1. Dynamic Control of dNTP Synthesis in Early Embryos
Yonghyun Song, Robert A. Marmion, Junyoung O. Park, Debopriyo Biswas, Joshua D. Rabinowitz, Stanislav Y. Shvartsman 
Developmental Cell 
Volume 42, Issue 3, Pages e3 (August 2017)
DOI: /j.devcel
Copyright © 2017 Elsevier Inc. Terms and Conditions

2. Figure 1
Embryos Synthesize dNTPs “On the Go” through RNR, which Is Allosterically Regulated by dNTPs
(A) Table for concentration of dNTPs in embryos in μM. Data for Drosophila are represented as mean ± 2SEM (n = 14). Numbers in parentheses represent the number of nuclear equivalents, the derivation of which is detailed in the text. Values for other organisms were obtained from the literature (Bray and Thomas, 1972; Woodland and Pestell, 1972; Mathews, 1975).
(B) Schematic of the injection experiments. Embryos were glued to a coverslip and injected with a needle. The injection solution contained the small molecule of interest and the red loading control dye (rhodamine B). The needle and injected solution immediately following an injection event are shown in red in the upper most embryo. The green dots represent nuclei. For all experiments, roughly 70 pL (1% of the embryo aqueous volume) was injected.
(C) The proportion of embryos that successfully completed 13 divisions after the injections. RhoB, rhodamine B; HU, hydroxyurea.
(D) Schematic for allosteric regulation of RNR, represented by dashed lines (adapted from Hofer et al., 2011).
(E) The proportion of embryos that successfully completed 13 divisions after the injections. n is the number of embryos imaged after injection.
See also Figure S1; Movies S1 and S2.
Developmental Cell  , e3DOI: ( /j.devcel )
Copyright © 2017 Elsevier Inc. Terms and Conditions

3. Figure 2 RNR Is Inhibited by Endogenous Concentrations of dATP
Concentration profiles of dATP, dCTP, and dTTP over time in fertilized and unfertilized embryos pooled by hours post fertilization (hpf). In fertilized embryos, replication consumes dNTPs and RNR produces it. In unfertilized embryos, there is no consumption of dNTPs. Each sample consisted of 20 or more embryos, and the horizontal bar depicts the mean value. n is the number of samples. The values inserted in the bottom left corner of the panels are the average enzyme activity levels during the 2 hr. The activity level is derived from the mass balance equation of dNTP over the first 2 hr, where total RNR activity = Δ[dNTP] − consumption by replication.
Asterisks denote p values from two-sided t-tests, where ∗p < 0.01, ∗∗p < 0.001, and ∗∗∗p <
Developmental Cell  , e3DOI: ( /j.devcel )
Copyright © 2017 Elsevier Inc. Terms and Conditions

4. Figure 3
Model for dATP Production Recapitulates the Inhibition of RNR by dATP
(A) Summary of the model. dATP is produced by RNR and consumed by DNA polymerization. dATP allosterically inhibits RNR.
(B) dATP consumption rate is proportional to the number of nuclei, which increases exponentially in time.
(C) Representative concentration profiles of dATP. In orange is the concentration profile that matches the measurements from Figure 2 as described in the text. The bars on the right represent mean ± 2SD of dATP measurements at the respective time intervals. In black are the profiles that were either too high or low in abundance to fit the data. The parameter tuples are also shown in (D) by matching shapes ×, +, and •.
(D) Set of feasible parameter tuples that agree with measurements in Figure 2. The color scale represents initial dATP values. Maximum V value was 1,000 μM/s, obtained by assuming the maximum concentration of RNR to be 1 mM, and the maximum kcat value to be 1/s (Kashlan et al., 2002). Ki values can be lower.
(E) Simulated concentration profiles of dATP, where Ki = 5 μM, V = 1.5 μM/s, and dATP(0) is varied from 0 to 256 μM. All the profiles converge by t = 120 min.
(F) Concentration of dNTPs in embryos that were injected with variable amounts of dNTPs. For all injections, all four dNTPs were injected at the same indicated concentration. The volume injected was approximately 70 pL, or 1% of the embryo's aqueous volume. 0–1 hr post fertilization (hpf) embryos are those that had just been injected and had not yet undergone major DNA synthesis events; 2–3 hpf embryos have undergone 6,000 nuclear synthesis events. Each dot represents a concentration measured from a pool of >15 embryos.
Developmental Cell  , e3DOI: ( /j.devcel )
Copyright © 2017 Elsevier Inc. Terms and Conditions

5. Figure 4
Perturbations to RNR Activity Lead to Predictable Changes in dNTP Concentration
(A) On the left is the protein sequence alignment of yeast, fly, and mouse RNRL. We introduced a point mutation at the 68th position in fly RNRL (57th position in yeast and mouse RNRL), which is located in the highly conserved dATP binding domain. On the right is the effect of D68N on RNR activity, which has been shown in yeast and mouse to abolish dATP-mediated feedback inhibition (Reichard et al., 2000; Chabes et al., 2003a).
(B) dNTP concentration of RNRLWT and RNRLD68N expressing embryos compared with that of Oregon R embryos. Expression was achieved by a UAS-Gal4 system, driven by a strong maternal driver (MTD-Gal4). Each sample consisted of 15 or more embryos, and the horizontal bar depicts the mean value. n is the number of samples. Asterisks denote p values from two-sided t tests, where ∗∗∗p <
(C) Comparison of relative total ion counts of metabolites between MTD>RNRLWT and MTD>RNRLD68N embryos at 0–1 hr post fertilization. Relative concentrations are plotted in log2 scale, and the error bars represent 2 SEM, where n = 5. dNTP metabolism-related metabolites are colored in red. x = y line is colored in orange. FBP, fructose 1,6-bisphosphate; HMP, hexose monophosphate.
(D) Cuticles of Oregon R, MTD>RNRLWT, and MTD>RNRLD68N embryos. RNRLD68N expression specifically leads to the loss of head structures.
See also Figure S3.
Developmental Cell  , e3DOI: ( /j.devcel )
Copyright © 2017 Elsevier Inc. Terms and Conditions