Electron microscopy of DNA replication in 3-D:: Evidence for similar-sized replication foci throughout S-phase
Electron microscopy of DNA replication in 3-D:: Evidence for similar-sized replication foci throughout S-phase
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DOI：
10.1002/jcb.20300
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发表时间：
2005-01-01
影响因子：
4
通讯作者：
Berezney, R
中科院分区：
文献类型：
--
作者：
Koberna, K;Ligasová, A;Berezney, R
同步 HeLa 细胞中的 DNA 复制位点 (RS) 已在电子显微镜水平上进行了研究。使用改进的检测方法，在体内掺入生物素-16-脱氧尿苷三磷酸后，在整个 S 期观察到离散的 RS 或病灶。特别是，通常通过荧光显微镜方法在 S 期中期和晚期观察到的更大的 RS 或焦点，被发现是由较小的离散焦点组成，这些焦点的大小实际上与早期 S 期观察到的 RS 相同。脉冲追踪实验表明，当追踪 S 期并进入下一代细胞时，早期 S 期的 RS 得以维持。体视学分析表明，在给定时间存在的较小尺寸病灶的相对数量从 S 期早期到中期保持恒定，而在 S 期晚期仅略有减少。连续切片的 3D 重建揭示了早期 S 期 RS 的网络状组织，并证实了许多较小尺寸的复制灶构成了晚期 S 期较大的 RS 特征。 (C) 2004 Wiley-Liss, Inc.
DNA replication sites (RS) in synchronized HeLa cells have been studied at the electron microscopic level. Using an improved method for detection following the in vivo incorporation of biotin-16-deoxyuridine triphosphate, discrete RS, or foci are observed throughout the S-phase. In particular, the much larger RS or foci typically observed by fluorescence microscopic approaches in mid- and late-S-phase, are found to be composed of smaller discrete foci that are virtually identical in size to the RS observed in early-S-phase. Pulse-chase experiments demonstrate that the RS of early-S-phase are maintained when chased through S-phase and into the next cell generation. Stereologic analysis demonstrates that the relative number of smaller sized foci present at a given time remains constant from early through mid-S-phase with only a slight decrease in late-S-phase. 3-D reconstruction of serial sections reveals a network-like organization of the RS in early-S-phase and confirms that numerous smaller-sized replication foci comprise the larger RS characteristic of late-S-phase. (C) 2004 Wiley-Liss, Inc.