Transcript Document

Chapter 15
The Replicon
15.1 Introduction
 Whether a cell has only one chromosome (as in prokaryotes) or has
many chromosomes (as in eukaryotes), the entire genome must be
replicated precisely once of every cell division.
 Two general principles: (1) Initiation of DNA replication commits
the cell to a further division. Replication is controlled at the stage of
initiation. Once replication has started, it continues until the entire
genome has been duplicated. (2) If replication proceeds, the
consequent division cannot be permitted to occur until the
replication event has been completed.
- replicon: the unit of DNA in which an individual act of replication
occurs.
- origin: at which replication is initiated.
- terminus: at which replication stops.
 A genome in a prokaryotic cell constitutes a single replicon; thus the
units of replication and segregation coincide.
 A plasmid is an autonomous circular DNA genome that constitutes a
separate replicon; may show single copy control or under
multicopy control. Any DNA molecule that contains an origin can
be replicated autonomously in the cell.
 Each eukaryotic chromosome contains a large number of replicons;
each must be activated no more than once in each cell cycle.
 The DNA of mitochondria and chloroplasts may be regulated more
like plasmids that exist in multiple copies per bacterium.
15.2 Replicons Can Be Linear or Circular
Key Concepts
 A replicated region appears as an eye 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.
 Fig. 15.1: replication eyes form bubbles.
 replication fork (or growing point): the point at which replication
occurs. A replication fork moves sequentially along the DNA from its
starting point at the origin. Unidirectional or bidirectional
replication.
 Fig. 15.2: replication eyes can be uni- or bidirectional
 Fig. 15.3: when a replicon is circular, the presence of an eye forms
the θ structure.
 Fig. 15.4: the successive stages of replication of the circular DNA of
polyoma virus.
Figure 15.1. Replicated DNA is seen as a replication eye
flanked by nonreplicated DNA.
Figure 15.2. Replicons may be
unidirectional or bidirectional,
depending on whether one or two
replication forks are formed at the
origin.
Figure 15.3. A replicatin eye forms a θ structure in circular DNA.
Figure 15.4. The replication eye
becomes larger as the
replication forks proceed along
the replicon.
15.3 Origins Can Be Mapped by Autoradiography and
Electrophoresis
Key Concepts
 Replication fork movement can be detected by autoradiography using
radioactive pulses.
 Replication forks create Y-shaped structures that change the
electrophoretic migration of DNA fragments.
 Whether a replicating eye has one or two replication forks can be
determined in two ways.
 Fig. 15.5: shows that the unidirectional replication causes one type
of label to be followed by the other at one end of the eye.
Bidirectional replication produces a (symmetrical) pattern at both
ends of the eye.
 Fig. 15.6: illustrates the two-dimensional mapping technique, in
which restriction fragments of replicating DNA are electrophoresed
in a first dimension that separates by mass and a second dimension
where movement is determined more by shape.
Figure 15.5. Different densities of radioactive labeling can be used to
distinguish unidirectional and bidirectional replication.
Figure 15.6. The position of the origin and the number of replicating forks
determine the shape of a replicating restriction fragment, which can be
followed by its electrophoretic path (solid line). The dashed line shows the
path for a linear DNA.
15.4 Does Methylation at the Origin Regulate Initiation?
Key Concepts
 oriC contains eleven GATC/CTAG repeats that are methylated on
adenine on both strands.
 Replication generates hemimethylated DNA, which cannot initiate
replication.
 There is a 13-minute delay before the GATC/CTAG repeats are
remethylated.
 What feature of a bacterial (or plasmid) origin ensures that it is used
to initiate replication only once per cycle?
 Some sequences that are used for this purpose are included in the
origin. oriC contains eleven copies of the sequence GATC/CTAG,
which is a target for methylation at the N6 position of adenine by the
Dam methylase (Figure 15.7).
 If the plasmid is methylated it undergoes a single round of
replication, and then the hemimethylated products accumulate
(Figure 15.8). Hemimethylated origins cannot initiate again until
the Dam methylase has converted them into fully methylated
origins.
Figure 15.7. Replication of methylated DNA gives hemimethylated DNA,
which maintains its state at GATC sites until the Dam methylase restores
the fully methylated condition.
Figure 15.8. Only fully methylated origins can initiate replication;
hemimethylated daughter origins cannot be used again until they have
been restored to the fully methylated state.
15.5 Origins May Be Sequestered after Replication
Key Concepts
 SeqA binds to hemimethylated DNA and is required for delaying
rereplication.
 SeqA may interact with DnaA.
 As the origins are hemimethylated they bind to the cell membrane and
may be unavailable to methylases.
 The nature of the connection between the origin and the membrane is
still unclear.
Figure 15.9. A membrane-bound inhibitor binds to hemimethylated DNA
at the origin and may function by preventing the binding of DnaA. It is
released when the DNA is remethylated.
15.6 Each Eukaryotic Chromosome Contains Many Replicons
Key Concepts
 Eukaryotic replicons are 40 to 100 kb in length.
 A chromosome is divided into many replicons.
 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.
 S phase usually lasts a few hours in a higher eukaryotic cell.
 Figure 15.10: Replicon sizes can be measured by adjacent eyes.
 Individual replicons in eukaryotic genomes are relatively small,
typically ~40 kb in yeast or fly and ~ 100 kb in animal cells. The
rate of replication is ~ 2000 bp/min, which is much slower than the
50,000 bp/min of bacterial replication fork movement.
 A mammalian genome could be replicated in ~1 hour if all replicons
functioned simultaneously. S phase actually lasts for >6 hours in a
typical somatic cell, implying that no more than 15% of the
replicons are likely to be active at any given moment.
