Factors Controlling Minisatellite Stability in Yeast

描述(申请人提供)：保持基因组的稳定性是最重要的；例如，基因组的不稳定性与人类的许多疾病状态相关。重复的DNA通常是基因组重排的来源。一类重复的DNA，小卫星序列，具有大约15到100个核苷酸的重复单位长度。哺乳动物基因组包含大量不同的小卫星区域。虽然这些区域在有丝分裂细胞周期中是稳定的，但在减数分裂期间它们会变得不稳定，在长度和序列组成上都发生了变化。不幸的是，控制小卫星区域稳定性的遗传和物理因素尚不清楚。小卫星区域可能具有遗传功能；与HRAS1癌基因相关的人类小卫星区域作为HRAS1的转录增强子，小卫星等位基因的改变与HRAS1肿瘤的发生相关。 我们建立了一个模型系统，将HRAS1小卫星引入酿酒酵母的HIS4基因座，在那里它展示了在哺乳动物细胞中观察到的所有表型。该束刺激转录和减数分裂重组，并在长度上经历减数分裂特异的改变。移除重组启动的内切酶可以消除肌束的改变，而移除减数分裂DNA环修复途径可以特别减少肌束扩张的频率。 这些初步研究将通过确定控制HRAS1小卫星稳定性的基因的完整互补来扩展。除了酵母缺失菌株Bank的定向筛选外，还启动了两个小卫星稳定性维持基因的一般筛选。这些筛查将确定小卫星稳定性所需的基因以及影响DNA环修复的基因。最后，我们将通过对本地人类HRAS1小卫星等位基因的遗传分析，确定观察到的HRAS1小卫星等位基因状态与乳腺癌发生相关的基础。许多拟议的实验不能在哺乳动物系统中进行；HIS4-HRAS1小卫星系统是获得这些重要数据的唯一手段。这些研究将提供对基因组维持、重组、DNA修复、转录启动和癌症易感性方面的见解。

DESCRIPTION (provided by applicant): Maintenance of genome stability is of paramount importance; genome instability has been correlated with numerous disease states in humans, for example. Repetitive DNA is often the source of genomic rearrangements. One class of repetitive DNA, minisatellite sequences, have an approximate repeat unit length ranging from 15 to 100 nucleotides. Mammalian genomes contain a large number of different minisatellite tracts. While these tracts are stable during the mitotic cell cycle, they destabilize during meiosis, altering in both length and sequence composition. Unfortunately the genetic and physical factors controlling the stability of minisatellite tracts are unknown. Minisatellite tracts can have genetic functions; a human minisatellite tract associated with the HRAS1 oncogene acts as a transcription enhancer for HRAS1, and altered minisatellite alleles have been correlated with HRAS1 oncogenesis. We established a model system by introducing the HRAS1 minisatellite into the HIS4 locus in the yeast S. cerevisiae, where it exhibits all of the phenotypes observed in mammalian cells. The tract stimulates transcription and meiotic recombination, and undergoes meiosis-specific alterations in length. Removal of a recombination-initiating endonuclease eliminates tract alterations, while removal of a meiotic DNA loop repair pathway specifically reduces the frequency of tract expansions. These initial studies will be extended by determining the complete complement of genes governing stability of the HRAS1 minisatellite. Two general screens for minisatellite stability maintenance genes have been initiated, in addition to a directed screen of the Yeast Deletion Strain Bank. The screens will identify genes required for minisatellite stability as well as genes affecting DNA loop repair. Finally, we will determine the basis for an observed correlation between HRAS1 minisatellite allele state and breast cancer oncogenesis, through genetic analysis of native human HRAS1 minisatellite alleles. Many of 1he proposed experiments cannot be done in a mammalian system; the HIS4-HRAS1 minisatellite system is the only means to gain these important data. These studies will provide insights into genome maintenance, recombination, DNA repair, transcription initiation, and aspects of cancer predisposition.