are essential for progression through cell
cycle. Excessive production of cyclins, or production at an inappropriate time may result
in abnormal or unrestrained cell division.
BACTERIAL DNA REPLICATION
Experimental systems Genetically simple bacteriophages and plasmids are useful
model systems for studying the in vitro replication of the large and fragile bacterial
chromosome. The simplest rely nearly exclusively on host cell replication proteins.
Larger phages encode many of their own replication factors.
1. Initiation Replication is regulated by the rate of initiation. Initiation at the E. coli
origin,

oriC, involves wrapping of the DNA around an initiator protein complex (DnaA–ATP)
and separation of the strands at AT-rich sequences. The helicase DnaB [DnaB is a DNA
helicase. Helicases are enzymes which use the energy of ATP hydrolysis to move into
and melt double-stranded DNA. The single-stranded bubble created in this way is
coated with single stranded binding protein (Ssb) to protect it from breakage and to
prevent the DNA renaturing.] then binds and extends the single-stranded region for

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 copying. The level of DnaA is linked to growth rate. Unwinding As the parental DNA is
unwound by DNA helicases and single-stranded binding protein, the resulting positive
supercoiling has to be relieved by the topoisomerase DNA gyrase. (DNA gyrase, a
topoisomerase inhibited by the quinolone family of antibiotics, is found only in
prokaryotes.)
2. Elongation Multiple primers are synthesized on the lagging strand. A dimer of DNA
polymerase III holoenzyme elongates both leading and lagging strands. The α subunits
polymerize the DNA and the ε subunits proofread it. The 5’→3’ exonuclease activity of
DNA
polymerase I removes the lagging strand RNA primers, and the polymerase function
simultaneously fills the gaps with DNA. DNA ligase joins the lagging strand fragments
together.
3. Termination and segregationIn E. coli, both replication forks meet at a terminus
region about 180° opposite the origin. The interlocked daughter molecules are
separated by a DNA topoisomerase.

The differences of replication between prokaryotes and eukaryotes

Prokaryotes Eukaryotes
1. single Initiation point specific (ori) 1. multiple specific Initiation points
2. DNA Polymerases are of three 2. DNA Polymerases are of five types, namely α, ε,
types, namely I, II and III β, γ and δ
3. Diverse functional variety specially 3. Functional variety of DNA polymerases is
that of DNA polymerase I specific
4. Not applicable 4. γ DNA polymerase is found in mitochondria
5. No repair function 5. β-Polymerase functions as repair enzyme
6. Only unwinding takes place in 6. Histone separation from DNA as well as
prokaryotes unwinding takes place
7. Not applicable 7. Synthesis of telomeres by Telomerase
8. Joining of Okazaki fragments DNA 8. DNA ligase
ligase
9. Removal of RNA primers by DNA 9. Removal of RNA primers by RNase H
polymerase I (5′→3′exonuclease) (5′→3′exonuclease)

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