Hua Brian L, Li Sharon, Orr-Weaver Terry L
Whitehead Institute, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142 Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142.
Whitehead Institute, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142.
G3 (Bethesda). 2014 Oct 14;4(12):2403-8. doi: 10.1534/g3.114.014050.
The mechanisms that underlie metazoan DNA replication initiation, especially the connection between transcription and replication origin activation, are not well understood. To probe the role of transcription in origin activation, we exploited a specific replication origin in Drosophila melanogaster follicle cells, ori62, which coincides with the yellow-g2 transcription unit and exhibits transcription-dependent origin firing. Within a 10-kb genomic fragment that contains ori62 and is sufficient for amplification, RNA-sequencing analysis revealed that all detected RNAs mapped solely to the yellow-g2 gene. To determine whether transcription is required in cis for ori62 firing, we generated a set of tagged yellow-g2 transgenes in which we could prevent local transcription across ori62 by deletions in the yellow-g2 promoter. Surprisingly, inhibition of yellow-g2 transcription by promoter deletions did not affect ori62 firing. Our results reveal that transcription in cis is not required for ori62 firing, raising the possibility that a trans-acting factor is required specifically for the activation of ori62. This finding illustrates that a diversity of mechanisms can be used in the regulation of metazoan DNA replication initiation.
后生动物DNA复制起始的机制，尤其是转录与复制起点激活之间的联系，目前还不太清楚。为了探究转录在起点激活中的作用，我们利用了果蝇卵泡细胞中的一个特定复制起点ori62，它与yellow-g2转录单元重合，并表现出转录依赖性的起点激发。在一个包含ori62且足以进行扩增的10 kb基因组片段内，RNA测序分析表明，所有检测到的RNA都仅映射到yellow-g2基因。为了确定顺式转录是否是ori62激发所必需的，我们生成了一组带有标签的yellow-g2转基因，通过在yellow-g2启动子中进行缺失，我们可以阻止ori62上的局部转录。令人惊讶的是，启动子缺失对yellow-g2转录的抑制并不影响ori62的激发。我们的结果表明，顺式转录不是ori62激发所必需的，这增加了一种可能性，即需要一种反式作用因子来特异性激活ori62。这一发现表明，后生动物DNA复制起始的调控可以使用多种机制。
