GENESIS OF CHROMOSOMAL TRANSLOCATIONS

染色体不稳定是癌细胞的一个特征。平衡易位，即非同源染色体的手臂在特定的断点处相互连接，是某些恶性肿瘤的显著特征。虽然通过研究易位断裂点存在的基因已经学到了很多东西，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。它们与脱氧核糖核酸修复的遗传性障碍(例如S综合征)、增加染色体断裂的环境条件(例如电离辐射)、针对脱氧核糖核酸断裂和重新连接的酶的药物(例如拓扑异构酶II抑制剂)以及经历程序性脱氧核糖核酸断裂和重新连接的细胞类型(例如淋巴样细胞中的V(D)J重组)有关。我们已经在真核生物酿酒酵母模型中开发了一种简单的遗传检测方法，它可以很容易地检测到非同源重组事件引起的染色体重排。在这种试验中，标记基因被诱导双链断裂，而通过染色体重排修复断裂是通过标记基因表达的变化来检测的。这种分析方法可以用来研究重排的频率和光谱，并且可以对系统进行修改，以适应遗传背景的变化、DSB来源的变化、重排的潜在靶序列的变化，或者细胞生长环境的变化。这项建议的具体目的是：1)研究拓扑异构酶在染色体易位发生中的体内作用；2)确定针对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的易位密切相关的人类DNA片段。我们的长期目标是了解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预防、高危个体识别和合理设计化疗药物具有重要意义。这项探索性/发展性(R21)建议的目的是开发这种新的分析方法的潜在应用，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.