Philimonenko Anatoly A, Jackson Dean A, Hodný Zdenek, Janácek Jirí, Cook Peter R, Hozák Pavel
Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Vídenská 1083, 142 20 Prague 4 Krc, Czech Republic.
J Struct Biol. 2004 Dec;148(3):279-89. doi: 10.1016/j.jsb.2004.08.001.
DNA replication in cells takes place in domains scattered throughout the nucleoplasm. We have characterized the dynamics of DNA synthesis in synchronized mid-S-phase HeLa cells. Saponin-permeabilized cells were allowed to elongate nascent DNA chains in presence of biotin-dUTP for 5, 15, and 30 min (a pulse experiment), or for 5 min followed by an incubation with unlabeled precursors for 10 or 25 min (a pulse-and-chase experiment). The replication foci were then identified in ultrathin sections using immunogold labeling of the incorporated biotin. Total number of particles per nucleus, total scanned area of the nucleus, size, shape, and gold particle number of each labeled cluster, and the density of clusters per nucleus were evaluated. We have demonstrated that as replication proceeds, the labeled sites increase in size up to 240 nm (30 min incorporation) while maintaining a broadly round shape. In pulse-and-chase experiments the labeled DNA was shown to spread to occupy DNA foci of approximately 400 nm in diameter. These results demonstrate that DNA replication is compartmentalized within cell nuclei at the level of DNA foci and support the view that the synthetic centers are spatially constrained while the chromatin loops are dynamic during DNA synthesis.
细胞中的DNA复制发生在分散于整个核质中的区域。我们已经对同步化处于S期中期的HeLa细胞中DNA合成的动力学进行了表征。用皂角苷通透处理的细胞在生物素-dUTP存在的情况下，使新生DNA链延伸5分钟、15分钟和30分钟（脉冲实验），或者延伸5分钟，随后与未标记的前体一起孵育10分钟或25分钟（脉冲追踪实验）。然后，通过对掺入的生物素进行免疫金标记，在超薄切片中鉴定复制灶。评估每个细胞核的颗粒总数、细胞核的总扫描面积、每个标记簇的大小、形状和金颗粒数量，以及每个细胞核中簇的密度。我们已经证明，随着复制的进行，标记位点的大小增加到240纳米（掺入30分钟），同时保持大致圆形。在脉冲追踪实验中，标记的DNA显示会扩散，占据直径约400纳米的DNA灶。这些结果表明，DNA复制在细胞核内以DNA灶的水平进行区室化，支持了这样一种观点，即合成中心在空间上受到限制，而染色质环在DNA合成过程中是动态的。
