Licensing Factor Binds to ORC

KEY CONCEPTS
-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.
-Cdt1 facilitates MCM loading on origins.
-When replication is initiated, Cdc6 and Cdt1 are displaced. The degradation of Cdc6 prevents reinitiation.
The key event in controlling replication is the behavior of the ORC complex at the origin. Recall that in S. cerevisiae, ORC is a 400-kD complex that binds to the ARS sequence . Its origin (ARS) consists of the A consensus sequence and three B elements

. The ORC complex of six proteins (all of which are encoded by essential genes) binds to the A and adjacent B1 element. Orc1 binds first, in G1 phase of the cell cycle and acts as a nucleating center; next, Orc2–5 binds strongly; Orc6 binds weakly and has a nuclear localization signal that must be activated by the cyclin/CDK kinase during the G1 to S transition . ATP is required for the binding, but is not hydrolyzed until a later stage. The transcription factor ABF1 binds to the B3 element; this assists initiation by affecting chromatin structure, but it is the events that occur at the A and B1 elements that actually cause initiation. Most origins are localized in regions between genes, which suggests that it might be important for the local chromatin structure to be in a nontranscribed condition.
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 binding of other proteins to the ORC-origin complex.
At the end of the cell cycle, ORC is bound to A–B1 elements of the origin. There is a change during G1 that results from the binding of Cdc6 and Cdt1 proteins to the ORC. In yeast, Cdc6 is a highly unstable protein, with a half-life of more than 5 minutes. It is synthesized during G1 and typically binds to ORC between the exit from mitosis and late G1. Its rapid degradation means that no protein is available later in the cycle. In mammalian cells, Cdc6 is controlled differently; it is phosphorylated during S phase, and as a result it is degraded by the ubiquitination pathway. Cdt1 is initially stabilized by the protein Geminin, which prevents its degradation, and subsequent Geminin binding prevents its reuse. These features make Cdc6 and Cdt1 the key licensing factors. These two proteins also provide the connection between ORC and a complex of proteins that is involved in initiation of replication. Cdc6 has an ATPase activity that is required for it to support initiation.
In yeast, the replication helicase MCM2-7 (minichromosome maintenance) complexes enter the nucleus as inactive double hexamers during mitosis. The presence of Cdc6 and Cdt1 at the yeast origin allows the two MCM complexes to bind to each of the two replication forks in G1 in the inactive state. Their presence is necessary for initiation. FIGURE 1 summarizes the cycle of the events that follow at the origin. The origin enters S phase in the condition of a prereplication complex, which contains ORC, Cdc6, Cdt1, and the inactive helicase, the MCM proteins. The MCM2–7 proteins form a six-member ring-shaped complex around DNA. MCM2,3,5 are regulatory, whereas MCM4,6,7 have the helicase activity. When initiation occurs, Cdc6 and Cdt1 are displaced, returning the origin to the state of the postreplication complex, which contains only ORC. Cdc6 is rapidly degraded during S phase and, as a result, it is not available to support reloading of MCM proteins. Thus, the origin cannot be used for a second cycle of initiation during S phase. In mammalian cells, Cdt1 is targeted for degradation by the action of a protein complex that is recruited to the origin of replication by PCNA, the eukaryotic counterpart of the bacterial β clamp.

FIGURE 1. Proteins at the origin control susceptibility to initiation.
Data from: Heller, R. C., et al. 2011. Cell 146:80–91.
If Cdc6 is made available to bind to the origin during G2 (by ectopic expression), MCM proteins do not bind until the following G1, which suggests 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. Some of the ORC proteins have similarities to replication proteins that load DNA polymerase onto DNA. It is possible that ORC uses hydrolysis of ATP to load the MCM ring onto DNA. In Xenopus extracts, replication can be initiated if ORC is removed after it has loaded Cdc6 and MCM proteins. This shows that the major role of ORC is to identify the origin to the Cdc6 and MCM proteins that control initiation and licensing.
As the transition from G1 to S phase begins, CDK/cyclins recruit cdc45 and the GINS complex to the MCM helicase, which then becomes known as the CMG complex (for Cdc45-MCM-GINS) for activation. This marks the transition from initiation to DNA replication, that is, the elongation phase of replication that entails the two different modes of synthesis on the leading (forward) strand and the lagging (discontinuous) strand. The MCM proteins, when activated, are required for elongation as well as for initiation, and they continue to function at the two bidirectional replication forks as the replication helicase.
 

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