A Novel DNA Re-replication Checkpoint

描述（由申请人提供）：这个项目的长期目标是了解限制DNA复制为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。我们将这些路径称为“重复复制检查点”。重复复制检查点确保在s期只发生一轮DNA合成，代表了维持基因组稳定性的基本机制。该项目的“具体目标”是：(1)验证Rad9-Radl-Husl（9-1-1）检查点蛋白复合物阻止DNA再复制的假设。(2)验证重复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摄动对重复制的影响。这些研究包括：过表达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进行靶向Rad9的RNA干扰实验，以及研究来自转基因Husl-null动物的细胞系。9-1-1阻止再复制的推测机制将通过生化方法确定，包括研究9-1-1介导的信号通路以及9-1-1与DNA复制因子之间的推测相互作用。甲氨蝶呤耐药性的获得将被用作研究特定基因组位点（DHFR基因）扩增的测定。我们将测试干扰重复制检查点对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缺陷细胞中重复复制的基因组位点。由于基因扩增，许多人类致癌基因在癌细胞中过度表达。因此，基因扩增是癌细胞的一个标志，有助于肿瘤的进展。此外，肿瘤细胞中扩增的基因可以赋予对化疗的抗性。因此，了解易导致基因扩增的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重新复制检查点的缺失造成的，那么这些检查点可以通过药物或基因疗法加以利用。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，并将与人类肿瘤的发生直接相关。

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.