Ma H, Samarabandu J, Devdhar R S, Acharya R, Cheng P C, Meng C, Berezney R
Department of Biological Sciences, State University of New York at Buffalo, Buffalo, New York 14260, USA.
J Cell Biol. 1998 Dec 14;143(6):1415-25. doi: 10.1083/jcb.143.6.1415.
Fluorescence microscopic analysis of newly replicated DNA has revealed discrete granular sites of replication (RS). The average size and number of replication sites from early to mid S-phase suggest that each RS contains numerous replicons clustered together. We are using fluorescence laser scanning confocal microscopy in conjunction with multidimensional image analysis to gain more precise information about RS and their spatial-temporal dynamics. Using a newly improved imaging segmentation program, we report an average of approximately 1,100 RS after a 5-min pulse labeling of 3T3 mouse fibroblast cells in early S-phase. Pulse-chase-pulse double labeling experiments reveal that RS take approximately 45 min to complete replication. Appropriate calculations suggest that each RS contains an average of 1 mbp of DNA or approximately 6 average-sized replicons. Double pulse-double chase experiments demonstrate that the DNA sequences replicated at individual RS are precisely maintained temporally and spatially as the cell progresses through the cell cycle and into subsequent generations. By labeling replicated DNA at the G1/S borders for two consecutive cell generations, we show that the DNA synthesized at early S-phase is replicated at the same time and sites in the next round of replication.
对新复制的DNA进行荧光显微镜分析，揭示了离散的颗粒状复制位点（RS）。从S期早期到中期，复制位点的平均大小和数量表明，每个RS包含许多聚集在一起的复制子。我们正在使用荧光激光扫描共聚焦显微镜结合多维图像分析，以获取有关RS及其时空动态的更精确信息。使用新改进的成像分割程序，我们报告在S期早期对3T3小鼠成纤维细胞进行5分钟脉冲标记后，平均约有1100个RS。脉冲-追踪-脉冲双标记实验表明，RS完成复制大约需要45分钟。适当的计算表明，每个RS平均包含1兆碱基对的DNA或大约6个平均大小的复制子。双脉冲-双追踪实验表明，随着细胞经历细胞周期并进入后代，在单个RS处复制的DNA序列在时间和空间上得到精确维持。通过在两个连续的细胞世代中在G1/S边界标记复制的DNA，我们表明在S期早期合成的DNA在下一轮复制中在相同的时间和位点进行复制。
