Alver Bonnie, Jauert Peter A, Brosnan Laura, O'Hehir Melissa, VanderSluis Benjamin, Myers Chad L, Kirkpatrick David T
Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, Minnesota 55455.
Department of Computer Science and Engineering, University of Minnesota, Minneapolis, Minnesota 55455.
G3 (Bethesda). 2013 Apr 9;3(4):741-756. doi: 10.1534/g3.112.005397.
Repetitive elements comprise a significant portion of most eukaryotic genomes. Minisatellites, a type of repetitive element composed of repeat units 15-100 bp in length, are stable in actively dividing cells but change in composition during meiosis and in stationary-phase cells. Alterations within minisatellite tracts have been correlated with the onset of a variety of diseases, including diabetes mellitus, myoclonus epilepsy, and several types of cancer. However, little is known about the factors preventing minisatellite alterations. Previously, our laboratory developed a color segregation assay in which a minisatellite was inserted into the ADE2 gene in the yeast Saccharomyces cerevisiae to monitor alteration events. We demonstrated that minisatellite alterations that occur in stationary-phase cells give rise to a specific colony morphology phenotype known as blebbing. Here, we performed a modified version of the synthetic genetic array analysis to screen for mutants that produce a blebbing phenotype. Screens were conducted using two distinctly different minisatellite tracts: the ade2-min3 construct consisting of three identical 20-bp repeats, and the ade2-h7.5 construct, consisting of seven-and-a-half 28-bp variable repeats. Mutations in 102 and 157 genes affect the stability of the ade2-min3 and ade2-h7.5 alleles, respectively. Only seven hits overlapped both screens, indicating that different factors regulate repeat stability depending upon minisatellite size and composition. Importantly, we demonstrate that mismatch repair influences the stability of the ade2-h7.5 allele, indicating that this type of DNA repair stabilizes complex minisatellites in stationary phase cells. Our work provides insight into the factors regulating minisatellite stability.
重复元件构成了大多数真核生物基因组的很大一部分。微卫星是一种由长度为15 - 100 bp的重复单元组成的重复元件，在活跃分裂的细胞中是稳定的，但在减数分裂和静止期细胞中其组成会发生变化。微卫星序列内的改变与多种疾病的发生有关，包括糖尿病、肌阵挛性癫痫和几种类型的癌症。然而，关于防止微卫星改变的因素却知之甚少。此前，我们实验室开发了一种颜色分离分析方法，其中将一个微卫星插入酿酒酵母的ADE2基因中以监测改变事件。我们证明，在静止期细胞中发生的微卫星改变会产生一种特定的菌落形态表型，称为泡状化。在这里，我们进行了改良版的合成基因阵列分析，以筛选产生泡状化表型的突变体。使用两种截然不同的微卫星序列进行筛选：由三个相同的20 bp重复序列组成的ade2 - min3构建体，以及由七个半28 bp可变重复序列组成的ade2 - h7.5构建体。分别有102个和157个基因的突变影响ade2 - min3和ade2 - h7.5等位基因的稳定性。只有七个命中结果在两个筛选中重叠，这表明取决于微卫星的大小和组成，不同的因素调节重复序列的稳定性。重要的是，我们证明错配修复影响ade2 - h7.5等位基因的稳定性，这表明这种类型的DNA修复在静止期细胞中稳定复杂的微卫星。我们的工作为调节微卫星稳定性的因素提供了见解。
