GENESIS OF CHROMOSOMAL TRANSLOCATIONS

染色体不稳定是癌细胞的一个特征。平衡易位，即非同源染色体臂在特定断点上相互连接，是某些恶性肿瘤的显著特征。虽然通过检查易位断点上存在的基因已经了解了很多，但关于染色体易位发生的分子机制几乎一无所知。易位似乎代表了不适当的双链断裂（DSB）修复事件。它们与DNA修复的遗传性疾病（如布鲁姆综合征）、增加染色体断裂的环境条件（如电离辐射）、靶向DNA断裂和重新连接酶的药物（如拓扑异构酶II抑制剂）以及经历程序性DNA断裂和重新连接的细胞类型（如淋巴样细胞中的V(D)J重组）有关。我们在模型真核生物酿酒酵母中开发了一种简单的遗传分析方法，可以很容易地检测由非同源重组事件引起的染色体重排。在这个实验中，在一个标记基因中诱导双链断裂，并通过标记基因表达的变化来检测染色体重排对断裂的修复。该试验可用于研究重排的频率和频谱，系统可以修改以适应遗传背景的变化，DSB来源的变化，重排的潜在目标序列的变化，或细胞生长环境的变化。本提案的具体目的是：1)检查拓扑异构酶在染色体易位发生中的体内作用；2)确定靶向拓扑异构酶的药物如何影响易位形成；3)在酵母中分析与癌症化疗相关易位密切相关的人类DNA片段。我们的长期目标是了解易使染色体易位的遗传和环境因素以及产生特定易位的分子机制。这些目标的实现将对癌症的预防、高危个体的识别和化疗药物的合理设计具有重要意义。本探索性/发展性（R21）提案的目的是开发这种新型检测方法的潜在应用，并产生足够的初步数据，以符合未来研究项目（即R01）的资助条件。

Chromosome instability is a hallmark of cancer cells. Balanced translocations, in which arms of nonhomologous chromosomes are joined to one another at specific breakpoints, are distinctive characteristics of certain malignancies. While much has been learned by examining the genes present at translocation breakpoints, almost nothing is known about the molecular mechanisms involved in the genesis of chromosomal translocations. Translocations appear to represent inappropriate double-strand break (DSB) repair events. They are associated with genetic disorders of DNA repair (e.g. Bloom s syndrome), environmental conditions which increase chromosomal breakage (e.g. ionizing radiation), drugs which target enzymes that break and rejoin DNA (e.g. topoisomerase II inhibitors) as well as with cell types that undergo programmed DNA breakage and rejoining (e.g. V(D)J recombination in lymphoid cells). We have developed a simple genetic assay in the model eukaryote Saccharomyces cerevisiae which can easily detect chromosomal rearrangements resulting from nonhomologous recombinational events. In this assay, a double strand break is induced in a marker gene and repair of the break by chromosomal rearrangements is detected by a change in marker gene expression. This assay can be used to study the frequency and spectrum of rearrangements and the system can be modified to accommodate changes in genetic background, changes in the source of DSB, changes in potential target sequences for rearrangements, or changes in the environment in which the cells are growing. The specific aims of this proposal are to 1)examine the in vivo role of topoisomerases in the genesis of chromosomal translocations; 2) determine how drugs that target topoisomerases influence translocation formation; and 3) analyze, in yeast, a segment of human DNA strongly linked to cancer chemotherapy associated translocations. Our long-term objectives are to understand the genetic and environmental factors that predispose chromosomes to translocate and the molecular mechanisms by which specific translocations are generated. Accomplishment of these goals will have great implications for cancer prevention, identification of at-risk individuals, and rational design of chemotherapeutic drugs. The purpose of this exploratory/developmental (R21) proposal is to develop the potential applications of this novel assay and generate sufficient preliminary data to qualify for future research project (i.e. R01) funding in the future.