Replication Fork Remodeling Curtails DNA Synthesis during Excessive Origin Firing

DNA replication is a faithful and accurate process during S-phase in each cell division. Errors in replication can potentially lead to cancer, highlighting the purpose of studying mechanisms of DNA replication. There are many challenges that cause DNA damage, making replication fork regulation essential to protect genome integrity. When cell misregulate during replication, it can result in re-replication, which replication origins are re-licensed and fired again. This leads to stress induced DNA breaks, activating cell cycle checkpoints and apoptosis, which cancer cells try to evade, making it a potential chemotherapy target. While re-replication is a source of genomic instability, re-replication regulatory pathways remain unclear, driving the aim to explore the mechanism of how cell respond to re-replication. Re-replication can be triggered by MLN4924 (pevonedistat), a drug that inhibits the neddylation activity of Cullin4A-RING E3 ubiquitin ligase (CRL4). One of the CRL4 targets is the replication licensing factor CDT1 for proteolytic degradation, in which the inhibition of CRL4 by MLN4924 leads to CDT1 accumulation. This promotes re-licensing and over-replication. We find that exposure of MLN4924 slows replication forks and induces replicative stress, which can lead to fork stalling and reversal. In normal cells, this reversal is mediated by fork translocases such as SMARCAL1, ZRANB3, and HLTF. However, in the MLN4924 treatment, HLTF shows its independency for fork reversal. This suggests unknown dynamics by which CDT1 alters replication dynamics. Our future work aims to explore the mechanism of how accumulation of CDT1 affects DNA replication and the regulatory involved in fork reversal.