Study on the novel S-phase specific proteolysis pathway

Cdt1是DNA复制许可的重要因子，在进入s期后很快被降解。我们发现两个泛素连接酶Skp2-Cul1和DDB1-Cul4可以独立识别Cdt1的n端进行泛素化。Cdt1的前10个氨基酸区包含一个保守序列，即PCNA相互作用基序（PIP-box）。在s期，PCNA和DDB1-Cul4协同使Cdt1泛素化。这个系统在DNA损伤后也能工作，比如紫外线照射。当PIP-box发生突变时，Cdt1在紫外线照射后变得稳定，并且在s期结合Skp2的沉默。分离出许多PCNA相互作用蛋白。其中，我们发现CDK抑制剂p21也通过PCNA和DDB1-Cul4系统降解。尽管Cdt1和p21在g1期发挥关键作用，但它们在s期开始后的存在并不适合正常的细胞周期进程。，因此在进入s相时应被降解。我们认为，依赖于PCNA的DDB1-Cul4泛素系统提供了一个反馈系统，帮助协调g1期结束和s期开始。

Cdt1, an essential factor for DNA replication licensing, is degraded soon after entry into S-phase. We discovered that two ubiquitin ligases, Skp2-Cul1 and DDB1-Cul4, independently recognize the N-terminus of Cdt1 for ubiquitination. The first 10 amino acid region of Cdt1 contains a conserved sequence, PCNA interacting motif (PIP-box). During S-phase, PCNA and DDB1-Cul4 co-operate to ubiquitinate Cdt1. This system also operates after DNA damage, such as UV irradiation. When PIP-box was mutated, Cdt1 became stable after UV irradiation, and during S-phase combined with silencing of Skp2. Many PCNA interacting proteins were isolated. Among them, we found that CDK inhibitor p21 is also degraded through PCNA and DDB1-Cul4 system. Though both Cdt1 and p21 play key roles during G1-phase, their presence after initiation of S-phase is not suitable for proper cell cycle progression., and thus should be degraded upon entry into S-phase. We propose that PCNA dependent DDB1-Cul4 ubiquitin system provides a feedback system that helps the coordination between end of G1-phase and initiation of S-phase.