During what phase would you expect the amount of DNA in the cell to change?

The division cycle of most cells consists of four coordinated processes: cell growth, DNA replication, distribution of the duplicated chromosomes to daughter cells, and cell division. In bacteria, cell growth and DNA replication take place throughout most of the cell cycle, and duplicated chromosomes are distributed to daughter cells in association with the plasma membrane. In eukaryotes, however, the cell cycle is more complex and consists of four discrete phases. Although cell growth is usually a continuous process, DNA is synthesized during only one phase of the cell cycle, and the replicated chromosomes are then distributed to daughter nuclei by a complex series of events preceding cell division. Progression between these stages of the cell cycle is controlled by a conserved regulatory apparatus, which not only coordinates the different events of the cell cycle but also links the cell cycle with extracellular signals that control cell proliferation.

A typical eukaryotic cell cycle is illustrated by human cells in culture, which divide approximately every 24 hours. As viewed in the microscope, the cell cycle is divided into two basic parts: mitosis and interphase. Mitosis (nuclear division) is the most dramatic stage of the cell cycle, corresponding to the separation of daughter chromosomes and usually ending with cell division (cytokinesis). However, mitosis and cytokinesis last only about an hour, so approximately 95% of the cell cycle is spent in interphase—the period between mitoses. During interphase, the chromosomes are decondensed and distributed throughout the nucleus, so the nucleus appears morphologically uniform. At the molecular level, however, interphase is the time during which both cell growth and DNA replication occur in an orderly manner in preparation for cell division.

The cell grows at a steady rate throughout interphase, with most dividing cells doubling in size between one mitosis and the next. In contrast, DNA is synthesized during only a portion of interphase. The timing of DNA synthesis thus divides the cycle of eukaryotic cells into four discrete phases (Figure 14.1). The M phase of the cycle corresponds to mitosis, which is usually followed by cytokinesis. This phase is followed by the G1 phase (gap 1), which corresponds to the interval (gap) between mitosis and initiation of DNA replication. During G1, the cell is metabolically active and continuously grows but does not replicate its DNA. G1 is followed by S phase (synthesis), during which DNA replication takes place. The completion of DNA synthesis is followed by the G2 phase (gap 2), during which cell growth continues and proteins are synthesized in preparation for mitosis.

The duration of these cell cycle phases varies considerably in different kinds of cells. For a typical rapidly proliferating human cell with a total cycle time of 24 hours, the G1 phase might last about 11 hours, S phase about 8 hours, G2 about 4 hours, and M about 1 hour. Other types of cells, however, can divide much more rapidly. Budding yeasts, for example, can progress through all four stages of the cell cycle in only about 90 minutes. Even shorter cell cycles (30 minutes or less) occur in early embryo cells shortly after fertilization of the egg (Figure 14.2). In this case, however, cell growth does not take place. Instead, these early embryonic cell cycles rapidly divide the egg cytoplasm into smaller cells. There is no G1 or G2 phase, and DNA replication occurs very rapidly in these early embryonic cell cycles, which therefore consist of very short S phases alternating with M phases.

In contrast to the rapid proliferation of embryonic cells, some cells in adult animals cease division altogether (e.g., nerve cells) and many other cells divide only occasionally, as needed to replace cells that have been lost because of injury or cell death. Cells of the latter type include skin fibroblasts, as well as the cells of many internal organs, such as the liver, kidney, and lung. As discussed further in the next section, these cells exit G1 to enter a quiescent stage of the cycle called G0, where they remain metabolically active but no longer proliferate unless called on to do so by appropriate extracellular signals.

Analysis of the cell cycle requires identification of cells at the different stages discussed above. Although mitotic cells can be distinguished microscopically, cells in other phases of the cycle (G1, S, and G2) must be identified by biochemical criteria. Cells in S phase can be readily identified because they incorporate radioactive thymidine, which is used exclusively for DNA synthesis (Figure 14.3). For example, if a population of rapidly proliferating human cells in culture is exposed to radioactive thymidine for a short period of time (e.g., 15 minutes) and then analyzed by autoradiography, about a third of the cells will be found to be radioactively labeled, corresponding to the fraction of cells in S phase.

Variations of such cell labeling experiments can also be used to determine the length of different stages of the cell cycle. For example, consider an experiment in which cells are exposed to radioactive thymidine for 15 minutes, after which the radioactive thymidine is removed and the cells are cultured for varying lengths of time prior to autoradiography. Radioactively labeled interphase cells that were in S phase during the time of exposure to radioactive thymidine will be observed for several hours as they progress through the remainder of S and G2. In contrast, radioactively labeled mitotic cells will not be observed until 4 hours after labeling. This 4-hour lag time corresponds to the length of G2—the minimum time required for a cell that incorporated radioactive thymidine at the end of S phase to enter mitosis.

