A novel fruitfly protein under developmental control degrades uracil-DNA

Békési, Angéla; Pukáncsik, Mária; Muha, Villő; Zagyva, Imre; Leveles, Ibolya; Hunyadi‐Gulyás, Éva; Klement, Éva; Medzihradszky, Katalin F.; Kele, Zoltán; Erdei, Anna; Felföldi, Ferenc; Kónya, Emese; Vértessy, Beáta G.

doi:10.1016/j.bbrc.2007.01.196

Cited by 23 publications

(15 citation statements)

References 18 publications

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“…First, similarly to the case of hypermethylated DNA, uracil–DNA may show a decreased response and interaction with transcriptional regulators, activators or other morphogenetic factors required specifically during pupal metamorphosis. Second, one or more factor(s), functional only in the pupal stage, may process uracil–DNA resulting in genome instability and defects in cell cycle progression or cell death [47], [48]. Beyond UNG that is missing from Drosophila , other uracil–DNA glycosylases would be suspect for this role.…”

Section: Resultsmentioning

confidence: 99%

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Uracil-Containing DNA in Drosophila: Stability, Stage-Specific Accumulation, and Developmental Involvement

et al. 2012

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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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Section: Resultsmentioning

confidence: 99%

“…Plasmids were purified with Plasmid Midi Kit (QIAGEN). Uracil content of the plasmids was checked with UDG and AP endonuclease treatment followed by standard agarose gel electrophoresis [48].…”

Section: Methodsmentioning

confidence: 99%

Uracil-Containing DNA in Drosophila: Stability, Stage-Specific Accumulation, and Developmental Involvement

et al. 2012

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“…The significance of UDE is two‐fold: (a) it may be developed into a versatile molecular biotechnological tool [39]; and (b) its targeting may yield species‐specific insecticides to be used against, for example, malaria mosquitoes. Here, we employed a multidisciplinary set of theoretical and experimental approaches (schematically described in Fig.…”

Section: Discussionmentioning

confidence: 99%

Physiological truncation and domain organization of a novel uracil‐DNA‐degrading factor

et al. 2010

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Uracil in DNA is usually considered to be an error, but it may be used for signaling in Drosophila development via recognition by a novel uracil‐DNA‐degrading factor (UDE) [(Bekesi A et al. (2007) Biochem Biophys Res Commun355, 643–648]. The UDE protein has no detectable similarity to any other uracil‐DNA‐binding factors, and has no structurally or functionally described homologs. Here, a combination of theoretical and experimental analyses reveals the domain organization and DNA‐binding pattern of UDE. Sequence alignments and limited proteolysis with different proteases show extensive protection by DNA at the N‐terminal duplicated conserved motif 1A/1B segment, and a well‐folded domain within the C‐terminal half encompassing conserved motifs 2–4. Theoretical structure prediction suggests that motifs 1A and 1B fold as similar α‐helical bundles, and reveals two conserved positively charged surface patches that may bind DNA. CD spectroscopy also supports the presence of α‐helices in UDE. Full functionality of a physiologically occurring truncated isoform in Tribolium castaneum lacking one copy of the N‐terminal conserved motif 1 is revealed by activity assays of a representative truncated construct of Drosophila melanogaster UDE. Gel filtration and analytical ultracentrifugation results, together with analysis of predicted structural models, suggest a possible dimerization mechanism for preserving functionality of the truncated isoform. Structured digital abstract http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7385914: UDE (uniprotkb:http://www.uniprot.org/uniprot/Q961C4?format=text&ascii) and UDE (uniprotkb:http://www.uniprot.org/uniprot/Q961C4?format=text&ascii) bind (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0407) by cosedimentation in solution (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0028)

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“…For example, Drosophila has no uracil-DNA glycosylase homologs, yet two poorly characterized activities removing uracil from DNA either as a free base or as part of short oligonucleotides have been described from larvae of various ages 44,45 . Larval tissues of Drosophila contain a greatly elevated level of uracil, whereas imaginal discs seem to support active uracil removal and prevention of dUMP incorporation 46 .…”

Section: Discussionmentioning

confidence: 99%

Base excision DNA repair in the embryonic development of the sea urchin, Strongylocentrotus intermedius

et al. 2016

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In actively proliferating cells, such as the cells of the developing embryo, DNA repair is crucial for preventing the accumulation of mutations and synchronizing cell division. Sea urchin embryo growth was analyzed and extracts were prepared. The relative activity of DNA polymerase, apurinic/apyrimidinic (AP) endonuclease, uracil-DNA glycosylase, 8-oxoguanine-DNA glycosylase, and other glycosylases was analyzed using specific oligonucleotide substrates of these enzymes; the reaction products were resolved by denaturing 20% polyacrylamide gel electrophoresis. We have characterized the profile of several key base excision repair activities in the developing embryos (2 blastomers to mid-pluteus) of the grey sea urchin, Strongylocentrotus intermedius. The uracil-DNA glycosylase specific activity sharply increased after blastula hatching, whereas the specific activity of 8-oxoguanine-DNA glycosylase steadily decreased over the course of the development. The AP-endonuclease activity gradually increased but dropped at the last sampled stage (mid-pluteus 2). The DNA polymerase activity was high at the first cleavage division and then quickly decreased, showing a transient peak at blastula hatching. It seems that the developing sea urchin embryo encounters different DNA-damaging factors early in development within the protective envelope and later as a free-floating larva, with hatching necessitating adaptation to the shift in genotoxic stress conditions. No correlation was observed between the dynamics of the enzyme activities and published gene expression data from developing congeneric species, S. purpuratus. The results suggest that base excision repair enzymes may be regulated in the sea urchin embryos at the level of covalent modification or protein stability.

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A novel fruitfly protein under developmental control degrades uracil-DNA

Cited by 23 publications

References 18 publications

Uracil-Containing DNA in Drosophila: Stability, Stage-Specific Accumulation, and Developmental Involvement

Uracil-Containing DNA in Drosophila: Stability, Stage-Specific Accumulation, and Developmental Involvement

Physiological truncation and domain organization of a novel uracil‐DNA‐degrading factor

Base excision DNA repair in the embryonic development of the sea urchin, Strongylocentrotus intermedius

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