Developmental regulation of DNA replication during programmed gene ampplification

描述（由申请人提供）： 基因组不稳定性是癌症的标志，可由染色体的不完全复制或部分重新复制引发。此外，原癌基因的自发扩增通常与肿瘤发生有关。真核生物的模式，果蝇和四膜虫，提供了独特的机会来研究基因扩增，因为这些事件是发育编程。虽然复制起始的DMA序列要求在人类中知之甚少，但顺式作用决定簇已在果蝇绒毛膜位点和四膜虫核糖体DMA（rDNA）复制子/扩增子中被遗传地定义。在这两种情况下，基因扩增都依赖于通常将复制限制为每个细胞周期一次的起始蛋白。这些扩增子如何逃脱细胞周期控制尚不清楚。这项提案的目标是开发新的方法来理解为什么特定的复制起点重新启动和放大DMA时，其他起点得到适当的监管。先前的努力集中在“扩增子”本身。这个休假计划的目标是开发研究沉默染色体起源的工具。大部分研究将采用四膜虫模型，采用导师实验室开发的方法来研究果蝇中的DNA复制。申请人还将获得使用果蝇作为模式生物所需的技能。他还将在哺乳动物复制实验室接受培训，该实验室开发了一种单分子方法来研究复制起始。目标是掌握尖端技术，这将提高研究者在他的家乡机构的研究计划。将采取几种不同但互补的实验方法，以开辟新的调查途径。具体目标是：（1）获得研究四膜虫发育过程中DNA复制的新技术，特别是实时新生链PCR和复制DNA的单分子分析（SMARD）。(2)发展免疫细胞学方法，以可视化的亚核定位的复制蛋白在四膜虫发育的三个特定阶段-内复制阶段1，基因扩增，和内复制阶段2。(3)学习并使用实时PCR染色质免疫沉淀分析来检查发育过程中复制蛋白与扩增和非扩增起点的时间关联。(4)获得与果蝇合作的专业知识，将其用作新的模型系统。逃避细胞周期控制的复制起点对染色体稳定性具有深远的不稳定影响。了解如何防止再复制的保障措施是在遗传上易处理的模式生物中被推翻的，可以增强我们对人类癌细胞基因组不稳定性的理解。

DESCRIPTION (provided by applicant): Genome instability, a hallmark of cancer, can be triggered by incomplete replication or partial re-replication of chromosomes. In addition, the spontaneous amplification of proto-oncogenes is commonly associated with tumorigenesis. The model eukaryotes, Drosophila and Tetrahymena, provide unique opportunities to study gene amplification because these events are developmentally programmed. While the DMA sequences requirements for replication initiation are poorly understood in humans, cis-acting determinants have been genetically defined in the Drosophila chorion locus and Tetrahymena ribosomal DMA (rDNA) replicons/amplicons. In both instances, gene amplification is dependent on initiator proteins that normally restrict replication to once per cell cycle. How these amplicons escape cell cycle control is not known. The goal of this proposal is to develop new approaches to understand why specific replication origins re-initiate and amplify DMA when other origins are properly regulated. Prior efforts have focused on the 'amplicons' themselves. The goal of this sabbatical proposal is to develop tools to study the silenced chromosomal origins. The bulk of the research will employ the Tetrahymena model, adapting approaches developed in the mentor's lab to study DNA replication in Drosophila. The applicant will also gain the skills required to use Drosophila as a model organism. He will also receive training in a mammalian replication lab that developed a single molecule approach to study replication initiation. The goal is to master cutting edge technologies that will enhance the investigator's research program at his home institution. Several distinct, but complementary experimental approaches will be pursued to open up new avenues of investigation. The specific aims are: (1) To acquire new skills to study DNA replication during Tetrahymena development, specifically, real-time nascent strand PCR, and single molecule analysis of replicating DNA (SMARD). (2) To develop immunocytological approaches to visualize the sub-nuclear localization of replication proteins during three specific stages of Tetrahymena development- endoreplication phase 1, gene amplification, and endo replication phase 2. (3) To learn and use a real-time PCR chromatin immunoprecipitation assay to examine the temporal association of replication proteins with amplified and non-amplified origins during development. (4) To obtain expertise working with Drosophila to employ it as a new model system. Replication origins that escape cell cycle control have profound destabilizing effects on chromosome stability. Understanding how the safeguards that prevent re-replication are over-ridden in genetically tractable model organisms can enhance our understanding of genome instability in human cancer cells.