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© The Rockefeller University Press, 0021-9525/1998//1415 $5.00
The Journal of Cell Biology, Volume 143, Number 6, , 1998 1415-1425

Spatial and Temporal Dynamics of DNA Replication Sites in Mammalian Cells

Hong Ma^*, Jagath Samarabandu^*, Rekandu S. Devdhar, Raj Acharya, Ping-chin Cheng, Chunling Meng^*, and Ronald Berezney^*

^* Department of Biological Sciences, Department of Computer Science and Engineering, and Department of Electrical Engineering, State University of New York at Buffalo, Buffalo, New York 14260

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.

Key Words: replication sites • replication timing • cell nucleus • chromosomes • computer image segmentation

Abbreviations used in this paper: BrdU, 5-bromo-2'-deoxyuridine; CldU, 5-chloro-2'-deoxyuridine; IdU, 5-iodo-2'-deoxyuridine; PI, propidium iodide; RS, replication sites; RT, replication timing.

The experiments were funded by National Institutes of Health Grant GM 23922 to R. Berezney, and a grant from the Mark Diamond Fund of the State University of New York at Buffalo Graduate School to H. Ma (27-S-98).

Dr. Ma's present address is Vollum Institute, L-474, Oregon Health Sciences University, 3181 SW Sam Jackson Park Rd., Portland, OR 97201-3098.

Dr. Samarabandu's present address is Life Imaging Systems Inc., 195 Dufferin Ave., Suite 300, London, ON N6A 1K7, Canada.

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