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

S 期特异性泛素连接酶 CRL4-Cdt2 的机制

基本信息

批准号：
8852625
负责人：
Johannes Walter
金额：
$ 25.14万
依托单位：
HARVARD MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-09-20 至 2016-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8852625
关键词：
Address Binding Binding Sites Biology Boxing Cell Cycle Cell division Cell physiology Cells Chromatin Complex Coupled Coupling DNA DNA Binding DNA Damage DNA Methylation DNA Repair DNA Repair Pathway DNA biosynthesis DNA-Directed DNA Polymerase Dependence Diffusion Disease Dissociation Event Foundations Funding Generations Genome Genomic Instability Homo Human Image Ligase Maintenance Mediating Mismatch Repair Modeling Molecular Organism Pathway interactions Peptide Initiation Factors Play Process Property Proteins Proteolysis Proteomics Recruitment Activity Regulation Replication Initiation Role S Phase System Testing Thymine DNA Glycosylase Vertebrates Work Xenopus base egg epigenetic regulation genetic information genome integrity insight multicatalytic endopeptidase complex novel photoactivation polypeptide prevent research study response single molecule tool transmission process ubiquitin ligase ubiquitin-protein ligase

项目摘要

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.

描述（由申请人提供）：遗传信息从一代细胞忠实地传递到下一代细胞是所有生命系统的基本特性，对于人类来说，它是预防疾病的主要障碍。为了避免基因组不稳定，细胞进化出了许多DNA修复途径，并且它们严格地将DNA复制限制在每个细胞周期的一轮。在过去的两次资助期间，我们使用非洲爪蟾卵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“PIP 降解决定子”，可介导与 DNA 上的 DNA 聚合酶持续合成因子 PCNA 的结合。一旦 PCNA 和底物的二元复合物形成，CRL4Cdt2 就会被招募来生成三元复合物，并且底物泛素化发生在染色质上。 CRL4Cdt2 活性对 DNA 结合 PCNA (PCNADNA) 的依赖性确保底物仅在 S 期和 DNA 损伤后被破坏。在脊椎动物中，CRL4Cdt2 促进至少三种控制细胞周期中起源放电的蛋白质（Cdt1、p21 和 Set8）的 S 期破坏。鉴于其作为基因组守护者的核心作用，确定 CRL4Cdt2 如何识别其底物的分子机制至关重要，特别是这种识别如何与 PCNADNA 偶联。事实上，CRL4Cdt2 是唯一已知的泛素连接酶，当底物展示在另一个多肽上时，它可以识别底物。因此，研究其机制有可能为调控蛋白水解建立新的范例。由于每个新的 CRL4Cdt2 底物的鉴定都为了解基因组维护过程提供了重要的见解，因此鉴定其他靶标也是重中之重。在本提案中，我们将：（1）表征参与 DNA 修复的新 CRL4Cdt2 底物，并使用蛋白质组学来发现其他 CRL4Cdt2 靶标。 (2) 使用一种新颖的、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，以便新的底物可以结合。 (3) 确定需要多少 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新的 S 期特异性蛋白水解途径的生物学和机制，该途径充当基因组完整性的重要守护者。