Causes and Consequences of Genomic Instability at Fragile Sites

描述(由申请人提供)： 6.项目摘要癌细胞中的染色体含有导致细胞不正常生长的变化。当细胞不能正确修复DNA中的断裂或在复制DNA的过程中遇到困难时，就会导致基因变化。当带有这些变化的肿瘤细胞分裂和繁殖时，它们会将这些基因变化传递给它们的子细胞，最终产生转移性肿瘤。染色体的某些区域比其他区域更容易发生基因变化。许多类型的肿瘤被证明在被称为“脆性部位”的区域含有异常。人类细胞中的脆弱部位通常是稳定的，但当复制DNA的过程减慢或停滞时，这些部位就是断裂的热点。酵母细胞也含有脆弱的部位，我们将使用酵母模型系统的强大遗传工具来设计在人类细胞中技术上困难的实验。在这个提议的第一个目的中，我们将研究在有丝分裂过程中通过同源重组修复断裂时发生的DNA复制的保真度和程度。肿瘤中细胞的异常生长通常是由与细胞生长和DNA修复有关的基因的扩增或缺失引起的。因此，在第二个目标中，我们将研究酵母脆弱部位断裂所刺激的拷贝数量的变化，使用一种系统，允许我们选择丢失或获得基因拷贝的酵母，使其能够在含有铜和甲醛的介质上生长。由于酵母脆性位点的结构比人类的更简单，在第三个目标中，我们将研究酵母染色体上携带的人类脆性位点序列中的DNA复制过程。这一目的的实验将检验关于已提出的模型的假设，这些模型被用来解释为什么人类脆弱部位会形成断裂。这些研究脆弱部位DNA损伤和修复停滞复制叉处的实验将帮助我们了解促进肿瘤细胞基因变化的情况和环境。

DESCRIPTION (provided by applicant): 6. PROJECT SUMMARY Chromosomes in cancer cells contain changes that cause improper cell growth. When a cell can't correctly repair breaks in its DNA or has difficulty during the process of copying its DNA, genetic changes can result. When tumor cells with these changes divide and reproduce, they pass these genetic changes on to their daughter cells, eventually creating a metastasized tumor. Some regions of chromosomes are more prone to genetic changes than others. Many types of tumors have been shown to contain abnormalities at regions called "fragile sites." Fragile sites in human cells are normally stable, but when the process of copying DNA is slowed or stalled, these sites are hotspots for breaks. Yeast cells also contain fragile sites, and we will use the powerful genetic tools of the yeast model system to design experiments that would be technically difficult in human cells. In the first aim of this proposal, we will study the fidelity and extent of DNA replication that occurs when breaks are repaired during mitosis by homologous recombination. Abnormal cell growth in tumors is often caused by amplification or deletion of genes involved in cell growth and DNA repair. Thus, in the second aim we will study variations in copy number stimulated by breaks at a yeast fragile site, using a system that allows us to select for yeast that have lost or gained copies of genes that enable it to grow on media containing copper and formaldehyde. Because yeast fragile sites have a simpler structure than human ones, in the third aim, we will study the process of DNA replication in human fragile site sequences that are carried on yeast chromosomes. The experiments in this aim will test hypotheses about the models that have been proposed to explain why breaks form in human fragile sites. These experiments to study DNA damage at fragile sites and repair at stalled replication forks will help us understand the situations and environments that promote genetic changes in tumor cells.