Paranjape Neha P, Calvi Brian R
Department of Biology, Indiana University, Bloomington, Indiana 47405.
Department of Biology, Indiana University, Bloomington, Indiana 47405
G3 (Bethesda). 2016 Jun 1;6(6):1661-71. doi: 10.1534/g3.116.028068.
Eukaryotic DNA replication begins from multiple origins. The origin recognition complex (ORC) binds origin DNA and scaffolds assembly of a prereplicative complex (pre-RC), which is subsequently activated to initiate DNA replication. In multicellular eukaryotes, origins do not share a strict DNA consensus sequence, and their activity changes in concert with chromatin status during development, but mechanisms are ill-defined. Previous genome-wide analyses in Drosophila and other organisms have revealed a correlation between ORC binding sites and the histone variant H3.3. This correlation suggests that H3.3 may designate origin sites, but this idea has remained untested. To address this question, we examined the enrichment and function of H3.3 at the origins responsible for developmental gene amplification in the somatic follicle cells of the Drosophila ovary. We found that H3.3 is abundant at these amplicon origins. H3.3 levels remained high when replication initiation was blocked, indicating that H3.3 is abundant at the origins before activation of the pre-RC. H3.3 was also enriched at the origins during early oogenesis, raising the possibility that H3.3 bookmarks sites for later amplification. However, flies null mutant for both of the H3.3 genes in Drosophila did not have overt defects in developmental gene amplification or genomic replication, suggesting that H3.3 is not essential for the assembly or activation of the pre-RC at origins. Instead, our results imply that the correlation between H3.3 and ORC sites reflects other chromatin attributes that are important for origin function.
真核生物的DNA复制起始于多个位点。起始识别复合体（ORC）结合起始DNA，并搭建前复制复合体（pre-RC）的组装框架，随后该复合体被激活以启动DNA复制。在多细胞真核生物中，起始位点并不共享严格的DNA共有序列，并且它们的活性在发育过程中会随着染色质状态的变化而协同改变，但其机制仍不清楚。先前在果蝇和其他生物体中进行的全基因组分析揭示了ORC结合位点与组蛋白变体H3.3之间的相关性。这种相关性表明H3.3可能指定了起始位点，但这一观点尚未得到验证。为了解决这个问题，我们研究了H3.3在果蝇卵巢体细胞卵泡细胞中负责发育基因扩增的起始位点处的富集情况及其功能。我们发现H3.3在这些扩增子起始位点处含量丰富。当复制起始被阻断时，H3.3水平仍然很高，这表明在pre-RC激活之前，H3.3在起始位点处就已经大量存在。在早期卵子发生过程中，H3.3在起始位点处也有富集，这增加了H3.3标记位点以供后期扩增的可能性。然而，果蝇中两个H3.3基因均为无效突变体的果蝇在发育基因扩增或基因组复制方面并没有明显缺陷，这表明H3.3对于起始位点处pre-RC的组装或激活并非必不可少。相反，我们的结果表明H3.3与ORC位点之间的相关性反映了对起始功能很重要的其他染色质属性。
