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）：从一个细胞产生到下一个细胞的忠实传播是所有生物系统的基本特性，在人类中，它代表了抵抗疾病的主要障碍。为避免基因组不稳定性，细胞已经进化出了许多DNA修复途径，并且严格将DNA复制限制为每个细胞周期的单个圆形。在最近的两个资金杂志中，我们使用爪蟾卵提取物来表征一种新型的E3泛素连接酶，称为CRL4CDT2。 CRL4CDT2底物包含一个“ PIP DEGRON”，该底物介导与DNA上的DNA聚合酶加工性因子PCNA结合。一旦形成了PCNA和底物的二元复合物，CRL4CDT2就会募集以产生三元复合物，并在染色质上进行底物泛素化。 CRL4CDT2活性对DNA结合PCNA（PCNADNA）的依赖性可确保仅在S相和DNA损伤后破坏底物。在脊椎动物中，CRL4CDT2促进了控制原点在细胞周期中控制原点发射的至少三种蛋白（CDT1，P21和SET8）的S相破坏。鉴于其作为Genoe的托管人的核心作用，必须确定CRL4CDT2如何识别其底物的分子机制至关重要，特别是如何与PCNADNA耦合。实际上，CRL4CDT2是唯一已知的泛素连接酶，它在另一个多肽上显示底物时识别底物。因此，研究其机制具有为调节蛋白水解的新范式的潜力。由于对每个新的CRL4CDT2底物的识别都具有对基因组维持方案的重要见解，因此识别其他目标也是最佳优先事项。在此提案中，我们将：（1）表征与DNA修复有关的新CRL4CDT2底物，并使用蛋白质组学发现其他CRL4CDT2靶标。（2）使用一种新型的，基于提取物的单分子方法来确定泛素化的底物如何从PCNA中解离，以使新的底物可以结合。（3）解决支持CRL4CDT2活性和DNA复制需要多少个PCNA亚基。（4）阐明CRL4CDT2活性如何耦合到DNA结合的PCNA。总之，这些实验将探索新的S相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托管人。