DYNAMIC CONTROL OF DNA PRECURSOR SYNTHESIS IN EARLY EMBRYOS

Abstract

Animal embryogenesis starts with multiple rounds of nuclear divisions. During and shortly after these divisions, the zygotic genome is activated, the body plan of the organism is established, and gastrulation initiates the formation of tissues and organs. For these events to occur, the embryo needs to generate energy and provide metabolic precursors for biosynthesis. In this thesis, we used quantitative mass spectrometry and genetic manipulation techniques to examine how the early Drosophila melanogaster embryo controls the synthesis of DNA precursors.

Early Drosophila embryos undergo 13 rapid and synchronous nuclear division cycles within two hours of fertilization. This exponential increase in the number of nuclei requires massive amounts of deoxynucleoside triphosphates (dNTPs). 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 (RNR), is inhibited by endogenous levels of dATP present at fertilization and is activated as dATP is depleted via DNA polymerization. In the absence of inhibition by dATP, dNTP levels increase dramatically and induce embryonic lethality with particularly severe structural defects in the anterior regions.

In conclusion, this thesis demonstrated that dNTP synthesis in early Drosophila embryos is controlled mainly through a single feedback inhibition loop at the end of the dNTP production pathway. In the process, we have also found that misregulation of RNR activity suprisingly confers tissue specific defects. Going forward, this thesis establishes Drosophila development as a platform for mechanistic and quantitative studies of dNTP metabolism.