Distribution of DNA replication proteins in Drosophila cells
Distribution of DNA replication proteins in Drosophila cells
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DOI：
10.1186/1471-2121-8-42
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发表时间：
2007-10-15
期刊：
影响因子：
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通讯作者：
Cardoso, M. Cristina
中科院分区：
文献类型：
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作者：
Easwaran, Hariharan P.;Leonhardt, Heinrich;Cardoso, M. Cristina
背景：高等真核细胞中的DNA复制是在称为复制点(Replica Focus，RF)的离散亚核位置上组织的。在S期，大多数复制蛋白通过增殖细胞核抗原结合域(PBD)与增殖细胞核抗原相互作用而在RF处组装。结果：哺乳动物复制蛋白Dnmt1、HsDNA Lig I和HsPCNA的荧光融合在果蝇细胞中分析了它们靶向RF的能力。除HsPCNA外，其他蛋白及其缺失均未在果蝇细胞的Rf处积聚。我们推测，在果蝇细胞中，可能还有其他一些肽序列负责将蛋白质靶向RF。为了验证这一点，我们鉴定了DmDNA Lig I，并将其蛋白质序列与HsDNA Lig I进行了比较。这两个同源基因共享PBD，表明该结构域在果蝇中具有功能保守的作用。对DmDNA Lig I的一系列缺失进行了分析，以了解它们在果蝇和哺乳动物细胞中聚集在RF的能力。令人惊讶的是，在果蝇细胞中没有观察到RF的蓄积，而在哺乳动物细胞中，DmDNA Lig I通过其PBD在RF蓄积。此外，与Dnmt1、HsDNA Lig I和DmDNA Lig I的PBD结构域融合的GFP只能在哺乳动物细胞中靶向RF，而在果蝇细胞中不能靶向RF。结论：在哺乳动物细胞中，以增殖细胞核抗原样标记物标记的RF的形成是果蝇细胞S期的特征。然而，除增殖细胞核抗原外，在果蝇细胞中，没有一种复制蛋白及其缺失在Rf处积累，而在哺乳动物细胞中，相同的蛋白和缺失能够在Rf处积累。我们假设，与哺乳动物细胞不同，在果蝇细胞中，复制蛋白不会与复制机制形成长期的相互作用，而是通过在RF上非常短暂的相互作用来执行其功能。
Background: DNA replication in higher eukaryotic cells is organized in discrete subnuclear sites called replication foci (RF). During the S phase, most replication proteins assemble at the RF by interacting with PCNA via a PCNA binding domain (PBD). This has been shown to occur for many mammalian replication proteins, but it is not known whether this mechanism is conserved in evolution.Results: Fluorescent fusions of mammalian replication proteins, Dnmt1, HsDNA Lig I and HsPCNA were analyzed for their ability to target to RF in Drosophila cells. Except for HsPCNA, none of the other proteins and their deletions showed any accumulation at RF in Drosophila cells. We hypothesized that in Drosophila cells there might be some other peptide sequence responsible for targeting proteins to RF. To test this, we identified the DmDNA Lig I and compared the protein sequence with HsDNA Lig I. The two orthologs shared the PBD suggesting a functionally conserved role for this domain in the Drosophila counterpart. A series of deletions of DmDNA Lig I were analyzed for their ability to accumulate at RF in Drosophila and mammalian cells. Surprisingly, no accumulation at RF was observed in Drosophila cells, while in mammalian cells DmDNA Lig I accumulated at RF via its PBD. Further, GFP fusions with the PBD domains from Dnmt1, HsDNA Lig I and DmDNA Lig I, were able to target to RF only in mammalian cells but not in Drosophila cells.Conclusion: We show that S phase in Drosophila cells is characterized by formation of RF marked by PCNA like in mammalian cells. However, other than PCNA none of the replication proteins and their deletions tested here showed accumulation at RF in Drosophila cells while the same proteins and deletions are capable of accumulating at RF in mammalian cells. We hypothesize that unlike mammalian cells, in Drosophila cells, replication proteins do not form long-lasting interactions with the replication machinery, and rather perform their functions via very transient interactions at the RF.