Chapter 12
The Replicon: Initiation of Replication
12.1 Introduction
• replicon – A unit of the genome in which DNA is
replicated. Each contains an origin for initiation of
replication.
• origin – A sequence of DNA at which replication is
initiated.
12.1 Introduction
• single-copy replication control – A control
system in which there is only one copy of a
replicon per unit bacterium.
– The bacterial chromosome and some plasmids have
this type of regulation.
• multicopy replication control – Replication
occurs when the control system allows the
plasmid to exist in more than one copy per
individual bacterial cell.
12.2 An Origin Usually Initiates
Bidirectional Replication
• semiconservative replication
– Replication accomplished by
separation of the strands of a
parental duplex, with each
strand then acting as a template
for synthesis of a
complementary strand.
• A replicated region appears as a
Figure 12.01: Replicated DNA is seen
replication bubble within
as a replication bubble flanked by
nonreplicated DNA.
nonreplicated DNA.
12.2 An Origin Usually Initiates
Bidirectional Replication
• 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 12.02: Replicons may be
unidirectional or bidirectional, depending
on whether one or two replication forks
are formed at the origin.
12.3 The Bacterial Genome Is (Usually) a
Single Circular Replicon
Figure 12.04: Bidirectional replication
of a circular bacterial chromosome is
initiated at a single origin.
• Bacterial replicons are usually
circles that replicate
bidirectionally from a single
origin.
• The origin of E. coli, oriC, is
245 bp in length.
• The two replication forks
usually meet halfway around
the circle, but there are ter
sites that cause termination if
the replication forks go too far.
12.4 Methylation of the Bacterial Origin
Regulates Initiation
• oriC also contains eleven GATC/CTAG repeats that are
methylated on adenine on both strands.
• Replication generates hemimethylated DNA, which
cannot initiate replication.
Figure 12.05: The E. coli origin of replication, oriC contains multiple binding sites for the
DnaA initiator protein. In a number of cases these sites overlap Dam methylation sites.
12.4 Methylation of the Bacterial Origin
Regulates Initiation
• There is a 13-minute delay
before the GATC/CTAG
repeats are remethylated.
• SeqA binds to
hemimethylated DNA and is
required for delaying
rereplication.
Figure 12.06: Only fully methylated origins
can initiate replication; hemimethylated
daughter origins cannot be used again
until they have been restored to the fully
methylated state.
12.5 Initiation: Creating the Replication
Forks at the Origin
• Initiation at oriC requires the sequential assembly of a
large protein complex on the membrane.
• DnaA is the licensing factor (a factor necessary for
replication; it is inactivated or destroyed after one round
of replication).
• oriC must be fully methylated for replication to initiate.
• DnaA-ATP binds to short repeated sequences and forms
an oligomeric complex that melts DNA.
12.5 Initiation: Creating the Replication
Forks at the Origin
• Six DnaC monomers bind each hexamer of DnaB, and
this complex binds the origin.
• A hexamer of DnaB forms the replication fork. Gyrase
and SSB are also required.
• DnaG is bound to the helicase complex and creates the
replication fork.
Figure 12.07: The minimal origin is defined by the distance between the outside members
of the 13-mer and 9-mer repeats.
12.5 Initiation: Creating the Replication
Forks at the Origin
Figure 12.08: Prepriming involves formation of a complex by sequential association of
proteins, which leads to the separation of DNA strands.
12.6 Each Eukaryotic Chromosome
Contains Many Replicons
• A eukaryotic chromosome is divided into many replicons.
• The progression into S phase is tightly controlled.
Figure 12.09: A eukaryotic chromosome contains multiple origins of replication that
ultimately merge during replication.
12.6 Each Eukaryotic Chromosome
Contains Many Replicons
• Eukaryotic replicons are 40 to 100 kb in length.
• Individual replicons are activated at characteristic times
during S phase.
• Regional activation patterns suggest that replicons near
one another are activated at the same time.
12.7 Replication Origins Bind the ORC
Figure 12.10: An ARS extends for ~50
bp and includes a consensus sequence
(A) and additional elements (B1–B3).
• Origins in S. cerevisiae are
short A-T sequences that
have an essential 11 bp
sequence.
• The ORC is a complex of six
proteins that binds to an ARS
(an origin for replication in
yeast). The common feature
among different examples of
these sequences is a
conserved 11 bp sequence
called the A domain.
12.7 Replication Origins Bind the ORC
• Related ORC complexes are found in multicellular
eukaryotes.
12.8 Licensing Factor Controls Eukaryotic
Rereplication
• Licensing factor is necessary for initiation of replication
at each origin.
• Licensing factor is present in the nucleus prior to
replication, but is removed, inactivated, or destroyed by
replication.
12.8 Licensing Factor Controls Eukaryotic
Rereplication
• Initiation of another replication cycle becomes possible only
after licensing factor re-enters the nucleus after mitosis.
Figure 12.12: Licensing factor in the
nucleus is inactivated after replication.
A new supply of licensing factor can
enter only when the nuclear
membrane breaks down at mitosis.
12.8 Licensing Factor Controls Eukaryotic
Rereplication
• The ORC is a protein complex
that is associated with yeast
origins throughout the cell
cycle.
• Cdc6 protein is an unstable
protein that is synthesized only
in G1.
• Cdc6 binds to ORC and allows
MCM proteins to bind.
Figure 12.13: Proteins at the origin
control susceptibility to initiation.
12.8 Licensing Factor Controls Eukaryotic
Rereplication
• When replication is initiated, Cdc6, Cdt1, and MCM
proteins are displaced. The degradation of Cdc6
prevents reinitiation.
• prereplication complex – A protein-DNA complex at
the origin in S. cerevisiae that is required for DNA
replication. The complex contains the ORC complex,
Cdc6, and the MCM proteins.
• postreplication complex – A protein-DNA complex in
S. cerevisiae that consists of the ORC complex bound to
the origin.
12.8 Licensing Factor Controls Eukaryotic
Rereplication
• Some MCM proteins are in the nucleus throughout the
cell cycle, but others may enter only after mitosis.