A Global Screen for Genes Regulating Cdt1 Turnover In Response To DNA Damage

针对 DNA 损伤调节 Cdt1 更新的基因的全局筛选

• 批准号: 7912605
• 负责人: Malavika Raman
• 金额: $ 5.22万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-01 至 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7912605
• 关键词: 26S proteasome; ATP phosphohydrolase; Binding; Cancer Biology; Cataloging; Catalogs; Cell Cycle; Cell Cycle Proteins; Cells; Chromatin; Chromosome abnormality; Complex; DNA; DNA Damage; DNA biosynthesis; DNA lesion; Ensure; Face; Failure; G1 Phase; Genes; Homeostasis; Licensing; Licensing Factor; Microscopy; Modeling; Molecular; Mutagens; Organism; Pathway interactions; Phase; Phosphotransferases; Play; Proteins; Regulation; Replication Initiation; Replication Licensing; Replication Origin; Role; Signal Transduction; System; Techniques; Testing; Ubiquitin; Ubiquitination; Xenopus; abstracting; base; cofactor; egg; genetic regulatory protein; genome-wide; loss of function; multicatalytic endopeptidase complex; novel; prevent; protein degradation; public health relevance; reconstitution; response; tumorigenesis

DESCRIPTION (provided by applicant): DNA replication is a crucial step in the cell cycle where cellular DNA must be faithfully copied only once. Failure to do so will result in either incomplete replication or multiple rounds of replication, both of which result in aberrant chromosome numbers. This proposal aims to understand how a protein known as Cdt1, which has been shown in all organisms to be the key player in controlling DNA replication, is regulated. Mis-regulation of Cdt1 levels plays a key role in tumorigenesis, underscoring the importance of this pathway in cancer biology. PUBLIC HEALTH RELEVANCE: The ubiquitin proteasome system (UPS) plays a key role in cellular homeostasis by regulating both bulk protein turnover as well as the degradation of key regulatory proteins. Its role is of particular importance in regulating the spatial and temporal aspects of the cell cycle. Key cell cycle proteins are degraded at precise points in the cell cycle to signal that the next phase may be initiated. One such UPS target is the evolutionarily conserved DNA replication licensing factor Cdt1. Cdt1 licences origins of replication early in th G1 phase of the cell cycle. To ensure that DNA is replicated only once, Cdt1 must promptly be degraded via the UPS. Moreover, Cdt1 is also a target of the DNA damage response and is rapidly degraded when cells are treated with genotoxic agents to prevent replication initiation in the face of unrepaired DNA lesions. Surprisingly, canonical DNA damage kinases such as ATM/ATR that take part in many aspects of the cellular response to DNA damage, do not appear to be involved in its degradation. While Cul4-Ddb1Cdt2 has been identified to ubiquitinate Cdt1 that is associated with chromatin-bound PCNA, there is still not a complete catalog of genes in this pathway. In order to identify the entire repertoire of genes that impinge on Cdt1 turnover, I have developed and completed a high-throughput, microscopy-based, quantitative genome-wide loss-of-function screen in DNA damaged cells. This screen has identified not only all the known complexes that regulate Cdt1 turnover, but also a large number of novel genes that appear to stabilize Cdt1 in DNA damaged cells. In many cases, I have recovered multiple subunits of macro-molecular complexes, increasing the confidence that these complexes have bona fide roles in the the Cdt1 pathway. This proposal aims to dissect the mechanism by which one of the strongest complexes identified in the screen impact Cdt1 turnover. I will focus in on the mechanism by which the p97/Ufd1-Npl4-Ufd2 complex delivers ubiquitinated Cdt1 to the 26S proteasome. I identified p97 as well as its cofactors Ufd1 and Ufd2 from the screen. This complex has been shown to extract proteins from multimeric complexes and deliver the ubiquitinated target to the proteasome. I will identify all the components of this complex that are required for Cdt1 turnover and test if this complex associates with ubiquitinated Cdt1 on chromatin. Importantly, I will determine if p97 and cofactors extract Cdt1 from PCNA and chromatin via p97's AAA-ATPase activity using techniques in mammlian cells and Xenopus egg extracts. Finally, I will attempt to reconstitute the ubiquitination and degradation of Cdt1 biochemically with purified components to rigorously test all the predictions made by the model. In summary, this proposal aims to ascribe molecular function to new genes in the Cdt1 destruction pathway, mis-regulation of which has been demonstrated to result in tumorigenesis in multiple settings.

描述（由申请人提供）：DNA复制是细胞周期中的关键步骤，其中细胞DNA必须仅忠实地复制一次。如果不这样做，将导致不完全复制或多轮复制，这两种情况都会导致染色体数目异常。该提案旨在了解一种被称为Cdt 1的蛋白质是如何被调节的，Cdt 1在所有生物体中都被证明是控制DNA复制的关键角色。Cdt 1水平的错误调节在肿瘤发生中起着关键作用，强调了该途径在癌症生物学中的重要性。 公共卫生关系：泛蛋白蛋白酶体系统（UPS）通过调节大量蛋白质的周转以及关键调节蛋白的降解，在细胞稳态中发挥着关键作用。其作用在调节细胞周期的空间和时间方面特别重要。细胞周期中的关键蛋白质在细胞周期中的精确点降解，以发出下一个阶段可能开始的信号。一个这样的UPS目标是进化上保守的DNA复制许可因子Cdt 1。Cdt 1在细胞周期的G1期早期许可复制起点。为了确保DNA只复制一次，Cdt 1必须通过UPS迅速降解。此外，Cdt 1也是DNA损伤反应的靶标，并且当细胞用遗传毒性剂处理以防止在未修复的DNA损伤面前的复制起始时，Cdt 1迅速降解。令人惊讶的是，典型的DNA损伤激酶，如ATM/ATR，参与细胞对DNA损伤的反应的许多方面，似乎不参与其降解。虽然Cul 4-Ddb 1Cdt 2已被确定为泛素化Cdt 1，这是与染色质结合的PCNA，仍然没有一个完整的目录中的基因在这一途径。为了确定影响Cdt 1营业额的整个基因库，我已经开发并完成了一个高通量的，基于显微镜的，定量的全基因组功能丧失的DNA损伤细胞的屏幕。该筛选不仅确定了所有已知的调节Cdt 1周转的复合物，而且还确定了大量似乎在DNA损伤细胞中稳定Cdt 1的新基因。在许多情况下，我已经恢复了大分子复合物的多个亚基，增加了这些复合物在Cdt 1途径中具有真正作用的信心。 该提案旨在剖析屏幕中确定的最强复合物之一影响Cdt 1营业额的机制。我将集中在p97/Ufd 1-Np 14-Ufd 2复合物传递泛素化Cdt 1到26 S蛋白酶体的机制。我从屏幕上识别出了p97及其辅因子Ufd 1和Ufd 2。这种复合物已被证明可以从多聚体复合物中提取蛋白质，并将泛素化的靶标递送到蛋白酶体。我将确定Cdt 1周转所需的复合物的所有组成部分，并测试该复合物是否与染色质上的泛素化Cdt 1相关。重要的是，我将确定是否p97和辅因子提取Cdt 1的PCNA和染色质通过p97的AAA-ATP酶活性使用技术在非洲爪蟾细胞和卵提取物。最后，我将尝试用纯化的组分以生物化学方式重建Cdt 1的泛素化和降解，以严格测试模型所做的所有预测。 总之，该提案旨在将分子功能归因于Cdt 1破坏途径中的新基因，其错误调节已被证明会导致多种环境中的肿瘤发生。