Dynamics of DNA replication factories in living cells.

Dynamics of DNA replication factories in living cells.
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DOI:
10.1083/jcb.149.2.271
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发表时间:
2000-04-17
期刊:
The Journal of cell biology
影响因子:
--
通讯作者:
Cardoso MC
Cardoso MC
中科院分区:
其他
文献类型:
--
作者:
Leonhardt H;Rahn HP;Weinzierl P;Sporbert A;Cremer T;Zink D;Cardoso MC

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DNA 复制发生在细胞核中离散位点(复制焦点)的显微镜可见复合物中。这些焦点由与复制机器相关的 DNA 组成,即参与 DNA 复制的大型蛋白质复合物。为了研究活细胞中这些核复制灶的动态,我们将复制机制的核心组成部分增殖细胞核抗原(PCNA)与绿色荧光蛋白(GFP)融合。表达低水平 GFP-PCNA 的稳定细胞系的成像显示复制灶在大小和寿命上是异质的。延时研究表明,复制焦点与核斑点和卷绕体明显不同,因为它们既不表现出定向运动,也不似乎合并或分裂。这四个维度的分析表明,复制工厂稳定地锚定在细胞核中,并且模式的变化是通过整个 S 阶段逐渐、协调但异步的组装和拆卸而发生的。
DNA replication occurs in microscopically visible complexes at discrete sites (replication foci) in the nucleus. These foci consist of DNA associated with replication machineries, i.e., large protein complexes involved in DNA replication. To study the dynamics of these nuclear replication foci in living cells, we fused proliferating cell nuclear antigen (PCNA), a central component of the replication machinery, with the green fluorescent protein (GFP). Imaging of stable cell lines expressing low levels of GFP-PCNA showed that replication foci are heterogeneous in size and lifetime. Time-lapse studies revealed that replication foci clearly differ from nuclear speckles and coiled bodies as they neither show directional movements, nor do they seem to merge or divide. These four dimensional analyses suggested that replication factories are stably anchored in the nucleus and that changes in the pattern occur through gradual, coordinated, but asynchronous, assembly and disassembly throughout S phase.
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