Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, School of Medicine, Birmingham, AL, USA.
Cell Cycle. 2010 Feb 15;9(4):815-9. doi: 10.4161/cc.9.4.10730. Epub 2010 Feb 23.
Using Drosophila early egg extracts we have developed an optimized cell free system to study DNA replication. The efficiency of replication depends on a cold treatment of Drosophila embryos before the extract preparation and a formation of nuclei facilitated by the addition of membrane fractions to the extracts. In vitro DNA replication is ORC and CDC6 dependent, as a removal of these proteins from the extracts abolishes DNA replication. The N-terminal part of Orc1 protein, which is important for non-replicative functions of ORC, is dispensable for the replication in vitro. We also show that the conserved ATP ase motif of CDC6 is crucial for the replication. Our studies indicate that a Drosophila cell free system proves to be an extremely useful tool for a functional dissection of the processes and factors involved in DNA replication in metazoans.
利用果蝇早期卵提取物，我们开发了一种优化的无细胞体系来研究 DNA 复制。复制效率取决于在提取物制备前对果蝇胚胎进行低温处理，以及通过向提取物中添加膜部分来促进核的形成。体外 DNA 复制依赖于 ORC 和 CDC6，因为从提取物中去除这些蛋白质会导致 DNA 复制的丧失。Orc1 蛋白的 N 端部分对于 ORC 的非复制功能很重要，但对于体外复制是可有可无的。我们还表明，CDC6 的保守 ATP 酶基序对于复制至关重要。我们的研究表明，果蝇无细胞体系是研究真核生物 DNA 复制过程和参与因素的功能分析的极其有用的工具。
