Mechanism of PCNA-Dependent Cdt1 Destruction in S phase

S 期 PCNA 依赖性 Cdt1 破​​坏机制

• 批准号: 7330201
• 负责人: Courtney G Havens
• 金额: $ 4.68万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-01 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7330201
• 关键词: Amino Acid Motifs; Amino Acids; Binding; Binding Proteins; Biochemical; Boxing; Cancer Biology; Cancer Etiology; Cell Cycle; Cell division; Cell-Free System; Cells; Chromatin; DNA Binding; DNA Replication Factor; DNA biosynthesis; DNA chemical synthesis; DNA replication fork; DNA-Directed DNA Polymerase; Disease; Ensure; Eukaryotic Cell; Event; Failure; Fire - disasters; Fission Yeast; G1 Phase; Geminin; Genome; Genomic Instability; Goals; Health; Human; In Vitro; Laboratories; Licensing; Malignant Neoplasms; Mediating; Mitosis; Modeling; Molecular; Mus; Oncogenic; PCNA gene; Peptide Initiation Factors; Phase; Play; Process; Protein Overexpression; Proteolysis; Reaction; Recruitment Activity; Replication Initiation; Replication Origin; Role; Set protein; Testing; Ubiquitin-mediated Proteolysis Pathway; Vertebrates; Work; Xenopus; base; egg; helicase; neoplastic cell; phosphatidylinositol phosphate; prevent; reconstitution; ubiquitin ligase; ubiquitin-protein ligase

DESCRIPTION (provided by applicant): In eukaryotic cells, initiation of DMA replication at each origin of replication is restricted to once per S phase, insuring that the genome is duplicated precisely once during each cell cycle. Failure to properly restrict origin re-firing results in genomic instability and probably causes cancer. Origins are licensed in G1 by ORC, Cdc6, and Cdt1 which cooperate to recruit the MCM2-7 helicase. Once the origin initiates replication in S phase, it is inactivated. After initiation, origins cannot be re-licensed until the following G1 phase due to inhibition of Cdt1 activity through Geminin and ubiquitin-mediated proteolysis of Cdt1. Using a cell-free system derived from Xenopus egg extracts, my host laboratory fond that Cdt1 is ubiquitylated during S phase by the Cul4-Ddb1-Cdt2 ubiquitin ligase in a manner that requires interaction of Cdt1 with PCNA at the DMA replication fork. However, many questions remain unanswered with regard to the mechanism of Cdt1 destruction. For example, the basis for selective binding of Cdt1 to the chromatin-bound pool of PCNA, which is important to insure S phase-specific Cdt1 destruction, is unknown. In addition, the sequences within Cdt1 which mediate its destruction are not well defined. In Aim 1 will use reconstitution strategies to recapitulate the selective binding of Cdt1 to DNA-bound PCNA and thereby elucidate its mechanism. In Aim 2 will perform a mutational analysis of Cdt1 to identify specific amino acids which are required for Cdt1 destruction during DNA replication in Xenopus egg extracts. In addition, I will ask whether these amino acids mediate binding of Cdt1 to Cul4-Ddb1-Cdt2 in the context of DNA-bound PCNA. Finally, to test the proposed mechanism of Cdt1 destruction, I will attempt to reconstitute PCNA- and Cul4-Ddb1-Cdt2-dependent ubiquitylation of Cdt1 in vitro using purified components. Cdt1 is overexpressed in several tumor cells, and artificial Cdt1 over expression in mice increases oncogenic potential. Therefore, understanding the molecular mechanism of Cdt1 destruction is important for cancer biology. To avoid diseases such as cancer, our cells must create a precise copy of their genomes before each cell division. To limit genome duplication to a single round, a DNA replication factor called Cdt1 is normally destroyed after the first round has occurred. I propose to study in molecular detail how this destruction process takes place. Because Cdt1 over expression can cause cancer, the work is highly relevant for human health.

描述(申请人提供)：在真核细胞中，在每个复制起始处的DMA复制被限制在每个S期一次，以确保在每个细胞周期中基因组精确复制一次。如果不能适当地限制原点重新启动，会导致基因组不稳定，并可能导致癌症。起始点在G1中由ORC、CDC6和CDT1许可，它们合作招募MCM2-7解旋酶。一旦起源在S阶段启动复制，它就被灭活。在启动后，由于通过双黄素和泛素介导的CDT1蛋白分解抑制了CDT1的活性，所以起始阶段直到下一个G1期才能重新许可。利用非洲爪哇卵提取物的无细胞体系，我的宿主实验室发现，在S阶段，CDT1被CUL4-DDB1-CdT2泛素连接酶泛素化，这种方式需要在DMA复制分叉上CDT1与增殖细胞核抗原相互作用。然而，关于CDT1破坏的机制，许多问题仍然没有得到回答。例如，cdt1选择性结合到染色质结合的增殖细胞核抗原池的基础是未知的，这对确保S阶段特异性cdt1的破坏是重要的。此外，CDT1中调节其破坏的序列没有很好的定义。在目标1中，我们将使用重组策略概述CDT1与DNA结合的增殖细胞核抗原的选择性结合，从而阐明其机制。在AIM 2中，将对CDT1进行突变分析，以确定在非洲爪哇卵提取液中DNA复制过程中破坏CDT1所需的特定氨基酸。此外，我还会问，在DNA结合的增殖细胞核抗原的背景下，这些氨基酸是否介导了CDT1与CUL4-DDB1-CDT2的结合。最后，为了验证所提出的破坏CDT1的机制，我将尝试用纯化的成分在体外重建依赖于增殖细胞核抗原和CUL4-DDB1-Cdt2的CDT1泛素化。CDT1在几个肿瘤细胞中过表达，而在小鼠中人工过表达的CDT1增加了致癌潜力。因此，了解CDT1破坏的分子机制对肿瘤生物学具有重要意义。 为了避免癌症等疾病，我们的细胞必须在每次细胞分裂之前创建其基因组的精确副本。为了将基因组复制限制在一轮，一种名为CDT1的DNA复制因子通常在第一轮发生后被破坏。我建议从分子上详细研究这种破坏过程是如何发生的。由于CDT1的过度表达会导致癌症，这项工作与人类健康高度相关。