Replication

BEB-102, Autumn 2024

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 Learning Objectives
By the end of this section, you will learn about:
• What replication is
• Replication machinery
• The replication process
• Okazaki fragments

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Basic cellular Processes: Gene Expression

DNA

Replication

Takes place
in Nucleus

DNA

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 Replication
• DNA Replication is the process by which a cell
makes an identical copy of its DNA.

• Each new DNA molecule consists of one original

strand and one newly synthesized strand. Original Parent Duplex
Two Daughter Duplexes

• The parental strands are permanently separated 5’ 3’ 5’ 3’ 5’ 3’

and each forms a duplex molecule with the A T A T A T
daughter strand base-paired to it. T A T A T A
Transcription
+
A T A T A T
G C G C G C
C G C G C G
A T A T A T
• DNA replication is essential for:
Cell Division: Ensures each daughter cell 3’ 5’ 3’ 5’ 3’ 5’
receives an identical copy of genetic material. Parent Parent Parent Daughter Daughter Parent
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 Replication Characteristics
Semi Conservative Bidirectional Participation of
both DNA strands

• Both strands serve as

• The process occurs in two templates, unlike in
opposite directions from a single translation
origin of replication.
• Both the daughter cells have half old
and half new DNA • Bidirectional replication
enhances the speed and accuracy 5
of DNA duplication
 Main Proteins and Enzymes in Replication
1. Initiator Proteins
• Recognizing Origins (Rich in adenine
(A) and thymine (T) bases): They identify
and bind to specific DNA sequences at
the origin of replication.
• Unwinding DNA: By binding to the origin,
they help unwind a short stretch of the
DNA double helix, creating a replication
bubble.
• Recruiting Enzymes: Initiator proteins
facilitate the assembly of the replication
machinery, including helicase and DNA
polymerase.
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Main Proteins and Enzymes in Replication
2. Helicase
DNA Helicase: Unwinds the DNA double helix, separating the two
strands by breaking hydrogen bonds.

3. Single-Strand Binding Proteins (SSBPs)

Bind to single-stranded DNA to stabilize it and prevent it from re-
annealing or forming secondary structures.

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 Main Proteins and Enzymes in Replication
4. DNA Polymerases (III and I)
Roles:
• DNA synthesis: DNA polymerase (DNAP)
adds nucleotides one by one to the growing
chain, using a template strand to construct a
complementary sequence of nucleotides.
• Proofreading: DNAP can proofread the new
strand as it is created to ensure accuracy.
Regions: Synthesizes new DNA strands in 5'→3' direction by
catalyzing the formation of phosphodiester bond
a. DNA binding domain: Binds to DNA b/w the 3′ hydroxyl group at the growing end of
b. Polymerizing domain: Attaches new DNA chain and the 5′ phosphate group of incoming
nucleotides to the growing chain deoxy-ribonucleoside triphosphate (dNTP).
c. Exonuclease domain: Repairs mistakes But it can NOT initiate the DNA synthesis by its own
made during DNA synthesis and requires a primer
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DNA Polymerase

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Replication occurs in 5' to 3' direction?
• Growth in the 5′-to-3′ direction, shown on the right,
allows the chain to continue to be elongated when a
mistake in polymerization has been removed by
exonucleolytic proofreading (Figure ).

• In contrast, exonucleolytic proofreading in the

hypothetical 3′-to-5′ polymerization scheme, shown
on the left, would block further chain elongation. For
convenience, only the primer strand of the DNA
double helix is shown.

Molecular Biology of the Cell. 4th edition.

Alberts B, Johnson A, Lewis J, et al. 10
 Main Proteins and Enzymes in Replication
5. DNA Primase
• Synthesizes short RNA primers (8-10 nucleotides long) to provide a starting point for
DNA synthesis.
• DNA primase is a type of RNA polymerase that creates RNA primers that are
complementary to a single-stranded DNA template
• After DNA polymerases elongate the primers, the RNA primers are removed and the gap
in the DNA sequence is filled in by DNA polymerase I and DNA ligase.
6. DNA Ligase
Joins Okazaki fragments on the lagging strand by sealing nicks in the sugar-phosphate
backbone.
7. Topoisomerase
Relieves tension that builds up ahead of the replication fork by cutting and rejoining DNA
strands.
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Replication bubble and replication fork

• Each replication bubble

has two forks

• Eukaryotes have
multiple Origin of
Replication

fork • The two forks move in

opposite directions,
away from one another,
and the origin.

