Electron microscopy of DNA replication in 3‐D: Evidence for similar‐sized replication foci throughout S‐phase

Abstract

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.