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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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dUTPase silencing results in cell death and DNA strand breaks in larval imaginal discs.(A) Imaginal discs were isolated from wild type and dUTPase silenced wandering 3rd larvae and stained for TUNEL assay (shown as red dots). Discs from silenced animals showed highly increased TUNEL staining. (B) TUNEL positive cell counts in imaginal discs from wild type and dUTPase silenced wandering 3rd larvae. Error bars represent the standard error of mean. (C) Imaginal discs from wild type and dUTPase silenced 3rd wandering larvae stained against phospho-H2Av foci (white dots, some of these are appointed by white arrowheads). dUTPase depleted discs showed several nuclei with phospho-H2Av foci indicating DNA damage. Scale bar represents 50 µm.
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pgen-1002738-g006: dUTPase silencing results in cell death and DNA strand breaks in larval imaginal discs.(A) Imaginal discs were isolated from wild type and dUTPase silenced wandering 3rd larvae and stained for TUNEL assay (shown as red dots). Discs from silenced animals showed highly increased TUNEL staining. (B) TUNEL positive cell counts in imaginal discs from wild type and dUTPase silenced wandering 3rd larvae. Error bars represent the standard error of mean. (C) Imaginal discs from wild type and dUTPase silenced 3rd wandering larvae stained against phospho-H2Av foci (white dots, some of these are appointed by white arrowheads). dUTPase depleted discs showed several nuclei with phospho-H2Av foci indicating DNA damage. Scale bar represents 50 µm.

Mentions: To analyze if the effects of dUTPase silencing may lead to DNA damage or cell death, we applied TUNEL and phospho-Histone H2Av assays [40], [41]. We observed that imaginal discs isolated from 3rd stage wandering larva of dUTPase-silenced animals show strong enrichment in TUNEL positive cells (Figure 6A and 6B). TUNEL staining in the tissues indicate primarily cell death in the phase of DNA fragmentation. To address the question whether dUTPase depletion violates genome integrity we stained nuclei for phospho-Histone H2Av. H2Av histone modification by the ATR/ATM kinases indicates DNA double strand breaks (DSBs) in the proximity of the foci [41]. We observed numerous phospho-H2Av foci in dUTPase silenced wing imaginal discs, while no such foci were visible in the wild type (Figure 6C). These results suggest a potential correlation between dUTPase activity and DNA integrity. The increased level of DNA damage observed in our experiments in the dUTPase-silenced animals may result from excessive processing of uracil-containing DNA that concludes to DNA fragmentation.


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)

dUTPase silencing results in cell death and DNA strand breaks in larval imaginal discs.(A) Imaginal discs were isolated from wild type and dUTPase silenced wandering 3rd larvae and stained for TUNEL assay (shown as red dots). Discs from silenced animals showed highly increased TUNEL staining. (B) TUNEL positive cell counts in imaginal discs from wild type and dUTPase silenced wandering 3rd larvae. Error bars represent the standard error of mean. (C) Imaginal discs from wild type and dUTPase silenced 3rd wandering larvae stained against phospho-H2Av foci (white dots, some of these are appointed by white arrowheads). dUTPase depleted discs showed several nuclei with phospho-H2Av foci indicating DNA damage. Scale bar represents 50 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1002738-g006: dUTPase silencing results in cell death and DNA strand breaks in larval imaginal discs.(A) Imaginal discs were isolated from wild type and dUTPase silenced wandering 3rd larvae and stained for TUNEL assay (shown as red dots). Discs from silenced animals showed highly increased TUNEL staining. (B) TUNEL positive cell counts in imaginal discs from wild type and dUTPase silenced wandering 3rd larvae. Error bars represent the standard error of mean. (C) Imaginal discs from wild type and dUTPase silenced 3rd wandering larvae stained against phospho-H2Av foci (white dots, some of these are appointed by white arrowheads). dUTPase depleted discs showed several nuclei with phospho-H2Av foci indicating DNA damage. Scale bar represents 50 µm.
Mentions: To analyze if the effects of dUTPase silencing may lead to DNA damage or cell death, we applied TUNEL and phospho-Histone H2Av assays [40], [41]. We observed that imaginal discs isolated from 3rd stage wandering larva of dUTPase-silenced animals show strong enrichment in TUNEL positive cells (Figure 6A and 6B). TUNEL staining in the tissues indicate primarily cell death in the phase of DNA fragmentation. To address the question whether dUTPase depletion violates genome integrity we stained nuclei for phospho-Histone H2Av. H2Av histone modification by the ATR/ATM kinases indicates DNA double strand breaks (DSBs) in the proximity of the foci [41]. We observed numerous phospho-H2Av foci in dUTPase silenced wing imaginal discs, while no such foci were visible in the wild type (Figure 6C). These results suggest a potential correlation between dUTPase activity and DNA integrity. The increased level of DNA damage observed in our experiments in the dUTPase-silenced animals may result from excessive processing of uracil-containing DNA that concludes to DNA fragmentation.

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