Electron microscopy of DNA replication in 3-D: evidence for similar-sized replication foci throughout S-phase.
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.
Koberna
Karel
K
Department of Cell Biology, Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Albertov 4, CZ-12800 Prague 2, Czech Republic.
Ligasová
Anna
A
Malínský
Jan
J
Pliss
Artem
A
Siegel
Alan J
AJ
Cvacková
Zuzana
Z
Fidlerová
Helena
H
Masata
Martin
M
Fialová
Markéta
M
Raska
Ivan
I
Berezney
Ronald
R
D004261:DNA Replication; D005453:Fluorescence; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008854:Microscopy, Electron; D016196:S Phase