Molecular Studies of Cancer Specific Fragile Sites

描述（由申请人提供）：染色体脆性位点的不稳定性与许多癌症直接相关。在三种不同的培养条件下产生三种类型的脆性位点：FRA3B，aphidicolin诱导的脆性位点，FRA11B，叶酸敏感位点，和FRA16B，偏端霉素A诱导位点，参与癌症的形成。在脆性部位发现了紧密的染色质结构和不寻常的DNA序列。此外，脆弱的网站显示复制延迟，可以逃脱ATR依赖的复制检查点。这些观察结果为脆性位点的性质提供了一个有趣的模型，其中这些位点的染色质和DNA结构将暂停复制叉的进展，并破坏细胞周期检查点途径，以允许染色体重排和病毒整合，导致脆性位点特异性肿瘤发生。提出了三个目标：（1）确定独特的决定因素，为脆弱的位点特异性染色质。将采用染色质免疫沉淀（CHIP）试验来鉴定这些位点涉及的表观遗传标记。此外，通过在所有三种脆性DNA上重建染色质，将分析脆性位点特异性染色质结构，并鉴定参与脆性染色质形成的必要组分。(2)建立参与脆性位点表达的细胞周期检查点通路。使用CHIP测定和通过RNA干扰产生的突变细胞，将检查几种细胞周期检查点蛋白在脆性位点表达中的参与，以确定它们与脆性DNA的关联。(3)通过使用SV40复制模型系统确定影响脆性位点不稳定性的顺式和反式因子。通过操作脆性DNA（长度、序列、复制方向和相对于复制起点的位置）和细胞周期检查点组分，评价脆性位点不稳定性（断裂位点的产生和重复序列长度的变化）。还将表征含有脆性DNA的复制中间体，以提供关于脆性位点的复制延迟的直接信息。 这个建议解决了脆弱的网站如何导致致癌病变在三个连续的步骤。因此，这些实验将进一步推进有关这些脆弱位点的性质及其在癌症形成中的作用的知识，并解决基本的生物学问题，如染色质结构的决定因素。

DESCRIPTION (provided by applicant): Instability of chromosomal fragile sites is directly related to many cancers. Three types of fragile sites are generated under three different culture conditions: FRA3B, an aphidicolin-inducible fragile site, FRA11B, a folate-sensitive site, and FRA16B, a distamycin-A-inducible site, are involved in the formation of cancers. Compact chromatin structures and unusual DNA sequences have been found in fragile sites. Also, fragile sites display replication delay and can escape the ATR-dependent replication checkpoint. These observations provide an intriguing model for the nature of fragile sites, in which chromatin and DNA structures at these sites would pause the progress of the replication fork, and disrupt cell cycle checkpoint pathways to allow chromosomal rearrangement and viral integration, resulting in fragile site-specific tumorigenesis. Three goals are proposed: (1) Identify unique determinants for the fragile site-specific chromatin. Chromatin immunoprecipitation (CHIP) assays will be employed to identify epigenetic marks involved in these sites. Further, by reconstituting chromatin over all three fragile DNAs, fragile site-specific chromatin structure will be analyzed, and the essential components involved in the formation of fragile chromatin will be identified. (2) Establish cell cycle checkpoint pathways involved in the expression of fragile sites. Using CHIP assay and mutant cells created by RNA interference, the involvement of several cell cycle checkpoint proteins in the expression of fragile sites will be examined for their association with fragile DNA. (3) Determine cis- and trans-factors affecting fragile site instability by using an SV40 replication model system. By manipulating fragile DNA (length, sequences, replication direction, and location relative to replication origin) and cell cycle checkpoint components, fragile site instability (generation of break sites and changes in repeat length) will be evaluated. Replication intermediates containing fragile DNAs will also be characterized to provide direct information about replication delay of fragile sites. This proposal addresses how fragile sites lead to oncogenic lesions at three sequential steps. Therefore, these experiments will further advance knowledge about the nature of these fragile sites and their role in the formation of cancer, and also address fundamental biological questions, such as determinants of chromatin structure.