The G₂/M DNA damage checkpoint plays an important role in the maintenance of genome stability. It tightly controls the cell cycle machinery, and its deregulation may lead to cancer. Upon DNA damage induced by ionizing radiation or other DNA damaging agents, the checkpoint is activated through phosphorylation of ATR/ATM and CHK1/CHK2, thereby preventing cells from entering mitosis. After DNA damage is repaired, the checkpoint is turned off to allow progression into mitosis. As the detailed mechanism of checkpoint recovery is largely unknown, the aim of this study is to discover some of the components of this process. UCN-01, a CHK1 inhibitor, was used to inactivate the checkpoint after DNA damage, thereby mimicking checkpoint recovery. Whether this process is affected after indi...[ Read more ]

The G₂/M DNA damage checkpoint plays an important role in the maintenance of genome stability. It tightly controls the cell cycle machinery, and its deregulation may lead to cancer. Upon DNA damage induced by ionizing radiation or other DNA damaging agents, the checkpoint is activated through phosphorylation of ATR/ATM and CHK1/CHK2, thereby preventing cells from entering mitosis. After DNA damage is repaired, the checkpoint is turned off to allow progression into mitosis. As the detailed mechanism of checkpoint recovery is largely unknown, the aim of this study is to discover some of the components of this process. UCN-01, a CHK1 inhibitor, was used to inactivate the checkpoint after DNA damage, thereby mimicking checkpoint recovery. Whether this process is affected after individual proteins were downregulated by RNA interference was evaluated using time-lapse microscopy. As known players that participate in cell cycle control may be involved in checkpoint recovery, I evaluated the functions of 19 kinases, 14 phosphatases and 21 proteins involved in regulation of mitotic spindles in this process. Among all the 54 candidates, knockdown of 14 proteins affected checkpoint recovery, and the roles of CDC14A and Greatwall were confirmed with other checkpoint abrogating agents. I also discovered that CDC14A and TBK1 were required for DNA damage checkpoint maintenance. By combining results from this study and other data from our group, we found that Greatwall may be involved in maintaining genome stability by multiple mechanisms. Further investigations demonstrated that Greatwall protected genome stability through both controlling checkpoint recovery and preventing genome reduplication after DNA damage. These findings provided a better understanding of DNA damage checkpoint recovery mechanism and new insights into novel anticancer therapeutic targets.