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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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D. melanogaster genomic DNA uracil content inversely correlates with dUTPase expression.(A) Changes of dUTPase mRNA level throughout fruitfly development: embryo (E), 1st larvae (L1), 2nd larvae (L2), late 3rd larvae (L3) and pupae (P). Note that dUTPase is down-regulated in larvae. (B) Comparison of dUTPase RNA level in the larval tissues salivary gland and imaginal tissue. Data are presented as mean of triplicates ± s.e.m. mRNA level was measured by RT-qPCR and dUTPase mRNA level was normalized to Rp49 mRNA level. (C) Uracil content of D. melanogaster genome in different developmental stages: embryo (E), 1st larvae (L1), 2nd larvae (L2), late 3rd larvae (L3) and pupae (P). Embryonic sample was used as reference since it was shown to contain undetectable levels of uracil in DNA (cf. Figure S2). (D) Comparison of genomic uracil content in wild type imaginal disc and salivary gland of 3rd larvae. Data are presented as mean ± s.e.m.
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pgen-1002738-g003: D. melanogaster genomic DNA uracil content inversely correlates with dUTPase expression.(A) Changes of dUTPase mRNA level throughout fruitfly development: embryo (E), 1st larvae (L1), 2nd larvae (L2), late 3rd larvae (L3) and pupae (P). Note that dUTPase is down-regulated in larvae. (B) Comparison of dUTPase RNA level in the larval tissues salivary gland and imaginal tissue. Data are presented as mean of triplicates ± s.e.m. mRNA level was measured by RT-qPCR and dUTPase mRNA level was normalized to Rp49 mRNA level. (C) Uracil content of D. melanogaster genome in different developmental stages: embryo (E), 1st larvae (L1), 2nd larvae (L2), late 3rd larvae (L3) and pupae (P). Embryonic sample was used as reference since it was shown to contain undetectable levels of uracil in DNA (cf. Figure S2). (D) Comparison of genomic uracil content in wild type imaginal disc and salivary gland of 3rd larvae. Data are presented as mean ± s.e.m.

Mentions: The other key factor responsible for keeping uracil out of DNA is the enzyme dUTPase, the importance of which is even more substantiated in D. melanogaster that lacks UNG. During development of D. melanogaster, high dUTPase protein levels can be observed only in embryonic stages (Figure 2A). As shown by Western blotting and immunohistochemistry, starting from late embryonic phase, dUTPase expression level is decreased drastically and remains low during postembryonic stages. Under normal physiological circumstances of larval development, dUTPase is predominantly expressed in imaginal discs, the central nervous system and in the testis (Figure 2) but not in most larval tissues, like salivary gland and gut. In the ventriculus and in the salivary gland, some cells are associated with dUTPase expression – these seem to correspond to the imaginal cells (Figure 2B). In the imago, dUTPase is present in the ovary of females. Cellular localization of dUTPase is usually nuclear [21], [28]–[31], however cytoplasmic occurrence is also evident in the nurse cells of mature follicles within ovaries, as described previously [21]. The present results are in agreement with our more limited earlier observations at the protein level [21]. We also quantified the dUTPase mRNA level by real-time PCR and found that protein and mRNA levels show similar tendencies during development (Figure 3A and 3B).


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)

D. melanogaster genomic DNA uracil content inversely correlates with dUTPase expression.(A) Changes of dUTPase mRNA level throughout fruitfly development: embryo (E), 1st larvae (L1), 2nd larvae (L2), late 3rd larvae (L3) and pupae (P). Note that dUTPase is down-regulated in larvae. (B) Comparison of dUTPase RNA level in the larval tissues salivary gland and imaginal tissue. Data are presented as mean of triplicates ± s.e.m. mRNA level was measured by RT-qPCR and dUTPase mRNA level was normalized to Rp49 mRNA level. (C) Uracil content of D. melanogaster genome in different developmental stages: embryo (E), 1st larvae (L1), 2nd larvae (L2), late 3rd larvae (L3) and pupae (P). Embryonic sample was used as reference since it was shown to contain undetectable levels of uracil in DNA (cf. Figure S2). (D) Comparison of genomic uracil content in wild type imaginal disc and salivary gland of 3rd larvae. Data are presented as mean ± s.e.m.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3369950&req=5

pgen-1002738-g003: D. melanogaster genomic DNA uracil content inversely correlates with dUTPase expression.(A) Changes of dUTPase mRNA level throughout fruitfly development: embryo (E), 1st larvae (L1), 2nd larvae (L2), late 3rd larvae (L3) and pupae (P). Note that dUTPase is down-regulated in larvae. (B) Comparison of dUTPase RNA level in the larval tissues salivary gland and imaginal tissue. Data are presented as mean of triplicates ± s.e.m. mRNA level was measured by RT-qPCR and dUTPase mRNA level was normalized to Rp49 mRNA level. (C) Uracil content of D. melanogaster genome in different developmental stages: embryo (E), 1st larvae (L1), 2nd larvae (L2), late 3rd larvae (L3) and pupae (P). Embryonic sample was used as reference since it was shown to contain undetectable levels of uracil in DNA (cf. Figure S2). (D) Comparison of genomic uracil content in wild type imaginal disc and salivary gland of 3rd larvae. Data are presented as mean ± s.e.m.
Mentions: The other key factor responsible for keeping uracil out of DNA is the enzyme dUTPase, the importance of which is even more substantiated in D. melanogaster that lacks UNG. During development of D. melanogaster, high dUTPase protein levels can be observed only in embryonic stages (Figure 2A). As shown by Western blotting and immunohistochemistry, starting from late embryonic phase, dUTPase expression level is decreased drastically and remains low during postembryonic stages. Under normal physiological circumstances of larval development, dUTPase is predominantly expressed in imaginal discs, the central nervous system and in the testis (Figure 2) but not in most larval tissues, like salivary gland and gut. In the ventriculus and in the salivary gland, some cells are associated with dUTPase expression – these seem to correspond to the imaginal cells (Figure 2B). In the imago, dUTPase is present in the ovary of females. Cellular localization of dUTPase is usually nuclear [21], [28]–[31], however cytoplasmic occurrence is also evident in the nurse cells of mature follicles within ovaries, as described previously [21]. The present results are in agreement with our more limited earlier observations at the protein level [21]. We also quantified the dUTPase mRNA level by real-time PCR and found that protein and mRNA levels show similar tendencies during development (Figure 3A and 3B).

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
Related in: MedlinePlus