Spatial and Temporal Dynamics of DNA Replication Sites in Mammalian Cells

• Published in: The Journal of cell biology Vol. 143; no. 6; pp. 1415 - 1425
• Main Authors: Ma, Hong, Samarabandu, Jagath, Devdhar, Rekandu S., Acharya, Raj, Cheng, Ping-chin, Meng, Chunling, Berezney, Ronald
• Format: Journal Article
• Language: English
• Published: United States Rockefeller University Press 14.12.1998; The Rockefeller University Press
• Subjects: 3T3 Cells; Animals; Cell cycle; Cell Cycle - physiology; Cell nucleus; Cells; Chromatin; Chromosomes; Chromosomes - genetics; Chromosomes - ultrastructure; Deoxyribonucleic acid; DNA; DNA - biosynthesis; DNA - chemistry; DNA - genetics; DNA Replication; G1 Phase; Kinetics; Mammals; Mice; Microscopy, Confocal; Reed Sternberg cells; Replicon; Replicon - genetics; S Phase; Time Factors
• ISSN: 0021-9525; 1540-8140
• 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 ∼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 ∼45 min to complete replication. Appropriate calculations suggest that each RS contains an average of 1 mbp of DNA or ∼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.