Mechanism of PCNA-Dependent Cdt1 Destruction in S phase

S 期 PCNA 依赖性 Cdt1 破​​坏机制

• 批准号: 7480940
• 负责人: Courtney G Havens
• 金额: $ 4.96万
• 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/7480940
• 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（由申请人提供）：在真核细胞中，每个复制起点处的DMA复制起始限于每个S期一次，确保基因组在每个细胞周期中精确复制一次。如果不能适当地限制起源再激发，会导致基因组不稳定，并可能导致癌症。在G1中由ORC、Cdc 6和Cdt 1许可起源，它们合作募集MCM 2 -7解旋酶。一旦起始点在S期启动复制，它就被灭活。启动后，由于通过Geminin和泛素介导的Cdt 1蛋白水解抑制Cdt 1活性，直到随后的G1期，起源不能被重新许可。使用来自非洲爪蟾卵提取物的无细胞系统，我的宿主实验室发现Cdt 1在S期被Cul 4-Ddb 1-Cdt 2泛素连接酶以需要Cdt 1与PCNA在DMA复制叉相互作用的方式泛素化。然而，关于Cdt 1的破坏机制仍有许多问题没有答案。例如，Cdt 1选择性结合PCNA染色质结合池的基础是未知的，这对于确保S期特异性Cdt 1破坏是重要的。此外，Cdt 1内介导其破坏的序列尚未明确。目的1将使用重建策略来重现Cdt 1与DNA结合的PCNA的选择性结合，从而阐明其机制。目的2将进行Cdt 1的突变分析，以确定在非洲爪蟾卵提取物中DNA复制过程中Cdt 1破坏所需的特定氨基酸。此外，我会问这些氨基酸介导的Cdt 1的结合Cul 4-Ddb 1-Cdt 2的背景下，DNA结合的PCNA。最后，为了测试Cdt 1破坏的拟议机制，我将尝试重建PCNA和Cul 4-Ddb 1-Cdt 2依赖的泛素化Cdt 1在体外使用纯化的组件。Cdt 1在几种肿瘤细胞中过表达，并且在小鼠中人工Cdt 1过表达增加致癌潜力。因此，了解Cdt 1破坏的分子机制对癌症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单轮，通常在第一轮发生后，称为Cdt 1的DNA复制因子被破坏。我打算从分子水平上详细研究这种破坏过程是如何发生的。由于Cdt 1过度表达可导致癌症，因此这项工作与人类健康高度相关。