A Novel DNA Re-replication Checkpoint

一种新的 DNA 再复制检查点

• 批准号: 7234366
• 负责人: Cyrus Vaziri
• 金额: $ 29.82万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-06-01 至 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7234366
• 关键词: A549; Adenovirus Vector; Adenoviruses; Alleles; Animals; Biochemical; Biological Assay; Cell Cycle; Cell Cycle Progression; Cell Line; Cell Survival; Cells; Clinic; Complex; Condition; DHFR gene; DNA; DNA Replication Factor; DNA biosynthesis; DNA chemical synthesis; Data; Dihydrofolate Reductase; Down-Regulation; Drug usage; Ensure; Failure; Fire - disasters; Fluorescent in Situ Hybridization; Gene Amplification; Gene Expression; Gene Silencing; Genes; Genome; Genome Stability; Genomic Instability; Genomics; Goals; H1299; Human; Lead; Link; Mediating; Methotrexate; Mus; Nuclear; Oncogenes; Pathway interactions; Phase; Protein p53; Proteins; RNA Interference; Research Personnel; Resistance; Role; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Signaling Protein; Small Interfering RNA; Somatic Cell; Testing; Tetracycline; Tetracyclines; Therapeutic; Transgenic Organisms; Viral; anticancer research; base; cancer cell; chemotherapy; comparative genomic hybridization; design; gene therapy; in vivo; neoplastic cell; novel; prevent; prognostic; programs; research study; response; tumor; tumor progression; tumorigenesis

DESCRIPTION (provided by applicant): The broad long-term goal of this project is to understand mechanisms that restrict DNA replication to once-per- cell cycle. Our preliminary studies have identified checkpoint signaling pathways that prevent DNA re-replication. We have termed these pathways 're-replication checkpoints'. Re-replication checkpoints ensure that only one round of DNA synthesis occurs during S-phase and represent a fundamental mechanism for maintaining genomic stability. The 'Specific Aims' of this project are: (1) To test the hypothesis that the Rad9-Radl-Husl (9-1-1) checkpoint protein complex prevents DNA re-replication. (2) To test the hypothesis that perturbation of re-replication checkpoints promotes gene amplification. We will study the effect of 9-1-1 perturbation on re-replication using several complementary approaches. These include: over-expressing the 9-1-1 antagonist, Husl B, RNA interference experiments targeting Rad9 using a novel adenovirus vector we constructed for gene silencing, and studying cell lines from transgenic Husl-null animals. The putative mechanism by which 9-1-1 prevents re-replication will be determined using biochemical approaches including studying 9-1-1-mediated signaling pathways and putative interactions between 9-1-1 and DNA replication factors. Acquisition of methotrexate resistance will be used as an assay to study amplification of a defined genomic locus (the DHFR gene). We will test the effects of perturbing the re-replication checkpoint on DHFR amplification. Microarray-based Comparative Genomic Hybridization (CGH) will be used to identify re-replicating genomic loci in 9-1-1-deficient cells. Many human oncogenes are over-expressed in cancer cells as a result of gene amplification. Therefore, gene amplification is a hallmark of cancer cells and contributes to tumor progression. Additionally, genes amplified in tumor cells can confer resistance to chemotherapies. Therefore, an understanding of factors that pre-dispose to gene amplification will be of prognostic and therapeutic value in the clinic. If gene amplification in tumors results from loss of re-replication checkpoints, these checkpoints could be exploited using drugs or gene therapies. The results of our studies may provide a novel paradigm for mechanisms of gene amplification and will be directly relevant to tumorigenesis in humans.

描述（由申请人提供）：该项目的广泛的长期目标是了解限制DNA复制到每个细胞周期一次的机制。我们的初步研究已经确定了阻止DNA再复制的检查点信号通路。我们将这些途径称为“再复制检查点”。再复制检查点确保在S期期间仅发生一轮DNA合成，并且代表维持基因组稳定性的基本机制。本项目的“具体目的”是：（1）检验Rad 9-Radl-Husl（9-1-1）检查点蛋白复合物阻止DNA再复制的假设。(2)为了验证再复制检查点的扰动促进基因扩增的假设。我们将使用几种互补的方法研究9-1-1扰动对再复制的影响。其中包括：过量表达9-1-1拮抗剂Husl B，使用我们构建的用于基因沉默的新型腺病毒载体靶向Rad 9的RNA干扰实验，以及研究来自转基因Husl缺失动物的细胞系。9-1-1阻止再复制的假定机制将使用生物化学方法来确定，包括研究9- 1-1介导的信号传导途径和9-1-1与DNA复制因子之间的假定相互作用。甲氨蝶呤耐药性的获得将用作研究确定的基因组位点（DHFR基因）扩增的试验。我们将测试干扰再复制检查点对DHFR扩增的影响。基于微阵列的比较基因组杂交（CGH）将用于鉴定9-1-1缺陷细胞中的重复复制基因组位点。由于基因扩增，许多人类癌基因在癌细胞中过度表达。因此，基因扩增是癌细胞的标志，并有助于肿瘤进展。此外，在肿瘤细胞中扩增的基因可以赋予对化疗的抗性。因此，了解基因扩增的易感因素将在临床上具有预后和治疗价值。如果肿瘤中的基因扩增是由于再复制检查点的丢失造成的，那么可以使用药物或基因疗法来利用这些检查点。我们的研究结果可能为基因扩增的机制提供一个新的范例，并将与人类肿瘤发生直接相关。