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

描述（由申请人提供）： 遗传信息从一代细胞到下一代细胞的忠实传递是所有生命系统的基本特性，在人类中，它是抵抗疾病的主要屏障。为了避免基因组的不稳定性，细胞已经进化出许多DNA修复途径，并且它们严格限制DNA复制到每个细胞周期的一轮。在过去的两个资助期间，我们使用爪蟾卵提取物来表征一种新的E3泛素连接酶，称为CRL 4Cdt2。CRL4Cdt2底物含有介导与DNA上的DNA聚合酶持续合成因子PCNA结合的“PIP降解决定子”。一旦PCNA和底物的二元复合物形成，CRL4Cdt2被募集以产生三元复合物，并且底物泛素化在染色质上发生。CRL4Cdt2活性对DNA结合的PCNA（PCNADNA）的依赖性确保了底物仅在S期和DNA损伤后被破坏。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源放电。鉴于其作为基因监护人的核心作用，确定CRL4Cdt2如何识别其底物的分子机制将是至关重要的，特别是这种识别如何与PCNADNA偶联。事实上，CRL4Cdt2是唯一已知的当底物展示在另一种多肽上时识别底物的泛素连接酶。因此，研究其机制有可能建立新的范式调节蛋白质水解。由于每个新的CRL4Cdt2底物的鉴定为基因组维持过程提供了重要的见解，因此鉴定其他靶标也是当务之急。在这个提议中，我们将：（1）表征一个新的参与DNA修复的CRL4Cdt2底物，并使用蛋白质组学来发现其他CRL4Cdt2靶点。(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从PCNA解离，以便新底物可以结合。(3)说明需要多少PCNA亚基来支持CRL4Cdt2活性和DNA复制。(4)阐明CRL4Cdt2活性如何与DNA结合的PCNA偶联。这些实验将共同探索一种新的S期特异性蛋白水解途径的生物学和机制，该途径是基因组完整性的重要监护人。 公共卫生相关性： 为了保持我们基因组的完整性，细胞在每次细胞分裂之前都要精确复制一份DNA（“DNA复制”）。以前，我们发现这种调节涉及破坏一个关键的复制起始因子Cdt1。破坏是由一种非常不寻常的“泛素连接酶”引发的，称为CRL 4Cdt2，它靶向特定的蛋白质， 杀伤性在这个建议中，我们扩大了我们的搜索这种连接酶的新目标，我们探索的分子机制，它识别其底物。

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. PUBLIC HEALTH RELEVANCE: Narrative To maintain the integrity of our genomes, it is crucial that cells make one precise copy of their DNA before each cell division ("DNA replication"). Previously, we discovered that this regulation involves the destruction of a key replication initiation factor called Cdt1. Destructionis triggered by a highly unusual "ubiquitin ligase" called CRL4Cdt2 that targets specific proteins for destruction. In this proposal, we expand our search for new targets of this ligase and we explore the molecular mechanism by which it recognizes its substrates.