Angéla Békési

Developmental Regulation of dUTPase in Drosophila melanogaster

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.

Catalytic and structural role of the metal ion in dUTP pyrophosphatase

The metal ion dependence of the catalytic activity of recombinant Escherichia coli dUTP pyrophosphatase (dUTPase), an essential enzyme preventing incorporation of uracil into DNA, has been investigated by steady-state kinetic, electron paramagnetic resonance, and electron nuclear double resonance methods. Values of kcat and kcat/Km were 4.5 ± 0.1 s−1 and 0.49 ± 0.1 × 106 M−1⋅s−1 in the absence of divalent metal ions, 14.7 ± 2.2 s−1 and 25.1 ± 7.4 × 106 M−1⋅s−1 in the presence of Mg2+ or Mn2+, and 24.2 ± 3.6 s−1 and 2.4 ± 0.7 × 106 M−1⋅s−1 when supported by VO2+ or bis(acetylacetonato)oxovanadium(IV). Binding of VO2+ to the enzyme in the presence of dUDP, a nonhydrolyzable substrate analog, was specific and competitive with Mg2+. Electron paramagnetic resonance spectra of the ternary enzyme–VO2+-chelate–dUDP complex revealed a pattern of 31P superhyperfine coupling specifying two structurally equivalent phosphate groups equatorially coordinated to the VO2+ ion. Proton electron nuclear double resonance spectra revealed an equatorial acetylacetonate ligand, indicating that one of the organic ligands had been displaced. By molecular graphics modeling, we show that the diphosphate group of enzyme-bound dUDP is sterically accessible to a hemi-chelate form of VO2+. We propose a similar location compatible with all kinetic and spectroscopic results to account for the reactivity of VO2+ and the VO2+-chelate in dUTP hydrolysis. In this location the metal ion could promote an ordered conformation of the C-terminal fragment that is obligatory for catalysis but dynamically flexible in the free enzyme.

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

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.

Methylene substitution at the α-β bridging position within the phosphate chain of dUDP profoundly perturbs ligand accommodation into the dUTPase active site

dUTP pyrophosphatase, a preventive DNA repair enzyme, contributes to maintain the appropriate cellular dUTP/dTTP ratio by catalyzing dUTP hydrolysis. dUTPase is essential for viability in bacteria and eukaryotes alike. Identification of species‐specific antagonists of bacterial dUTPases is expected to contribute to the development of novel antimicrobial agents. As a first general step, design of dUTPase inhibitors should be based on modifications of the substrate dUTP phosphate chain, as modifications in either base or sugar moieties strongly impair ligand binding. Based on structural differences between bacterial and human dUTPases, derivatization of dUTP‐analogous compounds will be required as a second step to invoke species‐specific character. Studies performed with dUTP analogues also offer insights into substrate binding characteristics of this important and structurally peculiar enzyme. In this study, α,β‐methylene‐dUDP was synthesized and its complex with dUTPase was characterized. Enzymatic phosphorylation of this substrate analogue by pyruvate kinase was not possible in contrast to the successful enzymatic phosphorylation of α,β‐imino‐dUDP. One explanation for this finding is that the different bond angles and the presence of the methylene group may preclude formation of a catalytically competent complex with the kinase. Crystal structure of E. coli dUTPase:α,β‐methylene‐dUDP and E. coli dUTPase:dUDP:Mn complexes were determined and analyzed in comparison with previous data. Results show that the “trans” α‐phosphate conformation of α,β‐methylene‐dUDP differs from the catalytically competent “gauche” α‐phosphate conformation of the imino analogue and the oxo substrate, manifested in the shifted position of the α‐phosphorus by more than 3 Å. The three‐dimensional structures determined in this work show that the binding of the methylene analogue with the α‐phosphorus in the “gauche” conformation would result in steric clash of the methylene group with the protein atoms. In addition, the metal ion cofactor was not bound in the crystal of the complex with the methylene analogue while it was clearly visible as coordinated to dUDP, arguing that the altered phosphate chain conformation also perturbs metal ion complexation. Isothermal calorimetry titrations indicate that the binding affinity of α,β‐methylene‐dUDP toward dUTPase is drastically decreased when compared with that of dUDP. In conclusion, the present data suggest that while α,β‐methylene‐dUDP seems to be practically nonhydrolyzable, it is not a strong binding inhibitor of dUTPase probably due to the altered binding mode of the phosphate chain. Results indicate that in some cases methylene analogues may not faithfully reflect the competent substrate ligand properties, especially if the methylene hydrogens are in steric conflict with the protein. Proteins 2008.

Structural disorder and local order of hNopp140.

Human nucleolar phosphoprotein p140 (hNopp 140) is a highly phosphorylated protein inhibitor of casein kinase 2 (CK2). As in the case of many kinase-inhibitor systems, the inhibitor has been described to belong to the family of intrinsically disordered proteins (IDPs), which often utilize transient structural elements to bind their cognate enzyme. Here we investigated the structural status of this protein both to provide distinct lines of evidence for its disorder and to point out its transient structure potentially involved in interactions and also its tendency to aggregate. Structural disorder of hNopp140 is apparent by its anomalous electrophoretic mobility, protease sensitivity, heat stability, hydrodynamic behavior on size-exclusion chromatography, (1)H NMR spectrum and differential scanning calorimetry scan. hNopp140 has a significant tendency to aggregate and the change of its circular dichroism spectrum in the presence of 0-80% TFE suggests a tendency to form local helical structures. Wide-line NMR measurements suggest the overall disordered character of the protein. In all, our data suggest that this protein falls into the pre-molten globule state of IDPs, with a significant tendency to become ordered in the presence of its partner as demonstrated in the presence of transcription factor IIB (TFIIB).

