De novo dNTP production is essential for normal postnatal murine heart development

Abstract

Edited by John M. Denu The building blocks of DNA, dNTPs, can be produced de novo or can be salvaged from deoxyribonucleosides. However, to what extent the absence of de novo dNTP production can be compensated for by the salvage pathway is unknown. Here, we eliminated de novo dNTP synthesis in the mouse heart and skeletal muscle by inactivating ribonucleotide reductase (RNR), a key enzyme for the de novo production of dNTPs, at embryonic day 13. All other tissues had normal de novo dNTP synthesis and theoretically could supply heart and skeletal muscle with deoxyribonucleosides needed for dNTP production by salvage. We observed that the dNTP and NTP pools in WT postnatal hearts are unexpectedly asymmetric, with unusually high dGTP and GTP levels compared with those in whole mouse embryos or murine cell cultures. We found that RNR inactivation in heart led to strongly decreased dGTP and increased dCTP, dTTP, and dATP pools; aberrant DNA replication; defective expression of muscle-specific proteins; progressive heart abnormalities; disturbance of the cardiac conduction system; and lethality between the second and fourth weeks after birth. We conclude that dNTP salvage cannot substitute for de novo dNTP synthesis in the heart and that cardiomyocytes and myocytes initiate DNA replication despite an inadequate dNTP supply. We discuss the possible reasons for the observed asymmetry in dNTP and NTP pools in WT hearts. This work was supported by grants from the Swedish Cancer Society and the Swedish Research Council (to A. C.). The authors declare that they have no conflicts of interest with the contents of this article. This article was selected as one of our Editors' Picks. This article contains Tables S1-S3, Figs. S1-S6, and Movies S1 and S2.

A sensitive assay for dNTPs based on long synthetic oligonucleotides, EvaGreen dye and inhibitor-resistant high-fidelity DNA polymerase

“…We speculate that the variation in dATP levels could be related to ischemia during sample collection and delayed quenching of enzymatic activity. Similar to two chromatographic quantifications ( 6 , 23 ), we found heart dGTP concentration to be several-fold higher than that of other three dNTPs. Mitochondrial dNTP pools are extremely asymmetric with dGTP concentration being more than 10-fold higher than the concentration of other dNTPs ( 16 ).…”

“…( 6 ) and Tran et al. ( 23 ). Despite deviation in the absolute values, this reference data and our data showed highly similar dNTP pool asymmetry patterns for the mouse heart (Table 3 ).…”

A sensitive assay for dNTPs based on long synthetic oligonucleotides, EvaGreen dye, and inhibitor-resistant high-fidelity DNA polymerase

“…We speculate that the variation in dATP levels could be related to ischemia during sample collection and delayed quenching of enzymatic activity. Similar to two chromatographic quantifications (6,22), we found heart dGTP concentration to be 5 to 15-fold higher than that of other three dNTPs. Mitochondrial dNTP pools are extremely asymmetric with dGTP concentration being more than 10-fold higher than the concentration of other dNTPs (23).…”

The Microenvironment Is a Critical Regulator of Muscle Stem Cell Activation and Proliferation

“…Nucleotide biosynthesis has received scant attention in skeletal MuSCs, but a recent study by Tran et al (2019) reported on ribonucleotide reductase (RNR) knockout mouse. In this study the authors developed a mouse model with exon 9 of the M1 subunit of RNR flanked by two loxP sites ( Rrm1 fl/fl ) and bred it with a mouse expressing Cre recombinase under the control of muscle creatine kinase ( Mck cre ), with the resulting mouse expressing a truncated and inactive form of RNR in cardiac and skeletal muscle from embryonic day 13 (Tran et al, 2019). As RNR is a key enzyme for de novo nucleotide synthesis, its conditional ablation allowed the investigators to study the importance of this pathway in skeletal and cardiac muscle.…”

“…While not described in detail, the authors found that muscle fibers in the gastrocnemius muscles of knockout mice were less than half the size of those observed in wildtype mice. Additionally, the number of nuclei per fiber was reduced by more than half (Tran et al, 2019). It will be critical in future studies to determine whether a similar defect in dGTP (as observed in cardiac muscle) is observed in skeletal muscle of RNR knockout mice.…”