Jaunin F, Visser A E, Cmarko D, Aten J A, Fakan S
Centre of Electron Microscopy, University of Lausanne, 27 Bugnon, Lausanne, CH-1005, Switzerland.
Exp Cell Res. 2000 Nov 1;260(2):313-23. doi: 10.1006/excr.2000.4999.
Nascent DNA (newly replicated DNA) was visualized in situ with regard to the position of the previously replicated DNA and to chromatin structure. Localization of nascent DNA at the replication sites can be achieved through pulse labeling of cells with labeled DNA precursors during very short periods of time. We were able to label V79 Chinese Hamster cells for as shortly as 2 min with BrdU; Br-DNA, detected by immunoelectron microscopy, occurs at the periphery of dense chromatin, at individual dispersed chromatin fibers, and within dispersed chromatin areas. In these regions DNA polymerase alpha was also visualized. After a 5-min BrdU pulse, condensed chromatin also became labeled. When the pulse was followed by a chase, a larger number of gold particles occurred on condensed chromatin. Double-labeling experiments, consisting in first incubating cells with IdU for 20 min, chased for 10 min and then labeled for 5 min with CldU, reveal CldU-labeled nascent DNA on the periphery of condensed chromatin, while previously replicated IdU-labeled DNA has been internalized into condensed chromatin. Altogether, these results show that the sites of DNA replication correspond essentially to perichromatin regions and that the newly replicated DNA moves rapidly from replication sites toward the interior of condensed chromatin areas.
就先前复制的DNA的位置和染色质结构而言，对新生DNA（新复制的DNA）进行了原位观察。通过在极短的时间内用标记的DNA前体对细胞进行脉冲标记，可以实现新生DNA在复制位点的定位。我们能够用BrdU对V79中国仓鼠细胞进行短至2分钟的标记；通过免疫电子显微镜检测到的Br-DNA出现在致密染色质的周边、单个分散的染色质纤维以及分散的染色质区域内。在这些区域也观察到了DNA聚合酶α。在进行5分钟的BrdU脉冲后，浓缩染色质也被标记。当脉冲后进行追踪时，在浓缩染色质上出现了更多的金颗粒。双标记实验包括首先用IdU孵育细胞20分钟，追踪10分钟，然后用CldU标记5分钟，结果显示在浓缩染色质的周边有CldU标记的新生DNA，而先前复制的IdU标记的DNA已内化到浓缩染色质中。总之，这些结果表明DNA复制位点基本上对应于染色质周边区域，并且新复制的DNA迅速从复制位点向浓缩染色质区域的内部移动。
