Factors Controlling Minisatellite Stability in Yeast

描述（由申请人提供）：基因组稳定性的维持至关重要;例如，基因组不稳定性与人类的许多疾病状态相关。重复DNA通常是基因组重排的来源。一类重复DNA，小卫星序列，具有范围从15到100个核苷酸的近似重复单元长度。哺乳动物基因组包含大量不同的小卫星区。虽然这些束在有丝分裂细胞周期中是稳定的，但它们在减数分裂期间不稳定，改变长度和序列组成。不幸的是，控制小卫星轨道稳定性的遗传和物理因素尚不清楚。小卫星束可以具有遗传功能;与HRAS 1癌基因相关的人类小卫星束充当HRAS 1的转录增强子，并且改变的小卫星等位基因与HRAS 1肿瘤发生相关。 我们将HRAS 1小卫星导入酵母S.酿酒酵母，其中它表现出在哺乳动物细胞中观察到的所有表型。这条通道刺激转录和减数分裂重组，并经历减数分裂特异性的长度改变。重组起始核酸内切酶的去除消除了道的改变，而减数分裂DNA环修复途径的去除特别降低了道扩张的频率。 这些初步研究将通过确定HRAS 1小卫星稳定性基因的完整互补来扩展。除了酵母缺失菌株库的定向筛选外，还启动了两个小卫星稳定性维持基因的一般筛选。筛选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，我们将通过对天然人类HRAS 1小卫星等位基因的遗传分析，确定HRAS 1小卫星等位基因状态与乳腺癌发生之间观察到的相关性的基础。他提出的许多实验不能在哺乳动物系统中进行; HIS 4-HRAS 1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、转录起始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.