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

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.

Detection of uracil within DNA using a sensitive labeling method for in vitro and cellular applications

The role of uracil in genomic DNA has been recently re-evaluated. It is now widely accepted to be a physiologically important DNA element in diverse systems from specific phages to antibody maturation and Drosophila development. Further relevant investigations would largely benefit from a novel reliable and fast method to gain quantitative and qualitative information on uracil levels in DNA both in vitro and in situ, especially since current techniques does not allow in situ cellular detection. Here, starting from a catalytically inactive uracil-DNA glycosylase protein, we have designed several uracil sensor fusion proteins. The designed constructs can be applied as molecular recognition tools that can be detected with conventional antibodies in dot-blot applications and may also serve as in situ uracil-DNA sensors in cellular techniques. Our method is verified on numerous prokaryotic and eukaryotic cellular systems. The method is easy to use and can be applied in a high-throughput manner. It does not require expensive equipment or complex know-how, facilitating its easy implementation in any basic molecular biology laboratory. Elevated genomic uracil levels from cells of diverse genetic backgrounds and/or treated with different drugs can be demonstrated also in situ, within the cell.

Association of RNA with the uracil-DNA-degrading factor has major conformational effects and is potentially involved in protein folding

Recently, a novel uracil‐DNA‐degrading factor protein (UDE) was identified in Drosophila melanogaster, with homologues only in pupating insects. Its unique uracil‐DNA‐degrading activity and a potential domain organization pattern have been described. UDE seems to be the first representative of a new protein family with unique enzyme activity that has a putative role in insect development. In addition, UDE may also serve as potential tool in molecular biological applications. Owing to lack of homology with other proteins with known structure and/or function, de novo data are required for a detailed characterization of UDE structure and function. Here, experimental evidence is provided that recombinant protein is present in two distinct conformers. One of these contains a significant amount of RNA strongly bound to the protein, influencing its conformation. Detailed biophysical characterization of the two distinct conformational states (termed UDE and RNA–UDE) revealed essential differences. UDE cannot be converted into RNA–UDE by addition of the same RNA, implying putatively joint processes of RNA binding and protein folding in this conformational species. By real‐time PCR and sequencing after random cloning, the bound RNA pool was shown to consist of UDE mRNA and the two ribosomal RNAs, also suggesting cotranslational RNA‐assisted folding. This finding, on the one hand, might open a way to obtain a conformationally homogeneous UDE preparation, promoting successful crystallization; on the other hand, it might imply a further molecular function of the protein. In fact, RNA‐dependent complexation of UDE was also demonstrated in a fruit fly pupal extract, suggesting physiological relevance of RNA binding of this DNA‐processing enzyme.

Expanding the DNA alphabet in the fruit fly: uracil enrichment in genomic DNA.

DNA integrity is under the control of multiple pathways of nucleotide metabolism and DNA damage recognition and repair. Unusual sets of protein factors involved in these control mechanisms may result in tolerance and accumulation of non-canonical bases within the DNA. We investigate the presence of uracil in genomic DNA of Drosophila melanogaster. Results indicate a developmental pattern and strong correlations between uracil-DNA levels, dUTPase expression and developmental fate of different tissues. The intriguing lack of the catalytically most efficient uracil-DNA glycosylase in Drosophila melanogaster may be a general attribute of Holometabola and is suggested to be involved in the specific characteristics of uracil-DNA metabolism in these insects.

Potential steps in the evolution of a fused trimeric all-β dUTPase involve a catalytically competent fused dimeric intermediate.

Deoxyuridine 5′‐triphosphate nucleotidohydrolase (dUTPase) is essential for genome integrity. Interestingly, this enzyme from Drosophila virilis has an unusual form, as three monomer repeats are merged with short linker sequences, yielding a fused trimer‐like dUTPase fold. Unlike homotrimeric dUTPases that are encoded by a single repeat dut gene copy, the three repeats of the D. virilis dut gene are not identical due to several point mutations. We investigated the potential evolutionary pathway that led to the emergence of this extant fused trimeric dUTPase in D. virilis. The herein proposed scenario involves two sequential gene duplications followed by sequence divergence amongst the dut repeats. This pathway thus requires the existence of a transient two‐repeat‐containing fused dimeric dUTPase intermediate. We identified the corresponding ancestral dUTPase single repeat enzyme together with its tandem repeat evolutionary intermediate and characterized their enzymatic function and structural stability. We additionally engineered and characterized artificial single or tandem repeat constructs from the extant enzyme form to investigate the influence of the emergent residue alterations on the formation of a functional assembly. The observed severely impaired stability and catalytic activity of these latter constructs provide a plausible explanation for evolutionary persistence of the extant fused trimeric D. virilis dUTPase form. For the ancestral homotrimeric and the fused dimeric intermediate forms, we observed strong catalytic and structural competence, verifying viability of the proposed evolutionary pathway. We conclude that the progression along the herein described evolutionary trajectory is determined by the retained potential of the enzyme for its conserved three‐fold structural symmetry.