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G2 phase, or Gap 2 phase, is the third subphase of interphase in the cell cycle directly preceding mitosis. It follows the successful completion of S phase, during which the cell’s DNA is replicated. G2 phase ends with the onset of prophase, the first phase of mitosis in which the cell’s chromatin condenses into chromosomes.

G2 phase is a period of rapid cell growth and protein synthesis during which the cell prepares itself for mitosis. Curiously, G2 phase is not a necessary part of the cell cycle, as some cell types (particularly young Xenopus embryos [1] and some cancers [2]) proceed directly from DNA replication to mitosis. Though much is known about the genetic network which regulates G2 phase and subsequent entry into mitosis, there is still much to be discovered concerning its significance and regulation, particularly in regards to cancer. One hypothesis is that the growth in G2 phase is regulated as a method of cell size control. Fission yeast (Schizosaccharomyces pombe) has been previously shown to employ such a mechanism, via Cdr2-mediated spatial regulation of Wee1 activity.[3] Though Wee1 is a fairly conserved negative regulator of mitotic entry, no general mechanism of cell size control in G2 has yet been elucidated.

Biochemically, the end of G2 phase occurs when a threshold level of active cyclin B1/CDK1 complex, also known as Maturation promoting factor (MPF) has been reached.[4] The activity of this complex is tightly regulated during G2. In particular, the G2 checkpoint arrests cells in G2 in response to DNA damage through inhibitory regulation of CDK1. It is false to say Go resides in G2 phase.


Homologous recombinational repairEdit

During mitotic S phase, DNA replication produces two nearly identical sister chromatids. DNA double-strand breaks that arise after replication has progressed or during the G2 phase can be repaired before cell division occurs (M-phase of the cell cycle). Thus, during the G2 phase, double-strand breaks in one sister chromatid may be repaired by homologous recombinational repair using the other intact sister chromatid as template.[5]

G2/M checkpointEdit

In vertebrate cells, the G2/M DNA damage checkpoint consists of an arrest of the cell in G2 just before mitotic entry in response to genotoxic stress (such as UV radiation, oxidative stress, DNA intercalating agents, etc.) in both a p53-dependent and p53-independent manner.[6] DNA damage signals cause activation of the transcription factor p53. CDK1 is directly inhibited by three transcriptional targets of p53: p21, Gadd45, and 14-3-3σ. Inactive Cyclin B1/CDK1 is sequestered in the nucleus by p21,[7] while active Cyclin B1/CDK1 complexes are sequestered in the cytoplasm by 14-3-3σ.[6] Gadd45 disrupts the binding of Cyclin B1 and CDK1 through direct interaction with CDK1. P53 also transcriptionally represses CDK1.[6]

P53-independent G2 arrest is mainly affected through the actions of Chk1 kinase. DNA damage is sensed by ATM and ATR (Rad3 and Mec1 in yeast), which then signal to Chk1 and Chk2. Chk1 then mediates the degradation of cdc25A, an activator of CDK1.[8] ATR/ATM also activate p53, indicating that these pathways may act synergistically in regulating G2 arrest.[6]

Both p53-dependent and p53-independent cell cycle arrest are not specific to G2; these same proteins function upstream in DNA damage checkpoints in G1 and S phase as well. In yeast, which has no p53 homolog, G2 arrest functions through the p53-independent pathway.[6]

End of G2/entry into mitosisEdit

Mitotic entry is determined by a threshold level of active cyclin B1/CDK1 complex. In vertebrates, there are five cyclin B isoforms (B1, B2, B3, B4, and B5), but specific role of each of these isoforms in regulating mitotic entry is still unclear. It is known that cyclin B1 can compensate for loss of both cyclin B2 (and vice versa in Drosophila). Cyclin B1/CDK1 activity is regulated both spatially and temporally during G2 phase to ensure proper entry into mitosis.[9]

Cyclin B1 transcription begins at the end of S phase after DNA replication. Its promoter contains consensus binding sequences for a number of transcription factors, including p53, p21, Ets, Ap-1, NF-Y, c-Myc, TFE3, and USF.[9] Cyclin B1 accumulates in the cytoplasm throughout G2, where it binds to and activates CDK1's kinase activity. CDK1 activity is modulated primarily through regulation of its inhibitory phosphorylation sites at Thr14 and Tyr15. Wee1 and Myt1 phosphorylate these two residues, with Wee1 acting on the Tyr15 site and Myt1 acting predominantly on the Thr14 site. However, Myt1 has a separate inhibitory effect on CDK1; it can also sequester CDK1 in the cytoplasm via interaction with Myt1's C-terminal domain.[10] CDK1 is dephosphorylated primarily through the actions of Cdc25, which can dephosphorylate both the Thr14 and Tyr15 residues of CDK1.[10] There are three isoforms of Cdc25 (A, B, and C) in mammalian cells, all of which have been shown to have roles in regulation of G2 phase.[9][10]

CDK1, in turn, phosphorylates and modulates the activity of Wee1 and the Cdc25 isoforms A and C. Specifically, CDK1 phosphorylation inhibits Wee1 kinase activity,[9] activates Cdc25C phosphatase activity, and stabilizes Cdc25A.[11] Thus, CDK1 forms a positive feedback loop with Cdc25 and a double negative feedback loop with Wee1 (essentially a net positive feedback loop). These loops encode a hysteretic bistable switch in CDK1 activity relative to cyclin B1 levels. It is thought that this hysteretic behavior ensures that cells commit to mitosis even if cyclin B1 levels falter.[4]

In mammals, cyclin B1/CDK1 translocation to the nucleus is activated by phosphorylation of five serine sites on cyclin B1's cytoplasmic retention site (CRS): S116, S26, S128, S133, and S147. In Xenopus laevis, cyclin B1 contains four analogous CRS serine phosphorylation sites (S94, S96, S101, and S113) indicating that this mechanism is highly conserved. Nuclear export is also inactivated by phosphorylation of cyclin B1's nuclear export signal (NES).[9] The regulators of these phosphorylation sites are still largely unknown but several factors have been identified, including extracellular signal-regulated kinases (ERKs), PLK1, and CDK1 itself.[9][12] Upon reaching some threshold level of phosphorylation, translocation of cyclin B1/CDK1 to the nucleus is extremely rapid.[13] Once in the nucleus, cyclin B1/CDK1 phosphorylates many targets in preparation for mitosis, including histone H1, nuclear lamins, centrosomal proteins, and microtubule associated proteins (MAPs).[9]

Recently, evidence has emerged suggesting a more important role for cyclin A2/CDK complexes in regulating entry into mitosis. Cyclin A2/CDK2 activity begins in early S phase and increases during G2.[14] Cdc25B has been shown to dephosphorylate Tyr15 on CDK2 in early-to-mid G2 in a manner similar to the aforementioned CDK1 mechanism.[15] Downregulation of cyclin A2 in U2OS cells increases Wee1 activity and lowers Plk1 and Cdc25C activity.[16] However, cyclin A2/CDK complexes do not function strictly as activators of cyclin B1/CDK1 in G2, as CDK2 has been shown to be required for activation of the p53-independent G2 checkpoint activity, perhaps through a stabilizing phosphorylation on Cdc6.[17] CDK2-/- cells also have aberrantly high levels of Cdc25A.[17] Cyclin A2/CDK1 has also been shown to mediate proteosomal destruction of Cdc25B.[18] These pathways are often deregulated in cancer.[16]


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