MECHANISM OF FORMATION OF TERMINAL DELETIONS

由染色体畸变引起的人类遗传疾病中，约有10%是由末端缺失引起的。迄今为止分析的所有导致遗传疾病的末端缺失都是由于新的端粒被直接添加到断裂染色体的末端。丢失的端粒的修复，被称为染色体愈合，已经在单细胞生物中进行了广泛的研究，然而，对哺乳动物细胞中的这一过程知之甚少。该项目涉及使用在小鼠胚胎干细胞（ES）中开发的测定系统来研究末端缺失的形成机制和参与染色体愈合的基因。该系统利用含有可选择标记基因的质粒序列和与端粒相邻的I-SceI内切酶识别序列。I-SceI识别序列用于特异性引入双链断裂，选择性标记基因用于识别在被标记染色体末端有末端缺失的细胞。含有I-SceI内切酶基因的表达载体电穿孔产生染色体愈合引起的末端缺失。新的端粒是在染色体末端与端粒重复序列具有短同源性的位点上损失很少或没有DNA的情况下添加的。新的端粒比亲本细胞系短得多，并且随着培养时间的延长而增加，与端粒酶从头合成一致。在目前的拨款申请中，我们将利用该检测系统来研究染色体愈合的机制，影响这一过程的因素，以及染色体愈合在细胞对DNA双链断裂的反应中的重要性。将在端粒酶、DNA双链断裂修复和细胞周期调节缺陷的小鼠细胞系中研究染色体愈合中末端缺失的形成，以确定这些途径在这一过程中的作用。胚胎干细胞克隆还将用于建立转基因小鼠，这将为研究各种不同细胞类型的染色体愈合以及研究不同遗传背景下的染色体愈合提供一种方法。利用这些方法，我们将能够验证染色体愈合是一种细胞类型特异性机制的假设，这种机制可以影响细胞对DNA双链断裂的反应。该系统监测端粒长度和端粒损失率的能力也将为研究不同基因在维持端粒稳定性中的作用提供一种手段。

Terminal deletions are responsible for approximately 10 percent of human genetic diseases resulting from chromosome aberrations. All terminal deletions resulting in genetic diseases that have been analyzed thus far have resulted from new telomeres being added directly onto the ends of broken chromosomes. The restoration of lost telomeres, termed chromosome healing, has been studied extensively in single cell organisms, however, very little is known about this process in mammalian cells. This project involves the use of an assay system developed in mouse embryonic stem (ES) cells to study the mechanism of formation of terminal deletions and the genes involved in chromosome healing. This system utilizes plasmid sequences containing selectable marker genes and an I-SceI endonuclease recognition sequence integrated immediately adjacent to a telomere. The I-SceI recognition sequence is used to specifically introduce double-strand breaks and the selectable marker genes are used to identify cells that have terminal deletions on the end of the marked chromosome. Electroporation of an expression vector containing the gene for the I-SceI endonuclease produced terminal deletions resulting from chromosome healing. The new telomeres are added on with the loss of little or no DNA from the end of the chromosome at sites with short homologies to the telomeric repeat sequences. The new telomeres are much shorter than in the parental cell line, and increase in length with time in culture, consistent with de novo synthesis by telomerase. In the current grant proposal we will utilize this assay system to investigate the mechanism of chromosome healing, the factors that influence this process, and the importance of chromosome healing in the cellular response to DNA double-strand breaks. The formation of terminal deletions by chromosome healing will be studied in mouse cell lines with deficiencies in telomerase, DNA double-strand-break repair and cell cycle regulation to determine the role of these pathways in this process. The ES cell clones will also be used to establish transgenic mice, which will provide a method for studying chromosome healing in a variety of different cell types and for investigating chromosome healing in different genetic backgrounds. Using these approaches we will be able to test the hypothesis that chromosome healing is a cell-type specific mechanism that can influence the cellular response to DNA double-strand breaks. The ability of this system to monitor telomere length and the rate of telomere loss will also provide a means of investigating the role of different genes in maintaining telomere stability.