To study the role of
To study the role of DNA–PK in the response to replication arrest, we used the DNA replication inhibitor aphidicolin (APH). APH, a mycotoxin isolated from Cephalosporium aphidicola, inhibits DNA replication by interacting with the replicating DNA polymerase α (pol α). APH specifically inhibits the activity of replicating DNA polymerases in eukaryotic cells while not affecting other metabolic pathways, such as RNA, protein, and nucleotide biosynthesis.20., 21., 22. APH forms a pol α-DNA–APH ternary complex that does not inhibit the primase activity of the pol α-primase complex but inhibits the elongation step of DNA pol α, δ, and ε.24., 25. APH preferentially blocks dCTP incorporation.22., 26., 27. APH inhibits S-phase progression but allows cells in G2, M, and G1 to continue their growth cycle. High levels of APH completely inhibit DNA replication and induce a DNA damage S-phase checkpoint that requires the activation of Chk1. However, lower levels of APH decrease the rate of fork progression without activating checkpoints.
Discussion Long treatments with APH are known to generate DSBs49., 50., 51. and activate fragile site expression. In contrast, short treatments with APH (less than 1 h) are thought to inhibit replication fork progression without induction of DSBs.53., 54. The data reported here reveal that a short (10 min) treatment with APH rapidly activated transient γ-H2AX foci, which mark DSBs, in an ATR-dependent manner. This surge of γ-H2AX foci could occur in cells that had been treated with a Chk1 inhibitor, suggesting that γ-H2AX foci formation did not depend on the activation of the S-phase checkpoint through the Chk1-mediated signaling cascade. In cells with normal DNA–PK, γ-H2AX foci induced by low level of APH rapidly disappeared, indicating that the initial group of DSBs was repaired without inducing a influenza m2 protein checkpoint and that the subsequent low progression of replication forks did not lead to further induction of DSBs. Our data further indicate that DSBs were recognized by the Ku protein and that the reduction of γ-H2AX levels after exposure to a low dose of APH required DNA–PK activity. These observations suggest that an activity of DNA-PK, most likely the activation of the NHEJ pathway, rapidly repaired APH-induced DNA DSBs when damage was low. It is likely that the induction of a cell cycle checkpoint by ATR only occurred when DNA–PK activity was absent or insufficient to tackle the damage. The data reported here suggest that the ATR kinase is primarily responsible for the surge of DSBs and the inhibition of DNA replication after exposure to low levels of APH. By contrast, the cellular response to other drugs that inhibit DNA replication such as CPT are mediated primarily by ATM, suggesting that the two responses are activated by different lesions. Exposure to CPT in actively replicating cells directly forms DSBs that trigger the ATM-mediated damage response and can be prevented by APH, which inhibits replication and prevents the formation of replication-mediated DNA breaks. As shown here, cells with functional DNA–PK do not exhibit DNA breaks in the presence of mild doses of APH. In those cells, replication is inhibited by APH and no further DSBs are formed after the repair of the first surge of DSBs. This repair process, coupled with APH-induced inhibition of DNA replication, facilitates prevention of replication-dependent DNA breaks after exposure to CPT. Cells deficient in DNA–PK were not deficient in the activation of the S-phase checkpoint, which suppresses origin firing and stalls replication forks. However, these cells exhibited an increased sensitivity to low doses of APH and a lower threshold (0.1 μg/ml) beyond which the ATR-Chk1-mediated S-phase checkpoint was activated. These studies are consistent with the observation that DNA–PK inhibition strongly activates Chk1 and Chk2 after ionizing radiation that leads to sustained G2 arrest after DNA damage. Our studies are also consistent with the observed phosphorylation of DNA–PK, which correlates with its activation. DNA–PK deficient cells were reported to be highly sensitive to low doses of ionizing radiation56., 57. and were also sensitive to the replication inhibitor hydroxyurea.7., 19. Our data demonstrate that DNA–PKcs-deficient cells were hypersensitive to APH but only at low doses (below 1 μg/ml). We further demonstrated that exposure to APH recruits the regulatory subunit of DNA–PK, Ku, to chromatin regardless of DNA–PKcs status. In cells with an active DNA–PK, exposure to APH triggered the phosphorylation of the catalytic subunit of DNA–PK. Activation of DNA–PK occurred before activation of Chk1 and was not inhibited by the Chk1 inhibitor, UCN-01. It is plausible that at low levels of APH, DNA–PK repaired DSBs by NHEJ before the activation of Chk1-mediated S-phase checkpoint. In DNA–PKcs-deficient cells, damage sensors detected persistent DSBs and triggered the S-phase checkpoint, possibly because the NHEJ pathway was not activated. Consistent with this, replication elongation continued slowly after exposure to low levels of APH in cells with an active DNA–PK but forks completely stalled when cells were exposed to 10 μg/ml APH, suggesting that high levels of DNA damage that were not repaired by NHEJ triggered the Chk1-mediated S-phase checkpoint. These data are in concert with the observation that the yeast S-phase tolerates a low level of damage-like structures and requires a threshold level of DNA damage to activate the checkpoint response that prevents late replication.