Abstract
Base-excision repair and control of nucleotide pools safe-guard against permanent uracil accumulation in DNA relying on two key enzymes: uracil–DNA glycosylase and dUTPase. Lack of the major uracil–DNA glycosylase UNG gene from the fruit fly genome and dUTPase from fruit fly larvae prompted the hypotheses that i) uracil may accumulate in Drosophila genomic DNA where it may be well tolerated, and ii) this accumulation may affect development. Here we show that i) Drosophila melanogaster tolerates high levels of uracil in DNA; ii) such DNA is correctly interpreted in cell culture and embryo; and iii) under physiological spatio-temporal control, DNA from fruit fly larvae, pupae, and imago contain greatly elevated levels of uracil (200–2,000 uracil/million bases, quantified using a novel real-time PCR–based assay). Uracil is accumulated in genomic DNA of larval tissues during larval development, whereas DNA from imaginal tissues contains much less uracil. Upon pupation and metamorphosis, uracil content in DNA is significantly decreased. We propose that the observed developmental pattern of uracil–DNA is due to the lack of the key repair enzyme UNG from the Drosophila genome together with down-regulation of dUTPase in larval tissues. In agreement, we show that dUTPase silencing increases the uracil content in DNA of imaginal tissues and induces strong lethality at the early pupal stages, indicating that tolerance of highly uracil-substituted DNA is also stage-specific. Silencing of dUTPase perturbs the physiological pattern of uracil–DNA accumulation in Drosophila and leads to a strongly lethal phenotype in early pupal stages. These findings suggest a novel role of uracil-containing DNA in Drosophila development and metamorphosis and present a novel example for developmental effects of dUTPase silencing in multicellular eukaryotes. Importantly, we also show lack of the UNG gene in all available genomes of other Holometabola insects, indicating a potentially general tolerance and developmental role of uracil–DNA in this evolutionary clade.
Highlights
In wild-type organisms, notably excepting some rare bacteriophages with thymineless DNA genomes [1,2], uracil in DNA is thought to occur only transiently and at very low frequency (,20/ million bases) as a damage product [3,4]
Efficient base-excision DNA repair together with fine-tuned control of nucleotide pools safe-guard against uracil in DNA relying on two key enzymes: uracil–DNA glycosylase (UDG) [5] and dUTPase [6]
We reported that uracil–DNA is tolerated and interpreted at least from embryonic to 3rd larval stages
Summary
In wild-type organisms, notably excepting some rare bacteriophages with thymineless DNA genomes [1,2], uracil in DNA is thought to occur only transiently and at very low frequency (,20/ million bases) as a damage product [3,4]. Efficient base-excision DNA repair together with fine-tuned control of nucleotide pools safe-guard against uracil in DNA relying on two key enzymes: uracil–DNA glycosylase (UDG) [5] and dUTPase [6]. UDG deficiency, in combination with thymidylate synthase inhibition or depleted dUTPase activity, was reported to lead into notable uracil accumulation in DNA. UNG is reported to be the most abundant one that possesses the highest activity in removing uracils from any context of both single-stranded and double-stranded DNA [5]. MBD4 and TDG recognize mismatched uracil or thymine bases that base-pair with guanine [16]. The latter enzyme is known to function within CpG islands, where thymine is formed after spontaneous methyl-cytosine deamination [17]. Eukaryotic and bacterial DNA polymerases incorporate deoxyuridine into DNA with a rate that depends on
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