G2-M DNA damage checkpoint
Encyclopedia
The G2-M DNA damage checkpoint is an important cell cycle
checkpoint in eukaryotic organisms ranging from yeast to mammals. This checkpoint insures that cells don't initiate mitosis
before they have a chance to repair damaged DNA after replication. Cells that have a defective G2-M checkpoint enter mitosis before repairing their DNA, leading to death after cell division.
The cell cycle is driven by proteins called cyclin dependent kinases that associate with cyclin
regulatory proteins at different points of the cell cycle. Accumulation of cyclin B
increases the activity of the cyclin dependent kinase cdc2 as cells prepare to enter mitosis. Cdc2 activity is further regulated by phosphorylation
of its tyrosine-15 residue by the kinase wee1
. Phosphorylation of tyrosine-15 inhibits cdc2 activity while dephosphorylation by the phosphatase cdc25
activates the mitotic kinase.
Proteins that localize to sites of DNA damage in G2
initiate a signaling cascade that regulates wee1 and cdc25 activity, therefore controlling mitotic entry via cdc2-cyclin B. Delay in mitotic entry is important for cells to repair any DNA damage that may have accumulated after S phase
. Absence of wee1 or removal of the tyrosine-15 site removes negative regulation of cdc2 activity and causes cells to enter mitosis without completing repair, which effectively abolishes the G2-M checkpoint Absence of cdc25 arrests cells in G2, but still allows activation of the G2-M checkpoint, implicating activation of wee1 and deactivation of cdc25 as important regulatory steps in the checkpoint.
Proteins that function in the G2-M checkpoint were originally identified in yeast screens that looked for mutants which show enhanced sensitivity to radiation, termed "rad" mutants . Inefficient repair of DNA damaged by ionizing radiation or chemical agents in these mutants revealed proteins essential in this pathway. Early signaling proteins in the checkpoint pathway are members of a family of phosphotidylinositol 3-kinases, rad3 in yeast and ATR
in vertebrates, that are believed to localize to sites of DNA damage. Rad3 phosphorylates rad26 which is required to initiate, but not maintain the checkpoint. Rad3 also phosphorylates a number of other proteins whose absence abolishes checkpoint DNA repair, including rad1, rad9, hus1 and rad17. It has been hypothesized that rad9, hus 1 and rad 17 are similar to proteins involved in forming the clamp that increases the processivity
of DNA polymerase
during DNA replication.. In agreement with this idea, rad17 is similar to proteins involved in loading the clamp onto DNA. This supports a model where phosphorylation by rad3 causes recruitment of these proteins
to sites of DNA damage where they mediate the activity of DNA polymerases involved in DNA repair
.
The main rad3 effector is the kinase chk1 which is required for the G2-M arrest in response to DNA damaging agents. This kinase is phosphorylated by rad3 between S phase and mitosis, implicating its specific role in G2 arrest. Overexpression of chk1 also rescues the radiation sensitivity of rad mutants, presumably by allowing DNA repair to take place before entry into mitosis. This effect is dependent on the presence of wee1 and cdc25 which places chk1 upstream of these direct regulators of cyclin dependent kinase. Phophorylation activates chk1 which then phosphorylates wee1, increasing its stability.. Activated chk1 also phosphorylates cdc25, either leading to its inactivation or preventing its localization to the nucleus. An increasingly stable wee1 further inhibits cdc2 activity while an inactive cdc25 prevents removal of this inhibition, resulting in a strong G2 arrest .
Rad3 is required for activation of chk1 and initiation of G2 arrest but different proteins are believed to maintain G2 arrest so that DNA repair can occur. One such protein is rad18 that is required for G2 arrest even when chk1 is phosphorylated and active. This protein is also involved in DNA repair since rad18 mutants aren't able to repair DNA even when G2 arrest is prolonged by other means.
Cell cycle
The cell cycle, or cell-division cycle, is the series of events that takes place in a cell leading to its division and duplication . In cells without a nucleus , the cell cycle occurs via a process termed binary fission...
checkpoint in eukaryotic organisms ranging from yeast to mammals. This checkpoint insures that cells don't initiate mitosis
Mitosis
Mitosis is the process by which a eukaryotic cell separates the chromosomes in its cell nucleus into two identical sets, in two separate nuclei. It is generally followed immediately by cytokinesis, which divides the nuclei, cytoplasm, organelles and cell membrane into two cells containing roughly...
before they have a chance to repair damaged DNA after replication. Cells that have a defective G2-M checkpoint enter mitosis before repairing their DNA, leading to death after cell division.
The cell cycle is driven by proteins called cyclin dependent kinases that associate with cyclin
Cyclin
Cyclins are a family of proteins that control the progression of cells through the cell cycle by activating cyclin-dependent kinase enzymes.- Function :...
regulatory proteins at different points of the cell cycle. Accumulation of cyclin B
Cyclin B
Cyclin B is a member of the cyclin family.Cyclin B is a mitotic cyclin. The amount of cyclin B and the activity of the cyclin B-Cdk complex rise through the cell cycle until mitosis, where they fall abruptly due to degradation of cyclin B...
increases the activity of the cyclin dependent kinase cdc2 as cells prepare to enter mitosis. Cdc2 activity is further regulated by phosphorylation
Phosphorylation
Phosphorylation is the addition of a phosphate group to a protein or other organic molecule. Phosphorylation activates or deactivates many protein enzymes....
of its tyrosine-15 residue by the kinase wee1
Wee1
Wee1 is a nuclear kinase belonging to the Ser/Thr family of protein kinases in the fission yeast Schizosaccharomyces pombe . It has a molecular mass of 96 kDa and it is a key regulator of cell cycle progression....
