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Uracil-containing DNA in Drosophila: stability, stage-specific accumulation, and developmental involvement.

Muha V, Horváth A, Békési A, Pukáncsik M, Hodoscsek B, Merényi G, Róna G, Batki J, Kiss I, Jankovics F, Vilmos P, Erdélyi M, Vértessy BG - PLoS Genet. (2012)

Bottom Line: Upon pupation and metamorphosis, uracil content in DNA is significantly decreased.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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Enzymology, Research Center for Natural Sciences, Hungarian Academy of Science, Budapest, Hungary.

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.

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Tolerance and stability of uracil-containing DNA in D. melanogaster.(A) Dose-response curve upon FdUR treatment followed by Alamar Blue assay. (B) FdUR leads to uracil accumulation in DNA of Drosophila S2 cells. Data indicate that increased level of uracil is well-tolerated in Drosophila, but not in human cells. Data are presented as mean ± s.e.m. (C) Drosophila S2 (top panels) and human HeLa cells (bottom panels) were transfected with normal plasmid (left panels) or uracil-containing plasmid (right panels). Expression of YFP in Drosophila S2 cells or dsRedMonomer in HeLa cells indicates stability of the DNA. (D) Microinjection of uracil-plasmid into Drosophila embryo. Non-injected embryos served as control sample.
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pgen-1002738-g001: Tolerance and stability of uracil-containing DNA in D. melanogaster.(A) Dose-response curve upon FdUR treatment followed by Alamar Blue assay. (B) FdUR leads to uracil accumulation in DNA of Drosophila S2 cells. Data indicate that increased level of uracil is well-tolerated in Drosophila, but not in human cells. Data are presented as mean ± s.e.m. (C) Drosophila S2 (top panels) and human HeLa cells (bottom panels) were transfected with normal plasmid (left panels) or uracil-containing plasmid (right panels). Expression of YFP in Drosophila S2 cells or dsRedMonomer in HeLa cells indicates stability of the DNA. (D) Microinjection of uracil-plasmid into Drosophila embryo. Non-injected embryos served as control sample.

Mentions: Since the Drosophila genome lacks UNG, we wished to test whether Drosophila cells show similar characteristics to UNG deficient organisms. The drug 5′-fluorodeoxyuridine (FdUR), frequently used as an inhibitor of thymidylate biosynthesis [23], [24], leads to perturbation of nucleotide levels and cell death. Loss of uracil–DNA glycosylase activity was found to lead to fluoropyrimidine resistance in E. coli[25], yeast [26] and C. elegans[27]. Deficient uracil–DNA glycosylase activity was also reported to be required for increased genomic uracil content after FdUR exposure in E. coli and mammalian cells [7]–[10]. Therefore, response to FdUR treatment may be an indicator for testing cellular uracil–DNA glycosylase activity. We observed that the Drosophila S2 cell line shows only small decrease in viability in the presence of 1 mM of FdUR, while HeLa cells, possessing the ung gene, show strong lethality at this drug concentration (Figure 1A). As a dose-dependent response to increasing concentrations of FdUR, uracil accumulation in genomic DNA of Drosophila S2 cells became highly elevated (up to approx. 450 uracil/million bases) (Figure 1B). Both the observed relatively high resistance for FdUR and the cellular response of genomic uracil incorporation may be explained by the fact that Drosophila cells lack significant uracil–DNA glycosylase activity.


Uracil-containing DNA in Drosophila: stability, stage-specific accumulation, and developmental involvement.

Muha V, Horváth A, Békési A, Pukáncsik M, Hodoscsek B, Merényi G, Róna G, Batki J, Kiss I, Jankovics F, Vilmos P, Erdélyi M, Vértessy BG - PLoS Genet. (2012)

Tolerance and stability of uracil-containing DNA in D. melanogaster.(A) Dose-response curve upon FdUR treatment followed by Alamar Blue assay. (B) FdUR leads to uracil accumulation in DNA of Drosophila S2 cells. Data indicate that increased level of uracil is well-tolerated in Drosophila, but not in human cells. Data are presented as mean ± s.e.m. (C) Drosophila S2 (top panels) and human HeLa cells (bottom panels) were transfected with normal plasmid (left panels) or uracil-containing plasmid (right panels). Expression of YFP in Drosophila S2 cells or dsRedMonomer in HeLa cells indicates stability of the DNA. (D) Microinjection of uracil-plasmid into Drosophila embryo. Non-injected embryos served as control sample.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3369950&req=5

pgen-1002738-g001: Tolerance and stability of uracil-containing DNA in D. melanogaster.(A) Dose-response curve upon FdUR treatment followed by Alamar Blue assay. (B) FdUR leads to uracil accumulation in DNA of Drosophila S2 cells. Data indicate that increased level of uracil is well-tolerated in Drosophila, but not in human cells. Data are presented as mean ± s.e.m. (C) Drosophila S2 (top panels) and human HeLa cells (bottom panels) were transfected with normal plasmid (left panels) or uracil-containing plasmid (right panels). Expression of YFP in Drosophila S2 cells or dsRedMonomer in HeLa cells indicates stability of the DNA. (D) Microinjection of uracil-plasmid into Drosophila embryo. Non-injected embryos served as control sample.
Mentions: Since the Drosophila genome lacks UNG, we wished to test whether Drosophila cells show similar characteristics to UNG deficient organisms. The drug 5′-fluorodeoxyuridine (FdUR), frequently used as an inhibitor of thymidylate biosynthesis [23], [24], leads to perturbation of nucleotide levels and cell death. Loss of uracil–DNA glycosylase activity was found to lead to fluoropyrimidine resistance in E. coli[25], yeast [26] and C. elegans[27]. Deficient uracil–DNA glycosylase activity was also reported to be required for increased genomic uracil content after FdUR exposure in E. coli and mammalian cells [7]–[10]. Therefore, response to FdUR treatment may be an indicator for testing cellular uracil–DNA glycosylase activity. We observed that the Drosophila S2 cell line shows only small decrease in viability in the presence of 1 mM of FdUR, while HeLa cells, possessing the ung gene, show strong lethality at this drug concentration (Figure 1A). As a dose-dependent response to increasing concentrations of FdUR, uracil accumulation in genomic DNA of Drosophila S2 cells became highly elevated (up to approx. 450 uracil/million bases) (Figure 1B). Both the observed relatively high resistance for FdUR and the cellular response of genomic uracil incorporation may be explained by the fact that Drosophila cells lack significant uracil–DNA glycosylase activity.

Bottom Line: Upon pupation and metamorphosis, uracil content in DNA is significantly decreased.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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Enzymology, Research Center for Natural Sciences, Hungarian Academy of Science, Budapest, Hungary.

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.

Show MeSH