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 Three Steps of Replication
Initiation, Elongation and Termination

Initiation
• DNA replication begins at specific locations called origins of
replication.
• Unwinding DNA: By binding to the origin, initiator proteins
help unwind a short stretch of the DNA double helix,
creating a replication bubble.
• DNA helicase unwinds the double helix, creating replication
forks.
• Single-strand binding proteins stabilize the separated
strands.
• RNA Primer Synthesis
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 Elongation
Synthesis:
Both DNA strands are synthesized differently by DNAP
Leading Strand:
• Synthesized continuously in the direction of the
replication fork, with DNA polymerase adding
nucleotides to the 3' end in a 5' to 3' direction.

• One primer required

Lagging Strand:
• Synthesized discontinuously in short segments
called Okazaki fragments, moving opposite to the
replication fork. DNA polymerase cannot synthesize
in a 3' to 5' direction (we discussed previously).
• Many primers required 14
 Elongation Cont.

• Replacement of RNA Primers:

DNA Polymerase I removes RNA primers and replaces them with DNA
nucleotides.

• Ligation:
DNA ligase joins the Okazaki fragments on the lagging strand, sealing nicks
in the sugar-phosphate backbone.

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 DNA Synthesis (Leading Strand)

3’ 5’
Template/parent Strand

Primase makes RNA

Origin Primase primers at the 3’ end
RNA primer of the parental strand
of
replica
tion
5’
3’ 3’

Newly Synthesized DNA DNA polymerase III uses the

RNA primer as a starting
point
Daughter Strand 5’
being synthesized
3’ 5’
DNA Primase III 16
 Synthesis Lagging Strand
• The lagging strand is extended discontinuously from each primer forming
Okazaki fragments.
• RNase removes the primer RNA fragments, and a low processivity DNA
polymerase distinct from the replicative polymerase enters to fill the gaps.
• Ligase enzymes then joins the Okazaki fragments thus filling the gaps.

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Bidirectional Synthesis

Although depicted as individual

steps for clarity, unwinding and
synthesis of leading and lagging
strands occur concurrently.

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 Termination
Prokaryotes

• Replication is terminated whenever two replication forks meet -as it contains only one origin of
replication- and the replication machinery encounters termination sites.

• A termination protein binds to termination site and blocks the movement of helicase thus stopping
the replication fork and preventing further DNA replication.

Eukaryotes

• More complex mechanisms involving multiple origins of replication and checkpoints are employed.

• Since the replication forks are moving, DNA replication initiates at multiple sites along the “lagging”
strand, until the DNAP that synthesizes the lagging strand collides with the existing leading strand.
Collision leads to the removal of the RNA primer and ligation (linkage) of the two DNA molecules
together.
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Difference b/w Replication & Transcription

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 Key Concepts: Replication
• Each parental DNA strand acts as a template for daughter strand synthesis, forming a daughter
duplex (semi-conservative mechanism).

• New strands are synthesized in the 5' to 3' direction, starting at replication origins; eukaryotic
chromosomes have multiple origins.

• DNA polymerases cannot unwind DNA or initiate synthesis of new strands.

• At the replication fork, the leading strand is elongated continuously, while the lagging strand forms
in discontinuous Okazaki fragments from primers every few hundred nucleotides.

• Ribonucleotides at the 5' end of each Okazaki fragment are replaced by the next fragment's 3' end,
with DNA ligase joining adjacent fragments.

• Helicases use ATP to separate parental strands; primase synthesizes RNA primers.

• DNA replication occurs bidirectionally, with two replication forks forming at an origin and moving in
opposite directions, copying both template strands at each fork. 21