15.5: Pre-mitotic Phases

G₁/G₀ Phase

The G₁ phase is the state a cell is in immediately following cytokinesis. At that point, the cells will be somewhat undersized, and need to take up materials and energy sources, and convert them to cellular components in order to support the eventual cell division. During this time, the cell goes about doing its “normal” business - an endocrine cell makes and secretes hormones, an intestinal epithelial cell absorbs nutrients from the gut and passes them on to the bloodstream, a neuron conducts signals, etc. Most types of cells spend the majority of their cycle in G₁, although there are exceptions, such as the frog oocytes mentioned earlier. The length of G₁ is generally constant for a given cell type under normal conditions, but can vary greatly between different cell types. Post-mitotic cells, which have left the cell cycle and will no longer divide, are in G₁ until they die, barring reactivation of the cell cycle by stress conditions. This continuous G₁-like state is referred to as G₀.

For those cells preparing to move from G₁ into S, cyclins D and E, and cdk 2, 4, and 6 predominate, with activation of cyclin D complexes preceding activation of cyclin E complexes. Two major questions are asked by the cell: is the DNA undamaged and complete, and is the extracellular environment favorable for cell division? The cellular sensors for these conditions then link to cyclin complexes effect restriction points on cell cycle progression. The extracellular environment questions can be a tricky one, because this can include more than just assessment of nutrient availability or predatory threats; it can also be a requirement for an external trigger such as a mitogenic hormone or paracrine signal. In fact, nearly all normal animal cells require an extracellular signal to progress through the G1/S checkpoint. The cyclin E/cdk2 combination is the principal regulator of entry into S phase and DNA replication.

The active cyclin E/cdk2 complex phosphorylates the tumor suppressor protein Rb (retinoblastoma), which causes E2F to translocate to the nucleus and turn on genes needed for entry into S phase.

S phase

The mechanisms of DNA replication were discussed in Chapter 7. It is important to note that once a cell has entered S phase, it has essentially committed to going through cell division. Cells do not cope well with extra copies of chromosomes, and a cell that went through S phase without going through mitosis would likely have major malfunctions in gene regulation. For similar reasons, the cell must only undergo DNA replication once per cell division. The cyclinA/ cdk2 complex plays a key role in initiation of repli- cation by activating the pre-replicative complex. It also phosphorylates cdc6, causing it to dissociate from the ORC, and consequently the rest of the pre-RC. This prevents immediate re-use of this origin of replication, and since the phosphorylation of cdc6 allows it to be recognized by a ubiquitin ligase complex, it is tagged for proteolysis In addition to DNA replication, S phase is also the cell cycle stage in which centrosomes are duplicated in animal cells. The cyclin E/cdk2 combination licenses the duplication of centrosome, phosphorylating nucleophosmin, which then dissociates from the cen- trosome. This helps to trigger the centrosome duplication. Nucleophosmin does not reassociate with centrosomes until telophase, when it is no longer phosphorylated. Plk4 (Polo-family kinase 4) activity is necessary for centriole duplication, and appears to initiate the centriole assembly mechanism.

The ubiquitin ligase complex, SCF, is made up of three major proteins and several minor species. Skp1 (S-phase kinase-associated protein 1) can be an RNA polymerase elongation factor, but in this complex links the other two proteins together. Cul1 (Cullin 1) is an E3 type ubiquitin ligase. Finally, an F-box family protein like Rbx1 (Ring-box 1), that heterodimerizes with cullin-1 and may also recruit E2 ubiquitinating enzyme.. In addition to cdc6, it also recognizes and ubiquitinates CKIs (cyclin complex kinase inhibitors) such as p27, which is involved in processes such as DNA repair and error-checking.

G₂ Phase

The G₂ phase begins when DNA replication has completed. Having said that, before the cell is allowed out of G₂ and on to M phase, it must pass a DNA fidelity checkpoint, ensuring that not only has replication been fully completed, but that there are no major errors. G₂ is a relatively short phase (compared to G₁) in most cell types, and it is spent building up energy and material stores for cell division and checking the DNA. If everything is ok, and the cyclin B/cdk1 complex has been activated, the cell proceeds to M phase.