 Visualization of replicating forks by labeling with DNA precursors
identifies 100 to 300 “foci” instead of uniform staining; each focus
shown in Figure 15.11 probably contains >300 replication forks.
Figure 15.11. Replication forks are organized into foci in the nucleus.
Cells were labeled with BrdU. The leftmost panel was stained with
propidium iodide to identify bulk DNA. The right panel was stained using
an antibody to BrdU to identify replicating DNA.
15.7 Replication Origins Can Be Isolated in Yeast
Key Concepts
 Origins in S. cerevisiae are short A-T-rich sequences that have an
essential 11-bp sequence.
 The ORC is a complex of six proteins that binds to an ARS.
 Any segment of DNA that has an origin should be able to replicate,
so although plasmids are rare in eukaryotes, it may be possible to
construct them by suitable manipulation in vivo. This has been
accomplished in yeast, although not in higher eukaryotes.
 The discovery of yeast origins resulted from the observation that
some yeast DNA fragments (when circularized) are able to
transform defective cells very efficiently. These fragments can
survive in the cell in the unintegrated (autonomous) state, that is, as
self-replicating plasmids.
 This segment is called as ARS (for autonomously replicating
sequence). ARS elements are derived from origins of replication.
 An ARS element consists of an A-T-rich region.
 Figure 15.12: shows a systematic mutational analysis along the
length of an origin.
 Origin function is abolished completely by mutations in a 14-bp
“core” region, called the A domain, which contains an 11-bp
consensus sequence consisting of A-T base pairs.
 This consensus sequence (called ACS for ARS Consensus Sequence)
is the only homology between known ARS elements.
 Mutations in three adjacent elements, numbered B1 to B3, reduce
origin function. An origin can function effectively with any two of
the B elements, so long as a functional A element is present.
 The ORC (origin recognition complex) is a complex of six proteins
with a mass of ~400 kD. ORC binds to the A and B1 elements.
 There are about 400 origins in the yeast genome, meaning that the
average length of a replicon is ~ 35,000 bp.
Figure 15.12. An ARS extends for ~50 bp and includes a
consensus sequence (A) and additional elements (B1-B3).
15.8 Licensing Factor Controls Eukaryotic Rereplication
Key Concepts
 Licensing factor is necessary for initiation of replication at each origin.
 It is present in the nucleus prior to replication, but is inactivated or
destroyed by replication.
 Initiation of another replication cycle becomes possible only after
licensing factor reenters the nucleus after mitosis.
 A eukaryotic genome is divided into multiple replicons, and the
origin in each replicon is activated once and only once in a single
division cycle.
 Figure 15.13: >1 replication cycle needs cytoplasmic factors.
 Figure 15.14: explains the control of reinitiation by proposing that
this protein is a licensing factor.
 It is present in the nucleus prior to replication. One round of
replication either inactivates or destroys the factor, and another
round cannot occur until further factor is provided. Factor in the
cytoplasm can gain access to the nuclear material only at the
subsequent mitosis when the nuclear envelope breaks down.
Figure 15.13. A nucleus injected into
a Xenopus egg can replicate only once
unless the nuclear membrane is
permeabilized to allow subsequent
replication cycles.
Figure 15.14. 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.
15.9 Licensing Factor Consists of MCM Proteins
Key Concepts
 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.
 When replication is initiated, Cdc6 and MCM proteins are displaced.
The degradation of Cdc6 prevents reinitiation.
 Some MCM proteins are in the nucleus throughout the cycle, but
others may enter only after mitosis.
 The key event in controlling replication is the behavior of the ORC
complex at the origin. The origin (ARS) consists of the A consensus
sequence and three B elements. The ORC complex of six proteins
binds to the A and adjacent B1 element. The transcription factor
ABF1 binds to the B3 element; this assists initiation.
 Most origins are localized in regions between genes.
 Figure 15.15; summarizes the cycle of events at the origin.
 In yeast, Cdc6 is a highly unstable protein, with a half-life of <5
minutes. It is synthesized during G1 and typically binds to the ORC
between the exit from mitosis and late G1.
 In yeast the presence of Cdc6 at the origin allows MCM (minichromosome maintenance) proteins to bind to the complex. The
origin therefore enters S phase in the condition of a prereplication
complex, which contains ORC, Cdc6, and MCM proteins. When
initiation occurs, Cdc6 and MCM are displaced, returning the origin
to the state of the postreplication complex, which contains only
ORC.
Figure 15.15. Proteins at the
origin control susceptibility to
initiation.
15.10 D Loops Maintain Mitochondrial Origins
Key Concepts
 Mitochondria use different origin sequences to initiate replication of
each DNA strand.
 Replication of the H strand is initiated in a D loop.
 Replication of the L strand is initiated when its origin is exposed by
the movement of the first replication fork.
 Initiation requires separating the DNA strands and commencing
bidirectional DNA synthesis. A different type of arrangement is
found in mitochondria.
 Replication starts at a specific origin in the circular duplex DNA.
Initially, though, only one of the two parental strands (the H strand
in mammalian mitochondrial DNA) is used as a template for
synthesis of a new strand. Synthesis proceeds for only a short
distance, displacing the original partner (L) strand, which remains
single-stranded, as illustrated in Figure 15.16. The condition of this
region gives rise to its name as the displacement loop, or D loop.
Figure 15.16. The D loop
maintains an opening in
mammalian mitochondrial
DNA, which has separate
origins for the replication of
each strand.
 The existence of D loops exposes a general principle: An origin can
be a sequence of DNA that serves to initiate DNA synthesis using
one strand as template.
 The opening of the duplex does not necessarily lead to the initiation
of replication on the other strand. In the case of mitochondrial DNA
replication, the origins for replicating the complementary strands lie
at different locations.
 Origins that sponsor replication of only one strand are also found in
the rolling circle mode of replication.