Spatial and Temporal Dynamics of DNA Replication Sites in Mammalian Cells

doi:10.1083/jcb.143.6.1415

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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

Article

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 hasrevealed discrete granular sites of replication (RS). The averagesize and number of replication sites from early to mid S-phasesuggest that each RS contains numerous replicons clusteredtogether. We are using fluorescence laser scanning confocalmicroscopy in conjunction with multidimensional image analysisto gain more precise information about RS and their spatial-temporaldynamics. Using a newly improved imaging segmentation program,we report an average of $~$ 1,100 RS after a 5-min pulse labelingof 3T3 mouse fibroblast cells in early S-phase. Pulse-chase-pulsedouble labeling experiments reveal that RS take $~$ 45 min to completereplication. Appropriate calculations suggest that each RS containsan 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 maintainedtemporally and spatially as the cell progresses through thecell cycle and into subsequent generations. By labeling replicatedDNA 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.

We are extremely grateful to Dr. R. Summers for his assistancein confocal microscopy and to A. Siegel for use of the MicroscopicImaging Facility in our department. Special thanks to Dr. G.Mayers and Dr. R. Bankert for the gift of mouse anti-BrdU IgGantibodies; S. Somanathan and V. Sarangan for help with computerimaging; and J. Stamos for illustrations and photography.

The experiments were funded by National Institutes of HealthGrant GM 23922 to R. Berezney, and a grant from the Mark DiamondFund of the State University of New York at Buffalo GraduateSchool to H. Ma (27-S-98).

Dr. Ma's present address is Vollum Institute, L-474, OregonHealth 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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