Binding of Drosophila Orc Proteins to Anaphase Chromosomes Requires Cessation of Mitotic Cyclin-Dependent Kinase Activity
Binding of Drosophila Orc Proteins to Anaphase Chromosomes Requires Cessation of Mitotic Cyclin-Dependent Kinase Activity
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DOI：
10.1128/mcb.00981-08
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发表时间：
2009-01-01
影响因子：
5.3
通讯作者：
Gossen, Manfred
中科院分区：
文献类型：
--
作者：
Baldinger, Tina;Gossen, Manfred
真核染色体获得复制能力的第一步是多亚基起始识别复合物ORC的结合。我们描述了一个转基因果蝇模型，使动态成像的绿色荧光蛋白（GFP）标记的果蝇ORC亚基，DmOrc 2-GFP。它在遗传互补中起作用，在生理水平上表达，并定量参与复合物的形成。因此，该融合蛋白能够描述由果蝇起始蛋白形成的全复合物DmOrc 1 -6和核心复合物DmOrc 2 -6。其定位可以在体内沿着细胞周期和发育进行监测。DmOrc 2-GFP在中期染色体上未检测到，但在果蝇胚胎中快速结合到后期染色质。稳定的细胞周期蛋白A、B或B3的表达阻止了这种再结合，表明有丝分裂细胞周期蛋白依赖性激酶活性的停止对于DmOrc蛋白与染色体的结合是必不可少的。
The initial step in the acquisition of replication competence by eukaryotic chromosomes is the binding of the multisubunit origin recognition complex, ORC. We describe a transgenic Drosophila model which enables dynamic imaging of a green fluorescent protein (GFP)-tagged Drosophila melanogaster ORC subunit, DmOrc2-GFP. It is functional in genetic complementation, expressed at physiological levels, and participates quantitatively in complex formation. This fusion protein is therefore able to depict both the holocomplex DmOrc1-6 and the core complex DmOrc2-6 formed by the Drosophila initiator proteins. Its localization can be monitored in vivo along the cell cycle and development. DmOrc2-GFP is not detected on metaphase chromosomes but binds rapidly to anaphase chromatin in Drosophila embryos. Expression of either stable cyclin A, B, or B3 prevents this reassociation, suggesting that cessation of mitotic cyclin-dependent kinase activity is essential for binding of the DmOrc proteins to chromosomes.