Question

Single-stranded DNA and unreplicated DNA are recognized by ATR, which leads to G2 arrest. What would likely occur with a single-stranded DNA break in a cell with a mutation that prevented phosphorylation of Chk1? What if the mutation prevented the phosphorylation of Chk2?

Answer 1

ATM and ATR are DNA damage response kinases (DDR kinases) belonging to phosphatidylinositol-3-kinase-like kinase family (PIKKs). ATR and ATM carry out phosphorylation of numerous proteins upon DNA damage that causes single stranded or double stranded breaks. ATR or ATM further phosphorylates proteins by activating protein kinases such as Chk1, Chk2 protein kinases.

When there is single stranded breaks in DNA, ATR is activated. It will localize to site of the damage. Its kinase activity is enhanced. Double stranded breaks will cause activation of ATM.

ATR activates CHK1 and not ChK2 in single stranded breaks. Cdc25A encodes a tyrosine phosphorylase that controls the phosphorylation of cdc2. Cdc2 is a catalytic subunit of maturation promoting factor MPF (CDK/cyclin B). In G2 phase, the cyclin-CDK prepares the cell to enter mitosis. Cyclin B increases the expression of cdc2. Cyclin B-Cdc2 will activate cdc25A, which then inactivates Wee1 and Myt1, which are inhibitors. Cdc25A will cause dephosphorylation, leading to activation of cyclin B-cdc2 complex. This activation will cause entry to M phase and induces mitosis. Phosphorylated Chk1 will induce phosphorylation of Cdc25 at an inhibitory site S216. This leads to inhibition of activity of cdc2. As a result, the cyclin B-cdc2 is inactive. Hence, mitosis will be inhibited and there is G2 arrest. The mutation in phosphorylation of chk1 and Chk2 by ATM will inhibit the phosphorylation on S216 site on cdc25. As a result, cyclin B-cdc2 complex will be activated via the cdc25. Cdc25 will inactivate Wee 1. This will cause entry to M phase. The cell cycle will continue as apoptosis is inhibited. Due to increased cell division, there are increase chances of tumorigenesis. There will be no DNA repair seen in this case.

ATM activates Chk2 and inhibits cDC25A during double stranded break. This ChK2 activation is not seen in single stranded break. Hence, phosphorylation of CDC25A by CHK2 is important for double stranded break. Hence, loss of phosphorylation of ChK2 will have no effect on single stranded break related G2 arrest. This is because there is not much role of ChK2 in DNA repair of single stranded breaks.