Genome wide decrease of DNA replication eye density at the midblastula transition ofXenopus laevis

Abstract

During the first rapid divisions of early development in many species, the DNA:cytoplasm ratio increases until the midblastula transition (MBT) when transcription resumes and cell cycles lengthen. S phase is very rapid in early embryos, about 20-30 times faster than in differentiated cells. Using a combination of DNA fiber studies and a Xenopus laevis embryonic in vitro replication system, we show that S phase slows down shortly after the MBT owing to a genome wide decrease of replication eye density. Increasing the dNTP pool did not accelerate S phase or increase replication eye density implying that dNTPs are not rate limiting for DNA replication at the Xenopus MBT. Increasing the ratio of DNA:cytoplasm in egg extracts faithfully recapitulates changes in the spatial replication program in embryos, supporting the hypothesis that titration of soluble limiting factors could explain the observed changes in the DNA replication program at the MBT in Xenopus laevis.

SSRP1-mediated histone H1 eviction promotes replication origin assembly and accelerated development

“…We observed that replication origins in post-MBT embryos injected with buffer had an average IOD of 17.6 ± 0.7 Kb and a density of~4-5 origins every 100 Kb ( Supplementary Fig. 8b, c), similar to what previously reported 32 . In embryos injected with SSRP1, instead, we observed a significant stimulation of replication origin assembly with origins placed at an average IOD of 10.1 ± 0.3 Kb and at density of~8-9 origins every 100 Kb (Supplementary Fig.…”

“…Embryonic nucleo-cytoplasmic extracts were prepared as described 32 with some modifications. Briefly, 400 dejellied late blastula (stage 9) embryos were washed in embryo extraction buffer (10 mM Hepes, pH 7.7 KOH, 100 mM KCl, 50 mM Sucrose, 1 mM MgCl 2 , 0.1 mM CaCl 2 and spun at 700 × g for 1 min to pack embryos without crushing.…”

“…8a). These extracts contain nuclei that are able to replicate their DNA in vitro in the presence of an ATP regeneration system, reproducing DNA replication dynamics typical of the stage they derived from 32 . We pulse labeled DNA replication for short time using biotin-dUTP and monitored DNA replication initiation events and IODs by DNA combing as in Figure 2d.…”

Polo-like kinase 1 (Plk1) is a positive regulator of DNA replication in the Xenopus in vitro system

“…To follow up on this idea, we looked at Plk1 expression during the first embryonic divisions, before midblastula transition (MBT) when the cell cycle is deprived of G1and G2 phases and lasts only 30 min. We found that the level of Plk1 is high during the first rapid cell cycles of development, then suddenly declines at the time when the zygotic transcription resumes and when the cell cycle and the DNA replication program slows down 76 .…”

The role of dNTP metabolites in control of the embryonic cell cycle

“…A report in Xenopus claimed that injecting dNTPs could increase the number of synchronous embryonic cell cycles [37]. Later studies showed that injecting dNTPs to 2.5-7.5 folds of their endogenous concentration, did neither change the number of synchronous cleavage cycles [20,32], nor the duration of S-phase [38]. Correspondingly, in Drosophila, injection of all four dNTPs did neither affect the number nor the lengths of the embryonic cell cycle.…”