Abstract A035: Checkpoint is the control mechanism to ensure the strict order of cellular events during cell cycle. Here, we show that cells enter mitosis without completing DNA replication although with transient activation of known ATR/Chk1 checkpoint
Authors: Huang Min , Chen Junjie 
Published: January 2024

Checkpoints are the control mechanisms that ensure the events of the cell cycle strictly ordered into dependent pathways. Notably, genome duplication and chromosome segregation are two major events of every cell cycle, which respectively occur in S phase and mitosis (M). Checkpoint either for the S/G2 or G2/M transition is considered of great importance to ensure the completion of DNA replication before the initiation of mitosis. Moreover, the degradation tag (dTAG) system allows precise and well-controlled analysis of essential genes by inducing immediate degradation of the protein of interest in a strictly defined time window such as a specific cell cycle phase. To answer what if cells cannot complete DNA replication in mammalian cells, we employed dTAG system targeting DNA replication machinery and generated homozygous dTAG C-terminal knockin at the CDC45/MCM2/GINS4 (CMG) locus in HEK293A cells. Our data show that the CDC45/MCM2/GINS4-dTAG-HA proteins as well as DNA replication rapidly decreased to significantly lower levels in the presence of dTAGv-1 only for 1 hour than that in cells treated with dTAG-NEG. Contrary to the textbook model, our data show that the HEK293A-CMG-dTAG cells treated with dTAGv-1 for 24 to 48 hours, which did not complete DNA replication, began to die instead of being arrested. Moreover, although we observed the transient ATR/Chk1 checkpoint activation when the HEK293A-CMG-dTAG cells treated with dTAGv-1 within 24 hours, the ATR/Chk1 checkpoint disappeared but followed by increased CyclinB1 and pH3S10 protein levels after 24 hours. These data imply that the completion of DNA replication is not under the surveillance of known ATR/Chk1 checkpoint. We then hypothesized that CMG-depleted cells could enter and die in mitosis regarding the increased levels of CyclinB1. To test this, we first transduced HEK293A-CMG-dTAG cells with the cell cycle biosensor, Fucci (CA). HEK293A-CMG-dTAG-Fucci (CA) cells were synchronized in S phase followed by dTAG-NEG or dTAGv-1 treatment. DTAG-NEG-treated cells divided repeatedly, while dTAGv-1-treated cells entered in G2 or M phase without cell division as well as achieving 4N DNA content. To address in which precise phase of CMG-depleted cells die, G2 or M phase, we also transduced HEK293A-CMG-dTAG cells with GFP-H2B. By synchronizing cells in S phase, we observed that dTAGv-1-treated cells either exhibited condensed DNA that occurring in early mitosis or failed in chromosome segregation at later M, suggesting that these cells entered and died in mitosis. Our data demonstrate that the completion of DNA replication is not the prerequisite for cells to enter mitosis in CMG-depleted cells, although with transient activation of the intact ATR/Chk1 checkpoint. However, it is possible that CMG complex is the sensor for monitoring the accomplishment of genome duplication. Therefore, it is necessary to generate HEK293A-PCNA/RPA(1-3)-dTAG cells to test whether PCNA/RPA-depleted cells could recapitulate the phenotypes of CMG-depleted cells.

Citation Format: Min Huang, Junjie Chen. Checkpoint is the control mechanism to ensure the strict order of cellular events during cell cycle. Here, we show that cells enter mitosis without completing DNA replication although with transient activation of known ATR/Chk1 checkpoint [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr A035.