A cytokinetic function of Drosophila ORC6 protein resides in-a domain distinct from its replication activity
A cytokinetic function of Drosophila ORC6 protein resides in-a domain distinct from its replication activity
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DOI：
10.1073/pnas.1633580100
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发表时间：
2003-08-05
影响因子：
11.1
通讯作者：
Botchan, M
中科院分区：
文献类型：
--
作者：
Chesnokov, IN;Chesnokova, ON;Botchan, M
对共同蛋白质因子的需求可能促进了不同途径之间的协调，这一发现与调节DNA复制的蛋白质与其他重要细胞过程之间的联系一致。我们报道了最小的果蝇起源识别复合物亚基Orc6，在胚胎和细胞培养中发现，定位于细胞膜和细胞分裂时的分裂沟以及细胞核中。双杂交筛选显示Orc6与果蝇花生（pnut）相互作用，pnut是septin家族的一员，对细胞分裂很重要。这种相互作用由Orc6的一个独特的c端结构域介导，通过萃取物的共免疫沉淀和细胞学方法在果蝇细胞中得到证实。用双链RNA沉默Orc6的表达导致多核细胞的形成，也减少了DNA的复制。c端Orc6-花生相互作用结构域的缺失和随后Orc6突变蛋白的过表达导致了DNA复制的多核细胞的形成。这些结果表明Orc6进化出了一个主要对细胞质分裂至关重要的结构域。
Coordination between separate pathways may be facilitated by the requirements for common protein factors, a finding congruent with the link between proteins regulating DNA replication with other important cellular processes. We report that the smallest of Drosophila origin recognition complex subunits, Orc6, was found in embryos and cell culture localized to the cell membrane and cleavage furrow during cell division as well as in the nucleus. A two-hybrid screen revealed that Orc6 interacts with the Drosophila peanut (pnut), a member of the septin family of proteins important for cell division. This interaction, mediated by a distinct C-terminal domain of Orc6, was substantiated in Drosophila cells by coimmunoprecipitation from extracts and cytological methods. Silencing of Orc6 expression with double-stranded RNA resulted in a formation of multinucleated cells and also reduced DNA replication. Deletion of the C-terminal Orc6-peanut interaction domain and subsequent overexpression of the Orc6 mutant protein resulted in the formation of multinucleated cells that had replicated DNA. This mutant protein does not localize to the membrane or cleavage furrows' These results suggest that Orc6 has evolved a domain critical mainly for cytokinesis.