Cells at different stages of the cell cycle can also be distinguished by their DNA content (Figure 14.4). For example, animal cells in G1 are diploid (containing two copies of each chromosome), so their DNA content is referred to as 2n (n designates the haploid DNA content of the genome). During S phase, replication increases the DNA content of the cell from 2n to 4n, so cells in S have DNA contents ranging from 2n to 4n. DNA content then remains at 4n for cells in G2 and M, decreasing to 2n after cytokinesis. Experimentally, cellular DNA content can be determined by incubation of cells with a fluorescent dye that binds to DNA, followed by analysis of the fluorescence intensity of individual cells in a flow cytometer or fluorescence-activated cell sorter, thereby distinguishing cells in the G1, S, and G2/M phases of the cell cycle.

The progression of cells through the division cycle is regulated by extracellular signals from the environment, as well as by internal signals that monitor and coordinate the various processes that take place during different cell cycle phases. An example of cell cycle regulation by extracellular signals is provided by the effect of growth factors on animal cell proliferation. In addition, different cellular processes, such as cell growth, DNA replication, and mitosis, all must be coordinated during cell cycle progression. This is accomplished by a series of control points that regulate progression through various phases of the cell cycle.

A major cell cycle regulatory point in many types of cells occurs late in G1 and controls progression from G1 to S. This regulatory point was first defined by studies of budding yeast (Saccharomyces cerevisiae), where it is known as START (Figure 14.5). Once cells have passed START, they are committed to entering S phase and undergoing one cell division cycle. However, passage through START is a highly regulated event in the yeast cell cycle, where it is controlled by external signals, such as the availability of nutrients, as well as by cell size. For example, if yeasts are faced with a shortage of nutrients, they arrest their cell cycle at START and enter a resting state rather than proceeding to S phase. Thus, START represents a decision point at which the cell determines whether sufficient nutrients are available to support progression through the rest of the division cycle. Polypeptide factors that signal yeast mating also arrest the cell cycle at START, allowing haploid yeast cells to fuse with one another instead of progressing to S phase.

In addition to serving as a decision point for monitoring extracellular signals, START is the point at which cell growth is coordinated with DNA replication and cell division. The importance of this regulation is particularly evident in budding yeasts, in which cell division produces progeny cells of very different sizes: a large mother cell and a small daughter cell. In order for yeast cells to maintain a constant size, the small daughter cell must grow more than the large mother cell does before they divide again. Thus, cell size must be monitored in order to coordinate cell growth with other cell cycle events. This regulation is accomplished by a control mechanism that requires each cell to reach a minimum size before it can pass START. Consequently, the small daughter cell spends a longer time in G1 and grows more than the mother cell.

The proliferation of most animal cells is similarly regulated in the G1 phase of the cell cycle. In particular, a decision point in late G1, called the restriction point in animal cells, functions analogously to START in yeasts (Figure 14.6). In contrast to yeasts, however, the passage of animal cells through the cell cycle is regulated primarily by the extracellular growth factors that signal cell proliferation, rather than by the availability of nutrients. In the presence of the appropriate growth factors, cells pass the restriction point and enter S phase. Once it has passed through the restriction point, the cell is committed to proceed through S phase and the rest of the cell cycle, even in the absence of further growth factor stimulation. On the other hand, if appropriate growth factors are not available in G1, progression through the cell cycle stops at the restriction point. Such arrested cells then enter a quiescent stage of the cell cycle called G0, in which they can remain for long periods of time without proliferating. G0 cells are metabolically active, although they cease growth and have reduced rates of protein synthesis. As already noted, many cells in animals remain in G0 unless called on to proliferate by appropriate growth factors or other extracellular signals. For example, skin fibroblasts are arrested in G0 until they are stimulated to divide as required to repair damage resulting from a wound. The proliferation of these cells is triggered by platelet-derived growth factor, which is released from blood platelets during clotting and signals the proliferation of fibroblasts in the vicinity of the injured tissue.

Although the proliferation of most cells is regulated primarily in G1, some cell cycles are instead controlled principally in G2. One example is the cell cycle of the fission yeast Schizosaccharomyces pombe (Figure 14.7). In contrast to Saccharomyces cerevisiae, the cell cycle of S. pombe is regulated primarily by control of the transition from G2 to M, which is the principal point at which cell size and nutrient availability are monitored. In animals, the primary example of cell cycle control in G2 is provided by oocytes. Vertebrate oocytes can remain arrested in G2 for long periods of time (several decades in humans) until their progression to M phase is triggered by hormonal stimulation. Extracellular signals can thus control cell proliferation by regulating progression from the G2 to M as well as the G1 to S phases of the cell cycle.

