Mechanism of CRL4-Cdt2, an S phase-specific ubiquitin ligase

描述(由申请人提供)： 将遗传信息从一代细胞忠实地传递给下一代细胞是所有生命系统的基本属性，在人类中，这是抵御疾病的主要障碍。为了避免基因组的不稳定性，细胞进化出了许多DNA修复途径，它们严格将DNA复制限制在每个细胞周期的一轮。在过去的两个资助周期中，我们使用非洲爪哇卵子提取物来表征一种名为CRL4CDt2的新型E3泛素连接酶。CRL4Cdt2底物含有一个“PIP降解子”，它介导与DNA上的DNA聚合酶处理因子--增殖细胞核抗原的结合。一旦这种增殖细胞核抗原和底物的二元复合体形成，CRL4Cdt2就被招募来产生一个三元复合体，底物在染色质上发生泛素化。CRL4CDt2的活性依赖于DNA结合的增殖细胞核抗原(PCNADNA)，这确保了底物只有在S期和DNA损伤后才被破坏。在脊椎动物中，CRL4Cdt2促进至少三种蛋白质(CDT1、p21和Set8)的S期破坏，这些蛋白质在细胞周期中控制起始放电。鉴于CRL4CDt2作为Gene保管者的核心作用，确定CRL4CDt2如何识别其底物的分子机制，特别是这种识别如何与PCNADNA结合将是至关重要的。事实上，CRL4Cdt2是已知的唯一一种泛素连接酶，当底物被展示在另一种多肽上时，它可以识别底物。因此，研究它的机制有可能建立新的调节蛋白分解的范式。由于每个新CRL4Cdt2底物的鉴定为基因组维护过程提供了重要的见解，因此识别更多的靶点也是首要任务。在这个方案中，我们将：(1)鉴定一种新的参与DNA修复的CRL4Cdt2底物，并使用蛋白质组学来发现其他CRL4Cdt2靶标。(2)使用一种新的、基于提取物的单分子方法来确定泛素化底物如何与增殖细胞核抗原解离，从而使新的底物可以结合。(3)解决支持CRL4CDt2活性和DNA复制所需的多少个增殖细胞核抗原亚基。(4)阐明CRL4CDt2活性如何与DNA结合的增殖细胞核抗原偶联。总之，这些实验将探索一种新的S阶段特异性蛋白分解途径的生物学和机制，该途径是基因组完整性的重要保管者。

DESCRIPTION (provided by applicant): The faithful transmission of genetic information from one cell generation to the next is a fundamental property of all living systems, and in humans, it represents a major barrier against disease. To avoid genome instability, cells have evolved numerous DNA repair pathways, and they strictly limit DNA replication to a single round per cell cycle. In the last two funding perios, we used Xenopus egg extracts to characterize a novel E3 ubiquitin ligase called CRL4Cdt2. CRL4Cdt2 substrates contain a "PIP degron" that mediates binding to the DNA polymerase processivity factor, PCNA, on DNA. Once this binary complex of PCNA and substrate has formed, CRL4Cdt2 is recruited to generate a ternary complex, and substrate ubiquitylation takes place on chromatin. The dependence of CRL4Cdt2 activity on DNA-bound PCNA (PCNADNA) insures that substrates are destroyed only in S phase and after DNA damage. In vertebrates, CRL4Cdt2 promotes the S phase destruction of at least three proteins (Cdt1, p21, and Set8) that control origin firing during the cell cycle. Given its central role as a custodian of the genoe, it will be crucial to determine the molecular mechanism of how CRL4Cdt2 recognizes its substrates, in particular how this recognition is coupled to PCNADNA. Indeed, CRL4Cdt2 is the only known ubiquitin ligase that recognizes substrates when they are displayed on another polypeptide. As such, studying its mechanism has the potential to establish new paradigms for regulated proteolysis. Because the identification of each new CRL4Cdt2 substrate has lent important insights into the proces of genome maintenance, identifying additional targets is also a top priority. In this proposal, we will: (1) characterize a new CRL4Cdt2 substrate involved in DNA repair and use proteomics to discover other CRL4Cdt2 targets. (2) Use a novel, extract-based single molecule approach to determine how ubiquitylated substrates dissociate from PCNA so that a new substrate may bind. (3) Address how many subunits of PCNA are required to support CRL4Cdt2 activity and DNA replication. (4) Elucidate how CRL4Cdt2 activity is coupled to DNA-bound PCNA. Together, the experiments will explore the biology and mechanism of a new S phase-specific proteolysis pathway that acts as an essential custodian of genome integrity.