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.

Rad51 Regulates Cell Cycle Progression by Preserving G2/M Transition in Mouse Embryonic Stem Cells

“…(B) Based on the pattern of nuclear foci formation, cells were divided into three groups and quantified as in (A). (C) Cell populations existing as an independent entity were subdivided into three groups based on the estimated replication factory size [50]. (D) Rad51 foci inside the replication factory.…”

The Organisation of Replisomes

“…Every such domain represents several tagged segments of DNA (Koberna et al, 2005; Figure 1). As the total volume of the cell nucleus in the early S phase was 260 ± 44 ǚm 3 , the number of labelled domains in one cell nucleus was 5460 ± 923.…”

“…In the most of experiments, the cells were synchronised at the G1/S border by means of the double block with 2′-deoxytymidine (dT; Koberna et al, 2005). After the release from the block, the cells were labelled with biotin-dUTP (Koberna et al, 1999;Ligasová et al, 2009).…”

“…The ultrastructural localisation of the biotin-tagged DNA was performed using the synchronised cells by means of the pre-embedding approach (Koberna et al, 2005). Briefly, the cells were fixed by 2% formaldehyde and subsequently permeabilised by 0.2% Triton X-100.…”

Activation of new replication foci under conditions of replication stress

“…A characteristic pattern of foci arranged along a curved line may arise from positioning at the surface of structures like 1Mbp domains, as would be expected if the replication process were performed at the edges of such domains. 31,32 Global DNA damage response and the size of cell nuclei Exposure to Tpt leads to induction of DSBs. It has been shown previously that DNA damage leads to loosening of chromatin structure and opening of regions of high DNA density.…”