Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA.
Cell Cycle and Cancer Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, USA.
Nucleic Acids Res. 2021 Oct 11;49(18):10507-10523. doi: 10.1093/nar/gkab805.
A DNA replication program, which ensures that the genome is accurately and wholly replicated, is established during G1, before the onset of S phase. In G1, replication origins are licensed, and upon S phase entry, a subset of these will form active replisomes. 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 level of TICRR and its phosphorylation determine the number of origins that initiate during S phase. However, the mechanisms regulating TICRR protein levels are unknown. Therefore, we set out to define the TICRR/TRESLIN protein dynamics throughout the cell cycle. Here, we show that TICRR levels are high during G1 and dramatically decrease as cells enter S phase and begin DNA replication. We show that degradation of TICRR occurs specifically during S phase and depends on ubiquitin ligases and proteasomal degradation. Using two targeted siRNA screens, we identify CRL4DTL as a cullin complex necessary for TICRR degradation. We propose that this mechanism moderates the level of TICRR protein available for replication initiation, ensuring the proper number of active origins as cells progress through S phase.
一个确保基因组准确而完整复制的 DNA 复制程序，是在 G1 期建立的，即在 S 期开始之前。在 G1 期，复制起点被许可，并且在 S 期进入时，其中一部分将形成活跃的复制体。严格调控活跃复制体的数量对于防止复制压力引起的 DNA 损伤至关重要。TICRR/TRESLIN 对于 DNA 复制起始是必不可少的，TICRR 的水平及其磷酸化决定了 S 期起始的起始原点的数量。然而，调节 TICRR 蛋白水平的机制尚不清楚。因此，我们着手定义整个细胞周期中 TICRR/TRESLIN 蛋白的动态变化。在这里，我们表明 TICRR 水平在 G1 期较高，并且随着细胞进入 S 期并开始 DNA 复制而急剧下降。我们表明 TICRR 的降解发生在 S 期特异性，并且依赖于泛素连接酶和蛋白酶体降解。通过两个靶向 siRNA 筛选，我们确定了 CRL4DTL 作为一种用于 TICRR 降解的必需的连接酶复合物。我们提出，这种机制调节了用于复制起始的 TICRR 蛋白的水平，确保了细胞在 S 期进展过程中适当数量的活跃起点。
