Direct Visualization of DNA Replication Dynamics in Zebrafish Cells

Abstract

Spatiotemporal regulation of DNA replication in the S-phase nucleus has been extensively studied in mammalian cells because it is tightly coupled with the regulation of other nuclear processes such as transcription. However, little is known about the replication dynamics in nonmammalian cells. Here, we analyzed the DNA replication processes of zebrafish (Danio rerio) cells through the direct visualization of replicating DNA in the nucleus and on DNA fiber molecules isolated from the nucleus. We found that zebrafish chromosomal DNA at the nuclear interior was replicated first, followed by replication of DNA at the nuclear periphery, which is reminiscent of the spatiotemporal regulation of mammalian DNA replication. However, the relative duration of interior DNA replication in zebrafish cells was longer compared to mammalian cells, possibly reflecting zebrafish-specific genomic organization. The rate of replication fork progression and ori-to-ori distance measured by the DNA combing technique were ∼ 1.4 kb/min and 100 kb, respectively, which are comparable to those in mammalian cells. To our knowledge, this is a first report that measures replication dynamics in zebrafish cells.

DNA replication timing during development anticipates transcriptional programs and parallels enhancer activation

“…When the pre-MBT embryos (15 min Sphase) were compared to ZTF cells (8 h S-phase), the pre-MBT timing profile appeared compressed and resembled random data ( Fig. 4A; Kane and Kimmel 1993;Kuriya et al 2015). However, consistent timing profiles between biological replicates were evident when the data were scaled to normalized units (Fig.…”

Direct visualization of replication dynamics in early zebrafish embryos

“…1(C)). Although the temporal order of these patterns was determined from our previous study, 9) the frequency of pattern II was particularly high, which was also observed in somatic cells. Similar results were obtained when we used embryos at 2.75-4 hpf (blastula stage) where cell division takes between 15 and 54 min.…”

“…DNA fibers from EdU-labeled cells were prepared as described previously. 9) A fluorescence microscope (Axioplan 2 MOT; Carl Zeiss, Inc. Jena, Germany) equipped with a 63×objective lens and an ORCA R2 camera (Hamamatsu; Shizuoka, Japan) was used for imaging. This experimental system allows us to collect single embryo-derived cells, thus enabling analysis of the dynamic nature of DNA replication within a single embryo.…”

The CDK-PLK1 axis targets the DNA damage checkpoint sensor protein RAD9 to promote cell proliferation and tolerance to genotoxic stress

“…The DNA fiber assay was performed as described ( Conti et al, 2007 ; Kuriya et al, 2015 ; Michalet et al, 1997 ). Briefly, cells were labeled with 100 µM of IdU or CldU for 30 min and harvested by trypsinization.…”