Visualization of replication initiation and elongation in Drosophila.
Visualization of replication initiation and elongation in Drosophila.
复制标题
果蝇中复制启动和伸长的可视化。
DOI：
10.1083/jcb.200207046
复制
发表时间：
2002-10-28
影响因子：
7.8
通讯作者：
Orr-Weaver, Terry L
中科院分区：
文献类型：
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作者：
Claycomb, Julie M;MacAlpine, David M;Evans, James G;Bell, Stephen P;Orr-Weaver, Terry L
果蝇卵巢绒毛膜基因扩增是研究后生动物体内DNA复制的有力系统。利用高分辨率共聚焦和去卷积显微镜以及实时定量聚合酶链式反应，我们发现起始和延伸发生在不同的发育阶段，因此可以在体内分析这两个复制阶段。对溴脱氧尿苷、来源识别复合体、延伸因子微小染色体维持蛋白(MCM)2-7和增殖细胞核抗原进行了精确定位，并对不同发育阶段第三染色体绒毛膜扩增片段的DNA拷贝数进行了定量。这些研究表明，起始发生在卵室发育的10B和11阶段，而现有复制叉只有在卵室第12和13阶段发生伸长。区分起始和伸长的能力使这一模型成为破解各种复制因子在后生动物DNA复制过程中作用的杰出模型。我们利用这个系统来证明复制前复杂成分，双停放的蛋白质/细胞分裂周期10依赖的转录本1，不仅是正确定位MCM2-7所必需的，而且出人意料地存在于伸长过程中。
Chorion gene amplification in the ovaries of Drosophila melanogaster is a powerful system for the study of metazoan DNA replication in vivo. Using a combination of high-resolution confocal and deconvolution microscopy and quantitative realtime PCR, we found that initiation and elongation occur during separate developmental stages, thus permitting analysis of these two phases of replication in vivo. Bromodeoxyuridine, origin recognition complex, and the elongation factors minichromosome maintenance proteins (MCM)2–7 and proliferating cell nuclear antigen were precisely localized, and the DNA copy number along the third chromosome chorion amplicon was quantified during multiple developmental stages. These studies revealed that initiation takes place during stages 10B and 11 of egg chamber development, whereas only elongation of existing replication forks occurs during egg chamber stages 12 and 13. The ability to distinguish initiation from elongation makes this an outstanding model to decipher the roles of various replication factors during metazoan DNA replication. We utilized this system to demonstrate that the pre–replication complex component, double-parked protein/cell division cycle 10–dependent transcript 1, is not only necessary for proper MCM2–7 localization, but, unexpectedly, is present during elongation.