. Phosphorylation of tyrosine-15 inhibits cdc2 activity while dephosphorylation by the phosphatase cdc25
Cdc25
Cdc25 is a dual-specificity phosphatase first isolated from the yeast Schizosaccharomyces pombe as a cell cycle defective mutant. As with other cell cycle proteins such as Cdc2 and Cdc4, the "cdc" in its name refers to "cell division cycle".Dual-specificity phosphatases are considered a sub-class...
activates the mitotic kinase.
Proteins that localize to sites of DNA damage in G2
G2
G2, G02, G.II, G II, or G-2 may refer to:-Media:* G2, The Guardian weekday supplement* Gladiators 2000, 1994 spinoff of American Gladiators-Fiction:...
initiate a signaling cascade that regulates wee1 and cdc25 activity, therefore controlling mitotic entry via cdc2-cyclin B. Delay in mitotic entry is important for cells to repair any DNA damage that may have accumulated after S phase
S phase
S-phase is the part of the cell cycle in which DNA is replicated, occurring between G1 phase and G2 phase. Precise and accurate DNA replication is necessary to prevent genetic abnormalities which often lead to cell death or disease. Due to the importance, the regulatory pathways that govern this...
. Absence of wee1 or removal of the tyrosine-15 site removes negative regulation of cdc2 activity and causes cells to enter mitosis without completing repair, which effectively abolishes the G2-M checkpoint Absence of cdc25 arrests cells in G2, but still allows activation of the G2-M checkpoint, implicating activation of wee1 and deactivation of cdc25 as important regulatory steps in the checkpoint.
Proteins that function in the G2-M checkpoint were originally identified in yeast screens that looked for mutants which show enhanced sensitivity to radiation, termed "rad" mutants . Inefficient repair of DNA damaged by ionizing radiation or chemical agents in these mutants revealed proteins essential in this pathway. Early signaling proteins in the checkpoint pathway are members of a family of phosphotidylinositol 3-kinases, rad3 in yeast and ATR
ATR
ATR is an Italian-French aircraft manufacturer headquartered on the grounds of Toulouse Blagnac International Airport in Blagnac, France surburb of Toulouse. It was formed in 1981 by Aérospatiale of France and Aeritalia of Italy...
in vertebrates, that are believed to localize to sites of DNA damage. Rad3 phosphorylates rad26 which is required to initiate, but not maintain the checkpoint. Rad3 also phosphorylates a number of other proteins whose absence abolishes checkpoint DNA repair, including rad1, rad9, hus1 and rad17. It has been hypothesized that rad9, hus 1 and rad 17 are similar to proteins involved in forming the clamp that increases the processivity
Processivity
In molecular biology, processivity is a measure of the average number of nucleotides added by a DNA polymerase enzyme per association/disassociation with the template. DNA polymerases associated with DNA replication tend to be highly processive, while those associated with DNA repair tend to have...
of DNA polymerase
DNA polymerase
A DNA polymerase is an enzyme that helps catalyze in the polymerization of deoxyribonucleotides into a DNA strand. DNA polymerases are best known for their feedback role in DNA replication, in which the polymerase "reads" an intact DNA strand as a template and uses it to synthesize the new strand....
during DNA replication.. In agreement with this idea, rad17 is similar to proteins involved in loading the clamp onto DNA. This supports a model where phosphorylation by rad3 causes recruitment of these proteins
to sites of DNA damage where they mediate the activity of DNA polymerases involved in DNA repair
DNA repair
DNA repair refers to a collection of processes by which a cell identifies and corrects damage to the DNA molecules that encode its genome. In human cells, both normal metabolic activities and environmental factors such as UV light and radiation can cause DNA damage, resulting in as many as 1...
.
The main rad3 effector is the kinase chk1 which is required for the G2-M arrest in response to DNA damaging agents. This kinase is phosphorylated by rad3 between S phase and mitosis, implicating its specific role in G2 arrest. Overexpression of chk1 also rescues the radiation sensitivity of rad mutants, presumably by allowing DNA repair to take place before entry into mitosis. This effect is dependent on the presence of wee1 and cdc25 which places chk1 upstream of these direct regulators of cyclin dependent kinase. Phophorylation activates chk1 which then phosphorylates wee1, increasing its stability.. Activated chk1 also phosphorylates cdc25, either leading to its inactivation or preventing its localization to the nucleus. An increasingly stable wee1 further inhibits cdc2 activity while an inactive cdc25 prevents removal of this inhibition, resulting in a strong G2 arrest .
Rad3 is required for activation of chk1 and initiation of G2 arrest but different proteins are believed to maintain G2 arrest so that DNA repair can occur. One such protein is rad18 that is required for G2 arrest even when chk1 is phosphorylated and active. This protein is also involved in DNA repair since rad18 mutants aren't able to repair DNA even when G2 arrest is prolonged by other means.