Characterization of conserved arginine residues on Cdt1 that affect licensing activity and interaction with Geminin or Mcm complex

All organisms ensure once and only once replication during S phase through a process called replication licensing. Cdt1 is a key component and crucial loading factor of Mcm complex, which is a central component for the eukaryotic replicative helicase. In higher eukaryotes, timely inhibition of Cdt1 by Geminin is essential to prevent rereplication. Here, we address the mechanism of DNA licensing using purified Cdt1, Mcm and Geminin proteins in combination with replication in <i>Xenopus</i> egg extracts. We mutagenized the 223th arginine of mouse Cdt1 (mCdt1) to cysteine or serine (R-S or R-C, respectively) and 342nd and 346th arginines constituting an arginine finger-like structure to alanine (RR-AA). The RR-AA mutant of Cdt1 could not only rescue the DNA replication activity in Cdt1-depleted extracts but also its specific activity for DNA replication and licensing was significantly increased compared to the wild-type protein. In contrast, the R223 mutants were partially defective in rescue of DNA replication and licensing. Biochemical analyses of these mutant Cdt1 proteins indicated that the RR-AA mutation disabled its functional interaction with Geminin, while R223 mutations resulted in ablation in interaction with the Mcm2∼7 complex. Intriguingly, the R223 mutants are more susceptible to the phosphorylation-induced inactivation or chromatin dissociation. Our results show that conserved arginine residues play critical roles in interaction with Geminin and Mcm that are crucial for proper conformation of the complexes and its licensing activity.

所有生物体均通过一种名为复制许可（replication licensing）的过程，确保在S期仅发生一次DNA复制。Cdt1是Mcm复合体（Mcm complex）的关键组分与重要加载因子，而Mcm复合体是真核生物复制解旋酶的核心组成部分。在高等真核生物中，Geminin（Geminin）对Cdt1的及时抑制，是防止DNA过度复制的必要条件。本研究利用纯化的Cdt1、Mcm与Geminin蛋白，结合非洲爪蟾（Xenopus）卵提取物中的复制反应，解析DNA复制许可的分子机制。我们对小鼠Cdt1（mCdt1）的第223位精氨酸进行诱变，将其突变为半胱氨酸或丝氨酸（分别记为R-S与R-C突变体）；同时将构成类精氨酸指结构的第342位与第346位精氨酸突变为丙氨酸（记为RR-AA突变体）。与野生型蛋白相比，Cdt1的RR-AA突变体不仅能够恢复Cdt1缺失提取物中的DNA复制活性，其DNA复制与复制许可的比活性还显著提升。与之相反，R223突变体在恢复DNA复制与复制许可活性方面存在部分缺陷。对这些突变型Cdt1蛋白的生化分析表明，RR-AA突变破坏了其与Geminin的功能性相互作用，而R223突变则使其丧失了与Mcm2-7复合体（Mcm2~7 complex）的结合能力。有趣的是，R223突变体更易受磷酸化诱导的失活或染色质解离影响。本研究结果表明，保守的精氨酸残基在与Geminin和Mcm的相互作用中发挥关键作用，而这些相互作用对于复合体的正确构象及其复制许可活性至关重要。