Cellular control of replication in eukaryotes
Actively growing (or dividing) eukaryotic cells pass through a cell cyclethat is divided into four phases (Fig. 6.17). Classic studies showed that cells in two of these phases are discernable in the light microscope. Cells undergoing mitosis have condensed chromosomes, and in most organisms (but not yeast), the nuclear membrane breaks down. Cells with this appearance are in M phase(for mitosis). The other observable phase is S phase (for DNA synthesis). Cells in S phase can be marked by the incorporation of labeled thymidine into the nuclear DNA. These two phases are separated by two "gaps", G1and G2. G1 is a time of preparation for DNA synthesis in S phase, building up dNTPs and other components needed for replication. G2 follows completion of DNA replication and precedes the initiation of mitosis. Nonreplicating, or quiescent cells, can be considered to be "out of the cycle" or in a state referred to as G0. One can now separate cells by DNA content in a flow cytometer, allowing one to distinguish cells in G1, with a 2n chromosomal content, from those in G2 and early M, which have a 4n chromosomal content. Cells at progressive times during S phase have increasing DNA content.
Figure 6.17. The eukaryotic cell cycle
Passage from one phase to the next is a highly regulated event. Critical control points, or checkpoints, are found at the G1 to S transition and at the G2 to M transition. The checkpoint in late G1 is the time for the cell to assess whether it has enough nucleotides, proteins and other materials to make two cells. The checkpoint prior to the G2/M transition allows any necessary repairs or corrections in the DNA to be made prior to mitosis. Loss of control of the G0 to G1 transition, or at the other checkpoints, generates cells that grow in an uncontrolled manner. This inappropriate expansion in the number of cells is fundamental to the progression of cancers, and hence the study of the molecular events at these checkpoints is an intensely active area of research in cell biology and biochemistry. A full treatment of this important topic is beyond the scope of this course. In general, cell cycle progression is regulated by environmental signals (such as extracellular growth factors) and intracellular monitors of metabolic state, intactness of DNA, and so forth. These disparate signals eventually impinge on highly regulated protein kinases. Activation of particular protein kinases is required for progression through each checkpoint. In general, two types of regulation have been seen.
- Control of the amount of key proteins. The concentration of proteins called cyclins rise and fall through the cell cycle. Some of the cyclins are components of protein kinases whose activity regulates passage through the checkpoints. The cyclins must be present at a sufficiently high concentration for the kinase to be active.
- Control of the state of phosphorylation. Proteins regulating the cell cycle (as is true of many regulatory proteins) can be covalently modified, e.g. by phosphorylation in a process catalyzed by protein kinases. The state of phosphorylation will determine the level of activity of the protein. So for instance a key protein kinase regulating passage through the G1 to S checkpoint must have its catalytic subunit in the correct state of phosphorylation, as well as having sufficient amounts of its cyclin subunit.
Many lines of investigation are being pursued to understand better the regulation of the cell cycle. One fundamental approach has been be isolation of scores of conditional yeast mutants that are defective in their progression through the cell cycle at the restrictive temperature. These mutants have particular phenotypes depending on which stage of the cell cycle they arrest in under nonpermissive conditions. The complementation groups defined by such mutants are called CDC, for cell division cycle phenotypes, followed by a number. For example, a protein kinase whose activity is needed for both the G1/S and the G2/M transition in S. cerevisiaeis the product of the CDC28 gene, and the polypeptide is called Cdc28p.
Once a cell has entered S phase, each origin of replication must fire once, but only once. As discussed above, the ORC is required for initiation of replication at an origin, but what determines when the origin fires? This is a matter of considerable current study, and many of the details are still unknown. In S. cerevisiae, the ORC binds to specific DNA sequences, the origins of replication, throughout the cell cycle, not just during S phase when the origins are active. During G1 phase, ORC recruits other proteins, such as Cdc6 and Mcm (minichromosome maintenace) proteins, to form a prereplication complex. At the G1/S transition, additional factors associate with this complex, and a cyclin-dependent kinase (CDK) activity stimulates intitiation of replication in S phase. After initiation, the Cdc6 and Mcm proteins are released from the prereplication complex, leaving the ORC still bound to the origin but unable to reinitiate replication until the next cell cycle. In mammals, an intact ORC is not stably bound to the origin, but rather one of the subunit, ORC1, is recruited to the origin at a defined time during G1. However, in both yeast and mammals, events in G1 involving the preinitiation complex mark an origin for firing in the next S phase.
As discussed above, many origins of replication, and hence many replicons, are used to replicate each chromosome. These origins do not all fire at the same time. In fact, replicons can initiate at different times during S phase. Replicons containing genes that are actively expressed in a given cells tend to replicate earlier in S phase than do replicons containing nonexpressed genes. This is an example of tissue-specific variation in replication timing. The time during S phase at which a particular origin will fire is determined early in G1, at the time that chromatin domains are repositioned in the nucleus following mitosis and before the preinitiation complex forms. Events important to the regulation of initiation at replication origins occur at various times during G1, but the full range of proteins and activities carrying out these events is still a matter of study.