Differential DNA Replication in Drosophila Development

描述（由申请人提供）：DNA复制必须精确调节以维持基因拷贝数。癌细胞包含许多基因拷贝数扩增的区域，这些区域与肿瘤进展相关，但导致基因扩增的主要机制尚不清楚。复制失败可能导致染色体脆性位点，导致染色体不稳定。复制起始所需的蛋白质突变与人类发育缺陷有关。确定复制起点是如何被激活或失活的以及复制叉的进展是如何被控制的是至关重要的 在后生动物细胞中。这就需要分析个体的起源，复制起始蛋白的定位，以及检查叉进展的方法。 这项研究将利用基因组区域的发育模型，这些基因组区域在果蝇中的分化反应中拷贝数扩增或复制不足。这些模型提供了定义的复制起点，其中复制起始蛋白，在人类细胞中复制所必需的，可以定位，并可以在分化的细胞中进行分析，因为它们经历DNA复制的起始。它们还定义了允许或阻止复制叉进展的特定域。这些模型提供了一个范例，描绘复制启动和延伸的机制不存在于另一个后生动物系统。 该提案的具体目标是利用基因组学，遗传学和生物化学的跨学科方法，使用这些模型来回答有关复制起始和叉进展的基本问题。将针对两个扩增的结构域定义导致起始激活的机制，将研究基因组中允许或限制起始的区域，以确定起始识别复合物（ORC）如何定位以及是否存在ORC非依赖性起始机制。在第二个目标中，阻断起始的基因组区域将用于描绘染色质构型对ORC结合和起始激活的影响。最后一个目的是确定如何复制叉的进展是由染色质控制，通过识别蛋白质，促进或阻止叉运动。将研究通过突变体和定位模式鉴定的候选蛋白。本研究还将分析分叉处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.