WRN helicase regulates the ATR–CHK1-induced S-phase checkpoint pathway in response to topoisomerase-I–DNA covalent complexes

Abstract

Checkpoints are cellular surveillance and signaling pathways that coordinate the response to DNA damage and replicative stress. Consequently, failure of cellular checkpoints increases susceptibility to DNA damage and can lead to profound genome instability. This study examines the role of a human RECQ helicase, WRN, in checkpoint activation in response to DNA damage. Mutations in WRN lead to genomic instability and the premature aging condition Werner syndrome. Here, the role of WRN in a DNA-damage-induced checkpoint was analyzed in U-2 OS (WRN wild type) and isogenic cells stably expressing WRN-targeted shRNA (WRN knockdown). The results of our studies suggest that WRN has a crucial role in inducing an S-phase checkpoint in cells exposed to the topoisomerase I inhibitor campthothecin (CPT), but not in cells exposed to hydroxyurea. Intriguingly, WRN decreases the rate of replication fork elongation, increases the accumulation of ssDNA and stimulates phosphorylation of CHK1, which releases CHK1 from chromatin in CPT-treated cells. Importantly, knockdown of WRN expression abolished or delayed all these processes in response to CPT. Together, our results strongly suggest an essential regulatory role for WRN in controlling the ATR–CHK1-mediated S-phase checkpoint in CPT-treated cells.