TICRR/TRESLIN protein expression is cell cycle regulated by the CUL4-DDB1 E3 Ligase Kimberlie A. Wittig1,2, Courtney G. Sansam2, Tyler D. Noble1,2, Duane Goins2, Christopher L. Sansam1,2* 1 Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, USA; 2 Cell Cycle and Cancer Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104, USA A DNA replication program, which ensures that the genome is accurately and completely replicated, is established during G1 prior to the onset of S-phase. Tight regulation of the number of active replisomes is crucial to prevent replication stress-induced DNA damage. TICRR/TRESLIN is essential for DNA replication initiation, and the expression level of TICRR and its phosphorylation determine the number of origins that initiate simultaneously during S-phase. However, the mechanisms regulating TICRR protein expression are unknown. Here, we aimed to evaluate TICRR protein dynamics around the G1/S-phase transition. We tagged the endogenous C-terminus of TICRR with mClover in HCT-116 cells using CRISPR/Cas9 and applied an established flow cytometry assay to detect how levels of both insoluble and total TICRR change. Total TICRR expression is highest in G2/M, decreases with cell division, and further decreases at the G1/S-phase transition. However, insoluble TICRR levels are highest in G1 and sharply decrease with S-phase entry. Although total TICRR expression decreases between G2/M and G1, insoluble TICRR levels increase demonstrating that insoluble TICRR accumulation in G1 is not due to changes in its expression. In contrast, both total and insoluble TICRR levels decrease with S-phase entry demonstrating this decrease is at least in part due to the degradation of TICRR protein. Utilizing proteasomal and neddylation inhibitors, we show that degradation of TICRR depends on Cullin E3 ligases and is specific to S-phase. Additionally, through a targeted siRNA screen we have identified CUL4-DDB1 as the Cullin complex necessary for TICRR degradation. Collectively, our results demonstrate how total and insoluble levels of TICRR change in distinct ways throughout the cell cycle, and we have elucidated a mechanism for TICRR degradation at the G1/S transition affecting the levels of protein available for DNA replication initiation during S-phase. These results suggest a mechanism to control the rate of origin firing to prevent replication stress and DNA damage.