MECHANISM OF FORMATION OF TERMINAL DELETIONS

染色体末端缺失导致约10%的人类遗传性疾病。到目前为止，所有导致遗传疾病的末端缺失都是由于新的端粒直接添加到断裂染色体的末端。 丢失的端粒的恢复，称为染色体愈合，已经在单细胞生物中被广泛研究，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（ES）细胞中开发的测定系统来研究末端缺失形成的机制和参与染色体愈合的基因。该系统利用含有选择性标记基因的质粒序列和紧邻端粒整合的I-SceI内切核酸酶识别序列。 I-SceI识别序列用于特异性地引入双链断裂，而选择性标记基因用于鉴定在标记染色体末端具有末端缺失的细胞。 含有I-SceI内切核酸酶基因的表达载体的电穿孔产生染色体愈合导致的末端缺失。 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增加的，几乎没有或没有DNA损失。 新的端粒比亲本细胞系中的短得多，并且在培养中随着时间的推移长度增加，与端粒酶的从头合成一致。 在目前的拨款提案中，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控缺陷的小鼠细胞系中研究通过染色体愈合形成末端缺失，以确定这些途径在此过程中的作用。 ES细胞克隆也将用于建立转基因小鼠，这将为研究各种不同细胞类型中的染色体愈合和研究不同遗传背景中的染色体愈合提供一种方法。 使用这些方法，我们将能够测试的假设，染色体愈合是一种细胞类型的具体机制，可以影响细胞的DNA双链断裂的反应。 该系统监测端粒长度和端粒丢失率的能力也将提供研究不同基因在维持端粒稳定性中的作用的手段。

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.