Role of Fragile Sites in Chromosome Breakage and Cancer

描述（由申请方提供）：常见的脆性位点是当DNA复制受到干扰时，中期染色体上容易出现缺口和断裂的区域。这些位点跨越数百个酶，不是由突变引起的，而是染色体结构的正常组成部分。在过去的十年中，我们和其他人已经表明，常见的脆性位点是易位，缺失，姐妹染色单体交换和整合转染的DNA在培养细胞中的“热点”。随着脆性位点基因座的克隆，一些研究表明，常见的脆性位点在体内许多肿瘤细胞中显示出相同的特征，并加强了一个长期持有的假设，即常见的脆性位点参与癌症的基因组重排。然而，很少有人知道的分子机制负责不稳定的脆弱网站。我们最近确定了影响脆性位点稳定性的主要分子途径。这些是S内和G2/M检查点通路，其中ATR和BRCA 1基因发挥核心作用。由此，我们假设脆性位点是通常激活S/G2-M检查点和修复途径的未复制区域，并且肿瘤细胞中的脆性位点和相关重排是停滞复制叉的“签名”。我们将在这些重要的新发现和过去十年中的其他发现的基础上，进一步解决什么样的分子机制负责脆性位点的维持和不稳定性，以及染色体结构的什么成分负责这种不稳定性的问题。本论文的主要目的是：（1）进一步阐明S期和G2/M期检查点通路相关基因在脆性位点维持和不稳定性中的作用;（2）研究这些基因在其他生物学相关因子诱导脆性位点和脆性X位点诱导中的作用;（3）研究新近发现的参与停滞复制叉修复的基因在脆性位点稳定性和修复中的作用;（4）确定负责脆性位点不稳定性的序列或染色质结构;和（5）使用常规和新的分子梳理方法来确定复制定时、复制起点的间距、复制叉速度和脆性位点处的起点发射定时。从这些研究中，我们将进一步阐明脆弱位点维持，不稳定性和修复的具体分子机制，并提供新的见解关键的基因的功能检查点控制和DNA修复，以应对停滞的复制叉及其对人类染色体完整性的影响。

DESCRIPTION (provided by applicant): Common fragile sites are regions prone to gaps and breaks on metaphase chromosomes when DNA replication is perturbed. These sites, which span hundreds of kilobases, do not arise from mutation but are a normal component of chromosome structure. Over the past decade, we and others have shown that common fragile sites are "hot spots" for translocations, deletions, sister chromatid exchanges and integration of transfected DNA in cultured cells. With the cloning of fragile site loci, several lines of investigation have shown that common fragile sites display the same characteristics in many tumor cells, in vivo, and have strengthened a long-held hypothesis that common fragile sites are involved in genome rearrangements in cancer. Yet, little is known about the molecular mechanisms responsible for instability at fragile sites. We have recently identified the major molecular pathways affecting fragile site stability. These are the intra-S and G2/M checkpoint pathways in which the ATR and BRCA1 genes play central roles. From this, we hypothesize that fragile sites are unreplicated regions that normally activate the S/G2-M checkpoint and repair pathways, and that fragile sites and associated rearrangements in tumor cells are "signatures" of stalled replication forks. We will build on these important new findings and others made over the past decade to further address the question of what molecular mechanisms are responsible for fragile site maintenance and instability and what components of chromosome structure are responsible for this instability. Five aims are proposed to address these questions: (1) to further elucidate the role of genes involved in the S-phase and G2/M checkpoint pathways on fragile site maintenance and instability; (2) to examine the role of these genes in the induction of fragile sites by additional biologically-relevant agents and in induction of the fragile X site; (3) to investigate the role of genes recently implicated in the repair of stalled replication forks in fragile site stability and repair; (4) to determine the sequences or chromatin structure responsible for fragile site instability; and (5) to determine replication timing, the spacing of replication origins, replication fork velocity and timing of origin firing at fragile sites, using conventional and new molecular combing approaches. From these studies, we will further elucidate the specific molecular mechanisms underlying fragile site maintenance, instability and repair, and provide new insights into the function of genes critical to checkpoint control and DNA repair in response to stalled replication forks and their effects on human chromosome integrity.