Chapter 15The 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

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.

Key Concepts

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 bidirectionalFig. 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

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.

Key Concepts

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?

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.

Key Concepts

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

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.

Key Concepts

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

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.

Key Concepts

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

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.

Key Concepts

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

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.

Key Concepts

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

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.

Key Concepts

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 (mini-chromosome 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

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.

Key Concepts

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.