Abstract
dUTPase prevents uracil incorporation into DNA by strict regulation of the cellular dUTP:dTTP ratio. Lack of the enzyme initiates thymineless cell death, prompting studies on enzyme regulation. We investigated expression pattern and localization of Drosophila dUTPase. Similarly to human, two isoforms of the fly enzyme were identified at both mRNA and protein levels. During larval stages, a drastic decrease of dUTPase expression was demonstrated at the protein level. In contrast, dUTPase mRNAs display constitutive character throughout development. A putative nuclear localization signal was identified in one of the two isoforms. However, immunohistochemistry of ovaries and embryos did not show a clear correlation between the presence of this signal and subcellular localization of the protein, suggesting that the latter may be perturbed by additional factors. Results are in agreement with a multilevel regulation of dUTPase in the Drosophila proteome, possibly involving several interacting protein partners of the enzyme. Using independent approaches, the existence of such macromolecular partners was verified.
Highlights
Lack of dUTPase leads to uracil-substituted DNA that perturbs base excision repair, resulting in DNA fragmentation and thymineless apoptosis of the cell
Putative accumulation of uracil-containing DNA under thymineless conditions in larval stages of Drosophila melanogaster may play a role in programmed cell death necessary for metamorphosis in the pupal stage (12)
For the two putative isoforms, 64 and 59% of the peptide masses detected in the mass spectra showed clear matches to predicted dUTPase tryptic fragments, providing 72 and 66% coverage (Fig. 2, compare underlines and overlines) of the dUTPase sequence, respectively
Summary
Schneider Line 2 (S2) Cell Procedures—Drosophila S2 cells (Invitrogen) were cultured at 22 °C in Drosophila SFM (Life Technologies, Inc.) medium. Extracts were prepared from cell pellets washed twice in PBS, resuspended in lysis buffer (10 mM TRIS, pH 7.2, containing 10% glycerol, 0.15 M NaCl, 1 mM dithiothreitol, and proteinase inhibitor mix (Sigma)), sonicated for 1 min, and centrifuged at 18,000 ϫ g. Subcellular Fractionation of Drosophila Embryos—Twelve-h embryos were collected, washed, and homogenized in 50 mM 1,4-piperazinediethanesulfonic acid buffer, pH 7.9, containing 50 mM KCl, 5 mM EDTA, 2 mM MgCl2, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride. The first pellet after centrifugation at 700 ϫ g for 10 min contained the nuclear fraction. For far-Western blotting, wet membranes were blocked with 5% bovine serum albumin in Tris buffered saline with 0.1% Tween 20 for 1 h and incubated with 30 g/ml of recombinant Drosophila dUTPase at 4 °C overnight. The tryptic digests were analyzed on a Reflex III matrix-assisted laser desorption/ionization, time-of-flight mass spectrometer (MALDI-TOF MS, Bruker, Germany) unfractionated as well as after reversed-phase high-
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