Genes VII

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. This could be achieved by providing some rate-limiting component that functions only once at an origin or by the presence of a repressor that prevents rereplication at origins that have been used. The critical questions about the nature of this regulatory system are how the system determines whether any particular origin has been replicated, and what protein components are involved.

Insights into the nature of the protein components have been provided by using a system in which a substrate DNA undergoes only one cycle of replication. Xenopus eggs have all the components needed to replicate DNA Xin the first few hours after fertilization they undertake 11 division cycles without new gene expression Xand they can replicate the DNA in a nucleus that is injected into the egg. Figure 13.33 summarizes the features of this system.

The injected material can take the form of a sperm or interphase nucleus. Its DNA is replicated only once (followed by use of a density label, just like the original experiment that characterized semiconservative replication, shown previously in Figure 1.10). If protein synthesis is blocked in the egg, the membrane around the injected material remains intact, and the DNA cannot replicate again. However, in the presence of protein synthesis, the nuclear membrane breaks down just as it would for a normal cell division, and in this case subsequent replication cycles can occur. The same result can be achieved by using agents that permeabilize the nuclear membrane. This suggests that the nucleus contains a protein(s) needed for replication that is used up in some way by a replication cycle; although more of the protein is present in the egg cytoplasm, it cannot enter the nucleus, but is allowed to do so if the nuclear membrane breaks down. The system can in principle be taken further by developing an in vitro extract that supports nuclear replication, thus allowing the components of the extract to be isolated, and the relevant factors identified.

Figure 13.34 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. This regulatory system achieves two purposes. By removing a necessary component after replication, it prevents more than one cycle of replication from occurring. And it provides a feedback loop that makes the initiation of replication dependent on passing through cell division.

Components of the licensing factor are provided by the S. cerevisiae proteins MCM2, 3, 5, which are required for replication and enter the nucleus only during mitosis. Homologues are found in animal cells, where MCM3 is bound to chromosomal material before replication, but is released after replication. The animal cell MCM2,3,5 complex remains in the nucleus throughout the cell cycle, suggesting that it may be one component of the licensing factor; another component, able to enter only at mitosis, may be necessary for MCM2,3,5 to associate with chromosomal material.

An insight into the system that controls availability of licensing factor is provided by certain mutants in yeast. Mutations in the licensing factor itself could prevent initiation of replication; this is the behavior of MCM2, 3, 5. Mutations in the system that inactivates licensing factor after the start of replication should allow the accumulation of excess quantities of DNA, because the continued presence of licensing factor allows rereplication to occur. Such mutations are found in genes that code for components of the ubiquitination system that is responsible for degrading certain proteins. This suggests that licensing factor may be destroyed after the start of a replication cycle.

The state of the origin during the replication cycle has been followed in S. cerevisiae. The origin (ARS) consists of the A consensus sequence and three B elements (see Figure 12.10). The ORC complex of 6 proteins (all of which are coded by essential genes) binds to the A and adjacent B1 element. The transcription factor ABF1 binds to the B3 element; this has a facilitative or enhancing role on initiation, but it is the events that occur at the A and B1 elements that actually cause initiation.

The striking feature is that ORC remains bound at the origin through the entire cell cycle. However, changes occur in the pattern of protection of DNA as a result of the binding of other proteins to the ORC-origin complex. Figure 13.35 summarizes the cycle of events at the origin.

At the end of the cell, ORC is bound to A/B1, and generates a pattern of protection in vivo that is similar to that found when it binds to free DNA in vitro. Basically the region across A-B1 is protected against DNAase, but there is a hypersensitive site in the center of B1.

During G1, this pattern changes, most strikingly by the loss of the hypersensitive site. This is due to the binding of Cdc6 protein to the ORC. Cdc6 is a highly unstable protein (half-life <5 minutes). It is synthesized from late G1 through G1, and typically binds to the ORC between the exit from mitosis and late G1. Its rapid degradation means that no protein is available later in the cycle.

The presence of Cdc6 in turn allows Mcm proteins to bind to the complex. Their presence is necessary for initiation to occur at the origin. The origin therefore enters S phase in the condition of a prereplication complex, containing 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. Because Cdc6 is rapidly degraded during S phase, it is not available to support reloading of Mcm proteins, and so the origin cannot be used for a second cycle of initiation during the S phase.

If Cdc6 is made available to bind to the origin during G2 (by ectopic expression), Mcm proteins do not bind until the following G1, suggesting that there is a secondary mechanism to ensure that they associate with origins only at the right time. This could be another part of licensing control. At least in S. cerevisiae, this control does not seem to be exercised at the level of nuclear entry, but this could be a difference between yeasts and animal cells. We discuss how the cell cycle control system regulates initiation (and reinitiation) of replication in 27 Cell cycle and growth regulation.