Abstract
Cdc7-Dbf4 kinase plays a key role in the initiation of DNA replication and contributes to the replication stress in cancer. The activity of human Cdc7-Dbf4 kinase remains active and acts as an effector of checkpoint under replication stress. However, the downstream targets of Cdc7-Dbf4 contributed to checkpoint regulation and replication stress-support function in cancer are not fully identified. In this work, we showed that aberrant Cdc7-Dbf4 induces DNA lesions that activate ATM/ATR-mediated checkpoint and homologous recombination (HR) DNA repair. Using a phosphoproteome approach, we identified HSP90-S164 as a target of Cdc7-Dbf4 in vitro and in vivo. The phosphorylation of HSP90-S164 by Cdc7-Dbf4 is required for the stability of HSP90-HCLK2-MRN complex and the function of ATM/ATR signaling cascade and HR DNA repair. In clinically, the phosphorylation of HSP90-S164 indeed is increased in oral cancer patients. Our results indicate that aberrant Cdc7-Dbf4 enhances replication stress tolerance by rewiring ATR/ATM mediated HR repair through HSP90-S164 phosphorylation and by promoting recovery from replication stress. We provide a new solution to a subtyping of cancer patients with dominant ATR/HSP90 expression by combining inhibitors of ATR-Chk1, HSP90, or Cdc7 in cancer combination therapy.
Highlights
Cell cycle checkpoints are complex surveillance mechanisms that are used to ensure genomic stability and successful repair of DNA damage before entering phase
We used the adenovirus expressing Cdc[7] (Ad-myc-Cdc7) to increase Cdc[7] activity and Cdc[7] kinase dead mutant (Ad-mycCdc7KD) to inhibit Cdc[7] activity in cells, which was confirmed by the status of phosphorylation of MCM2 at S53 or S40/S41 (Fig. 1B and Supplementary Figure 1A) and the Cdc7-dependent chromatin loading of initiation factor Cdc[45] (Supplementary Figure 1B)
Overexpression of HSP90 increased the homologous recombination (HR) DNA repair caused by Cdc[7], but the S164A mutant inhibited the HR DNA repair (Fig. 4H). These results indicate that the phosphorylation of HSP90-Ser[164] by Cdc7-Dbf[4] is required for the maintenance of ATM/ATR-mediated S-phase checkpoint activation, HR DNA repair, and the stability of ATM/ATR signaling component proteins under replication stress
Summary
Cell cycle checkpoints are complex surveillance mechanisms that are used to ensure genomic stability and successful repair of DNA damage before entering phase. How replication stress induces extensive activation of ATR-Chk[1] signaling for DNA damage tolerance in cancer still is needed to explore. Cdc7-Dbf[4] kinase from yeast was considered as a target of S-phase checkpoint because the activity of Cdc7-Dbf[4] was inhibited upon replication stress[17,20]. Accumulating evidences suggested that human Cdc7-Dbf[4] remains active upon replication stress[18,19,21] These conflicting findings indicate that human Cdc7-Dbf[4] acts as an upstream effector to monitor S-phase checkpoint signaling. We unveiled a novel role of Cdc7-Dbf[4] in the regulation of ATR/ATM checkpoint signaling and HR DNA repair in cancer by phosphorylating HSP90 at S164
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