CHK1 activity is required for continuous replication fork elongation but not stabilization of post-replicative gaps after UV irradiation

Abstract

Ultraviolet (UV)-induced DNA damage causes an efficient block of elongating replication forks. The checkpoint kinase, CHK1 has been shown to stabilize replication forks following hydroxyurea treatment. Therefore, we wanted to test if the increased UV sensitivity caused by the unspecific kinase inhibitor caffeine—inhibiting ATM and ATR amongst other kinases—is explained by inability to activate the CHK1 kinase to stabilize replicative structures. For this, we used cells deficient in polymerase η (Polη), a translesion synthesis polymerase capable of properly bypassing the UV-induced cis–syn TT pyrimidine dimer, which blocks replication. These cells accumulate gaps behind progressing replication forks after UV exposure. We demonstrate that both caffeine and CHK1 inhibition, equally retards continuous replication fork elongation after UV treatment. Interestingly, we found more pronounced UV-sensitization by caffeine than with the CHK1 inhibitor in clonogenic survival experiments. Furthermore, we demonstrate an increased collapse of replicative structures after caffeine treatment, but not after CHK1 inhibition, in UV-irradiated cells. This demonstrates that CHK1 activity is not required for stabilization of gaps induced during replication of UV-damaged DNA. These data suggest that elongation and stabilization of replicative structures at UV-induced DNA damage are distinct mechanisms, and that CHK1 is only involved in replication elongation.

RECQ1 is required for cellular resistance to replication stress and catalyzes strand exchange on stalled replication fork structures

“…40 CHK1 suppresses origin firing during S-phase in response to replication blocks 41 and is required for continuous replication fork elongation. 42 Consistently, a defect in the frequency of origin firing was reported after transient depletion of RECQ1 along with a decrease in replication fork progression rate. 10 Phosphorylation of RPA and activation of CHK1 are also considered important consequences for the cellular signaling in response to DNA damage.…”

Targeting the ATR/CHK1 Axis with PARP Inhibition Results in Tumor Regression in BRCA-Mutant Ovarian Cancer Models

“…The direct substrates of ATR include RPA, CLSPN, MCM2, p53 and many other factors that play roles in replication fork progression, DNA repair, and the cell cycle(19,20). Thus, ATR may be able to stabilize replication forks independent of CHK1(40), and permit cell survival when CHK1 is inhibited(41). Additionally, ATR can suppress origin firing and the intra-S checkpoint independent of CHK1(42,43).…”

The natural anticancer compound rocaglamide selectively inhibits the G1-S-phase transition in cancer cells through the ATM/ATR-mediated Chk1/2 cell cycle checkpoints

“…To measure the time resolved gene responses to Roc-A, RNA was isolated from Roc-A-treated (50 nM) normal T and Jurkat T cells at t 5 [0, 1,2,3,4,6,8,10,14,20,28,36] and t 5 [0. 5,1,2,3,4,5,6,7,8,10,12,14,16,20,24,28,32,36] hr, respectively. The quality of total RNA was controlled with an Agilent 2100 Bioanalyzer (Agilent Technologies GmbH, Berlin, Germany).…”

“…To further verify the role of ATM/ATR-Chk1/Chk2 in Roc-A-mediated degradation of Cdc25A, we carried out an experiment using the ATM/ATR inhibitor caffeine 20,21 (Fig. 3a and 3b).…”