Differential DNA Replication in Drosophila Development

描述（由申请人提供）：控制DNA复制对于确保准确的基因拷贝数至关重要，因为基因拷贝的丢失和扩增的拷贝数增加都与癌症的发生和转移状态的进展有关。后生动物DNA复制的调控机制的阐明一直受到难以确定复制的具体起源和监测其激活的阻碍。大多数动物，包括人类，都含有多倍体组织，在多倍体组织中，细胞的DNA含量通过修饰的细胞周期（内切周期）增加，缺乏有丝分裂。除了基因组DNA的整体增加外，在许多多倍体细胞中，差异DNA复制发生在特定基因组间隔不复制或在某些情况下过度复制和扩增的情况下。这些差异DNA复制的实例为阐明后生动物DNA复制起源的结构和调控提供了极好的模型。利用基因组学方法，在果蝇卵巢卵泡细胞中发现了扩增的基因组区域，在果蝇幼虫的唾液腺中发现了单拷贝、共染的欠复制区域。扩增区域内的复制起点受发育控制，并允许对起点激活和抑制的因素进行分析，以及发现后生动物DNA复制启动的机制。本研究的目的是利用这些果蝇复制模型来定义在卵泡细胞分化过程中激活起始点的机制，确定复制起始点是如何失活的，并利用突变体来识别新的调节蛋白，并分析控制复制叉进展的机制。定义复制起源的识别，其复制可以量化，检测的能力

DESCRIPTION (provided by applicant): Control of DNA replication is critical to ensure accurate copy number of genes, because both loss of gene copies and increased copy number by amplification are associated with the onset of cancer and progression to metastatic states. Elucidation of regulatory mechanisms for metazoan DNA replication has been hampered by difficulty in defining specific origins of replication and monitoring their activation. Most animals, including humans, contain polyploid tissues in which the DNA content of the cells is increased by a modified cell cycle, the endo cycle, lacking mitosis. In addition to the overall increase in genomic DNA, in many polyploid cells differential DNA replication occurs in which specific genomic intervals are not replicated or in some cases are over-replicated and amplified. These instances of differential DNA replication provide superb models for elucidating the structure and regulation of metazoan DNA replication origins. Using genomic methodologies, amplified genomic regions were identified in the Drosophila ovarian follicle cells and single copy, euchromatic underreplicated regions were found in the larval salivary gland. The replication origins within the amplified regions are subject to developmental control and permit analysis of the factors responsible for origin activation and repression, as well as discovery of mechanisms by which metazoan DNA replication is initiated. The aims of this research are to exploit these Drosophila replication models to define the mechanisms that activate initiation at the origins during follicle cell differentiation, to determine how replication origins are inactivated and to use mutants to identify new regulatory proteins, and to analyze mechanisms controlling replication fork progression. The identification of defined replication origins whose duplication can be quantified, the ability to detect replication proteins bound at these origins and moving with the replication forks, combined with a collection of mutants affecting these processes permits these experimental goals to be achieved.