The controls discussed in the previous section regulate cell cycle progression in response to cell size and extracellular signals, such as nutrients and growth factors. In addition, the events that take place during different stages of the cell cycle must be coordinated with one another so that they occur in the appropriate order. For example, it is critically important that the cell not begin mitosis until replication of the genome has been completed. The alternative would be a catastrophic cell division, in which the daughter cells failed to inherit complete copies of the genetic material. In most cells, this coordination between different phases of the cell cycle is dependent on a system of checkpoints and feedback controls that prevent entry into the next phase of the cell cycle until the events of the preceding phase have been completed.

Several cell cycle checkpoints function to ensure that incomplete or damaged chromosomes are not replicated and passed on to daughter cells (Figure 14.8). One of the most clearly defined of these checkpoints occurs in G2 and prevents the initiation of mitosis until DNA replication is completed. This G2 checkpoint senses unreplicated DNA, which generates a signal that leads to cell cycle arrest. Operation of the G2 checkpoint therefore prevents the initiation of M phase before completion of S phase, so cells remain in G2 until the genome has been completely replicated. Only then is the inhibition of G2 progression relieved, allowing the cell to initiate mitosis and distribute the completely replicated chromosomes to daughter cells.

Progression through the cell cycle is also arrested at the G2 checkpoint in response to DNA damage, such as that resulting from irradiation. This arrest allows time for the damage to be repaired, rather than being passed on to daughter cells. Studies of yeast mutants have shown that the same cell cycle checkpoint is responsible for G2 arrest induced by either unreplicated or damaged DNA, both of which signal cell cycle arrest through related pathways.

DNA damage not only arrests the cell cycle in G2, but also slows the progression of cells through S phase and arrests cell cycle progression at a checkpoint in G1. This G1 arrest may allow repair of the damage to take place before the cell enters S phase, where the damaged DNA would be replicated. In mammalian cells, arrest at the G1 checkpoint is mediated by the action of a protein known as p53, which is rapidly induced in response to damaged DNA (Figure 14.9). Interestingly, the gene encoding p53 is frequently mutated in human cancers. Loss of p53 function as a result of these mutations prevents G1 arrest in response to DNA damage, so the damaged DNA is replicated and passed on to daughter cells instead of being repaired. This inheritance of damaged DNA results in an increased frequency of mutations and general instability of the cellular genome, which contributes to cancer development. Mutations in the p53 gene are the most common genetic alterations in human cancers (see Chapter 15), illustrating the critical importance of cell cycle regulation in the life of multicellular organisms.

Another important cell cycle checkpoint that maintains the integrity of the genome occurs toward the end of mitosis (see Figure 14.8). This checkpoint monitors the alignment of chromosomes on the mitotic spindle, thus ensuring that a complete set of chromosomes is distributed accurately to the daughter cells. For example, the failure of one or more chromosomes to align properly on the spindle causes mitosis to arrest at metaphase, prior to the segregation of the newly replicated chromosomes to daughter nuclei. As a result of this checkpoint, the chromosomes do not separate until a complete complement of chromosomes has been organized for distribution to each daughter cell.

The G2 checkpoint prevents the initiation of mitosis prior to the completion of S phase, thereby ensuring that incompletely replicated DNA is not distributed to daughter cells. It is equally important to ensure that the genome is replicated only once per cell cycle. Thus, once DNA has been replicated, control mechanisms must exist to prevent initiation of a new S phase prior to mitosis. These controls prevent cells in G2 from reentering S phase and block the initiation of another round of DNA replication until after mitosis, at which point the cell has entered the G1 phase of the next cell cycle.

Initial insights into this dependence of S phase on M phase came from cell fusion experiments of Potu Rao and Robert Johnson in 1970 (Figure 14.10). These investigators isolated cells in different phases of the cycle and then fused these cells to each other to form cell hybrids. When G1 cells were fused with S phase cells, the G1 nucleus immediately began to synthesize DNA. Thus, the cytoplasm of S phase cells contained factors that initiated DNA synthesis in the G1 nucleus. Fusing G2 cells with S phase cells, however, yielded a quite different result: The G2 nucleus was unable to initiate DNA synthesis even in the presence of an S phase cytoplasm. It thus appeared that DNA synthesis in the G2 nucleus was prevented by a mechanism that blocked rereplication of the genome until after mitosis had taken place.

The molecular mechanism that restricts DNA replication to once per cell cycle involves the action of a family of proteins (called MCM proteins) that bind to replication origins together with the origin replication complex (ORC) proteins (see Figure 5.17). The MCM proteins act as “licensing factors” that allow replication to initiate (Figure 14.11). Their binding to DNA is regulated during the cell cycle such that the MCM proteins are only able to bind to replication origins during G1, allowing DNA replication to initiate when the cell enters S phase. Once initiation has occurred, however, the MCM proteins are displaced from the origin, so replication cannot initiate again until the cell passes through mitosis and enters G1 phase of the next cell cycle.