Differential DNA Replication in Drosophila Development

描述(申请人提供)：DNA复制必须被精确调控以保持基因拷贝数。癌细胞包含许多基因拷贝数被放大的区域，这些区域与肿瘤的进展有关，但导致基因扩增的主要机制尚不清楚。复制失败可能会导致染色体脆性部位，从而导致染色体不稳定。复制启动所需蛋白质的突变与人类发育缺陷有关。确定如何激活或停用复制源以及如何控制复制分叉进度至关重要 在后生动物细胞中。这就需要对个体起源的分析，复制起始蛋白的定位，以及检查分叉进展的方法。这项研究将利用果蝇基因组区域的发育模型，这些区域因果蝇的分化而在拷贝数上被放大或复制不足。这些模型提供了明确的复制起点，对人类细胞复制至关重要的复制起始蛋白可以被定位，并可以在分化的细胞中进行分析，因为它们经历了DNA复制的启动。它们还定义了允许或阻碍复制分叉进展的特定域。这些模型为描述其他后生动物系统中不存在的复制启动和延长机制提供了一个范例。这项建议的具体目的是利用基因组学、遗传学和生物化学的跨学科方法，使用这些模型来回答关于复制启动和分叉进展的基本问题。导致起源激活的机制将被定义为两个扩增结构域，基因组中允许或限制起始的区域将被调查，以确定起源识别复合体(ORC)是如何定位的，以及是否存在ORC不依赖于ORC的起始机制。在第二个目的中，阻止起始的基因组区域将被用来描述染色质配置对ORC结合和起始激活的影响。最后一个目的是通过识别促进或阻止叉子运动的蛋白质来确定染色质是如何控制复制叉子的进程的。将对突变体确定的候选蛋白质和定位模式进行研究。这一目标还将分析叉口双链断裂修复的要求。

DESCRIPTION (provided by applicant): DNA replication must be regulated precisely to maintain gene copy number. Cancer cells contain many regions in which gene copy number has been amplified and these are associated with tumor progression, but the primary mechanisms causing gene amplification are unknown. Failure of replication may contribute to chromosome fragile sites that lead to chromosomal instability. Mutations in proteins needed for replication initiation are associated with human developmental defects. It is crucial to determine how replication origins are activated or inactivated and how replication fork progression is controlled in metazoan cells. This necessitates the analysis of individual origins, the localization of replication initiation proteins, and ways to examine fork progression. This research will exploit developmental models of genomic regions that become amplified in copy number or under-replicated in response to differentiation in Drosophila. These models provide defined replication origins to which replication initiation proteins, essential for replication in human cells, can be localized and that can be analyzed in differentiated cells as they undergo initiation of DNA replication. They also define specific domains through which replication fork progression is permitted or impeded. These models provide a paradigm for delineating the mechanisms of replication initiation and elongation not present in another metazoan system. The Specific Aims of this proposal are to use these models to answer fundamental questions about replication initiation and fork progression, utilizing interdisciplinary approaches of genomics, genetics and biochemistry. The mechanisms leading to origin activation will be defined for two amplified domains, regions of the genome permissive or restrictive for initiation will be investigated to determine how the Origin Recognition Complex (ORC) is localized and whether there are ORC- independent mechanisms of initiation. In the second aim, genomic regions that block initiation will be used to delineate the effects of chromatin configuration on ORC binding and origin activation. The last aim is to determine how replication fork progression is controlled by chromatin by identifying proteins that promote or block fork movement. Candidate proteins identified by mutants and localization patterns will be investigated. This aim also will analyze th requirement for double-strand break repair at the forks.