Dynamic Control of dNTP Synthesis in Early Embryos

Abstract

SUMMARY Animal embryogenesis begins with cell divisions, which require large amounts of DNA precursors (deoxyribonucleoside triphosphates (dNTPs)). Little is understood about how embryos satisfy this demand. We examined dNTP metabolism in the early Drosophila embryo, in which gastrulation is preceded by 13 sequential nuclear cleavages within two hours of fertilization. Surprisingly, despite the breakneck speed at which Drosophila embryos synthesize DNA, maternally deposited dNTPs can generate less than half of the genomes needed to reach gastrulation. The rest of the dNTPs are synthesized “on the go”. The rate-limiting enzyme of dNTP synthesis, ribonucleotide reductase, is inhibited by endogenous levels of dATP present at fertilization and is activated as dATP is depleted via DNA polymerization. This feedback inhibition renders the concentration of dNTPs at gastrulation robust with respect to large variations in maternal supplies, and is essential for normal progression of embryogenesis.

Fast and sensitive HPLC–MS/MS method for direct quantification of intracellular deoxyribonucleoside triphosphates from tissue and cells

“…This cross talk was most noted by ATP having a peak matching the retention time in the ATP channel however showing up in the in the dGTP channel as seen in Figure 1. This phenomenon has been reported by other groups [10,30,28]. Subsequently, the dNTPs were run as pure standards with adjustments to the chromatography to provide separation and to minimize cross talk.…”

“…It is well known that due to similarity between dNTPs, there is cross talk between the monitoring ion channels [28,29]. During the initial optimization of the chromatography protocol, the standards were prepared as a mixture of the four nucleotides, dATP, dGTP, dCTP, and dTTP, as well as ATP.…”

“…We have demonstrated the samples can be successfully diluted, which can be useful in measuring samples with higher concertation than the upper limit of our calibration curve. Additionally, this method was able to chromatographically separate ATP and dGTP, whereas previous methods were unable to achieve separation [10,19,28,35], had to use run times of up to 1 hour and switch ion modes [20], or utilize ion-pairing reagents [36] for successful separation. In summary, the assay is a fast, reproducible, and robust method that efficiently extracts nucleotide from small tissue samples and quantifies four intracellular deoxynucleosides triphosphates simultaneously.…”

Cellular metabolic reprogramming controls sugar appetite

“…Both low and high levels of nucleotides negatively affect morphogenetic processes in the early embryo (Djabrayan et al, 2019;Liu et al, 2019). Cells rely on the exquisitely precise regulatory control of ribonucleotide reductase enzymatic activity to ensure a precise regulation of nucleotide levels in the egg (Djabrayan et al, 2019;Song et al, 2017). It is therefore tempting to speculate that the germline induces PPP activity not only to ensure the availability of this critical nutrient but also to be able to regulate the levels of this metabolite in a diet-independent manner.…”

Still the most interesting enzyme in the world

“…Obviously, there is tremendous demand for dNTPs early after fertilization; what is their source? Song et al (16) noted that in Drosophila, some 13 nuclear cleavages occur within 2 h after fertilization. Storage of dNTPs in the egg accounted for only one-half of the total amount required, and the rest was shown to be synthesized "on the go."…”