Module 4b: Replication and cell cycle:

Initiation of replication

Copyright © 2018 Jones and Bartlett Learning, LLC, ALC

Learning objectives
By the end of this module, you should be able to:
• Understand the importance of replication initiation.
• Describe bidirectional replication initiation.
• Explain the role of methylation in regulating initiation.
• Comprehend the process of creating replication forks at the origin.
• Explore mechanisms to prevent premature replication reinitiation.
An origin usually initiates bidirectional replication

• A replicated region appears as a bubble within

nonreplicated DNA.
• A replication fork is initiated at the origin and then
moves sequentially along DNA.
• Replication is unidirectional when a single
replication fork is created at an origin.
• Replication is bidirectional when an origin creates
two replication forks that move in opposite
directions.

Figure 10.3: Replicons can be unidirectional or bidirectional,

depending on whether one or two replication forks are formed at the
origin.
DNA Replication in Bacterial Cells
Semidiscontinuous Replication
▪ Both daughter strands are synthesized simultaneously.
▪ DNA polymerase molecules move along a template only
in a 3′ → 5′ direction.
• The leading strand is synthesized continuously.
• The lagging strand is synthesized discontinuously (as
Okazaki fragments)
▪ The two newly assembled DNA strands grow in opposite
directions, one growing toward the replication fork and
the other growing away from it.

The two strands of a double helix are

synthesized by different sequences of events.
Copyright ©2020 John Wiley & Sons, Inc.
The Bacterial Genome is a Single Circular Replicon

• Bacterial replicons are usually circles

that replicate bidirectionally from a
single origin.
• The origin of Escherichia coli, oriC, is
245 base pairs (bp) in length, long AT-
rich sequence.
• Mostly two types of sequences are
present in this region, three repeats of
13bp called a 13mer and five repeats of
9bp called a 9mer.

Figure 10.5: Bidirectional replication of a circular bacterial

chromosome is initiated at a single origin.
Methylation of the Bacterial origin Regulates Initiation
• DnaA is an ATP-binding protein and the replication initiator
• oriC contains binding sites for DnaA: dnaA boxes.
• oriC also contains 11 GATC/CTAG repeats that are methylated on adenine on both strands
by Dam methylase enzyme.
• DnaA DNA binding is influenced by ATP, ADP, or no nucleotide binding.
• DNA methylation controls the activity of the replication origin. Only 1replication/Chromosome at a time

Figure 10.6: The E. coli origin of replication, oriC,

contains multiple binding sites for the DnaA initiator
protein.
 Methylation of the bacterial origin regulates initiation

• Replication generates hemimethylated

DNA, which cannot initiate replication
until they have been restored to the fully
methylated state.
• There is a 13-minute delay before the
GATC/CTAG repeats are remethylated.

Figure 10.7: Only fully methylated origins can initiate replication..

Methylation of the bacterial origin regulates initiation

• DNA methylation in bacteria aids in

distinguishing old and new DNA strands
during repair.
• When DNA polymerase makes a mistake,
the repair system uses the methylated
strand as a template to correct errors,
ensuring accurate identification of the new
strand.

Figure 10.7: Only fully methylated origins can initiate replication..

Initiation: Creating the replication forks at the origin oriC
• Requirements for initiation of replication:
• Protein synthesis is needed to produce the origin recognition protein,
DnaA, required for each round of replication.
• Licensing factor in E. coli, made anew for each round of replication.

• Transcription activation is necessary, involving the transcription of genes

flanking oriC. This aids DnaA in opening the origin.

• Membrane/cell wall synthesis is vital.

Initiation: Creating the replication forks at the origin oriC
• Initiation of replication at oriC involves six proteins: DnaA, DnaB, DnaC, HU,
gyrase, and SSB.
• DnaA- plays a unique role in the initiation process; it
recognizes oriC sequences for initiation of replication.

• DnaB- an ATP-dependent helicase, unwinds DNA after the origin opens, but
this requires full methylation on both DNA strands.

• DnaC- at the origin of replication it helps helicase (DnaB) to recognize the

site for its action
Initiation: Creating the replication forks at the origin oriC
• Initiation of replication at oriC involves six proteins: DnaA, DnaB, DnaC, HU,
gyrase, and SSB.
• HU- not directly involved in the initiation of replication, however had the
capacity to stimulate the reaction because bends DNA and builds the
structure that leads to the formation of the open complex

• DNA gyrase– it is a DNA topoisomerase II that helps in the unwinding of

DNA.

• SSB- The single-stranded binding protein binds to the single strand of

DNA and protects it from rejoining. When helicase unwinds DNA, SSB
proteins bind the single strand near helicase.
Initiation: Creating the replication forks at the origin oriC

• DnaG primase is bound to the helicase complex and creates

the replication forks.
Initiation: Creating the replication forks at the origin oriC

https://geneticeducation.co.in/prokaryotic-dna-replication-initiation-elongation-and-
termination/
DNA Replication in Bacterial Cells
The Machinery Operating at the Replication Fork

The roles of the DNA helicase, single-stranded DNA-binding proteins, and primase at the replication fork

Copyright ©2020 John Wiley & Sons, Inc.

Multiple mechanisms exist to prevent premature reinitiation of
replication
Mechanisms for controlling reinitiation involve:
1.Physical sequestration of the origin
2.Delay in remethylation
3.Competition for DnaA binding and ATP hydrolysis
4.Repression of DnaA transcription
Multiple mechanisms exist to prevent premature reinitiation of
replication
• SeqA binds to hemimethylated DNA and is required for delaying rereplication.
• SeqA can interact with DnaA.
• As the origins are hemimethylated they bind to the cell membrane and might be
unavailable to methylases.

• DnaA-ATP----DnaA-ADP feedback loop: Hda protein is recruited to the

replication origin to convert DnaA-ATP to DnaA-ADP.
 Each eukaryotic chromosome contains many replicons
• Eukaryotes replicate their genome in small
portions called replicons.
• 40 to 100 kilobases (kb) in length.
• Each replicon has its own origin from which
replication forks proceed outward in both
directions.
• Individual replicons are activated during S
phase.
• Replication in eukaryotes is slower at about
2,000 bp/min due to chromatin structure, while
bacterial replication moves faster at 50,000
Figure 10.10: A eukaryotic chromosome contains multiple
bp/min. origins of replication that ultimately merge during
replication.
Figure 10.0.11.0: Replication forks are organized into foci in the nucleus. Cells were labeled
with BrdU. The left panel was stained with propidium iodide to identify bulk DNA. The right panel
was stained using an antibody to BrdU to identify replicating DNA.
DNA Replication in Eukaryotic Cells
Initiation of Replication in Eukaryotic Cells
▪ Origins in Saccharomyces cerevisiae are short A-
T sequences that have an essential 11-bp
sequence.
▪ Origin of replication: Autonomous replicating
sequence (ARS)
• Associated with a multiprotein complex called the
origin recognition complex (ORC)
• Contain 6 proteins

Replication in eukaryotic chromosomes

begins at many sites along the DNA.
Licensing Factor Binds to ORC
• Cdc6 and Cdt1 are essential licensing factors
connecting ORC to the replication initiation
protein complex.
• Cdc6 is synthesized exclusively in G1 phase.
• Cdc6 binds to ORC, enabling MCM protein
binding.
• Cdt1 helps load MCM proteins onto origins.
• Both Cdc6 and Cdt1 are displaced during
replication initiation to prevent reinitiation.

Figure 10.15: Proteins at the origin control

susceptibility to initiation.