Trading in cooperativity for specificity to maintain uracil-free DNA

Members of the dUTPase superfamily play an important role in the maintenance of the pyrimidine nucleotide balance and of genome integrity. dCTP deaminases and the bifunctional dCTP deaminase-dUTPases are cooperatively regulated by dTTP. However, the manifestation of allosteric behavior within the same trimeric protein architecture of dUTPases, the third member of the superfamily, has been a question of debate for decades. Therefore, we designed hybrid dUTPase trimers to access conformational states potentially mimicking the ones observed in the cooperative relatives. We studied how the interruption of different steps of the enzyme cycle affects the active site cross talk. We found that subunits work independently in dUTPase. The experimental results combined with a comparative structural analysis of dUTPase superfamily enzymes revealed that subtile structural differences within the allosteric loop and the central channel in these enzymes give rise to their dramatically different cooperative behavior. We demonstrate that the lack of allosteric regulation in dUTPase is related to the functional adaptation to more efficient dUTP hydrolysis which is advantageous in uracil-DNA prevention

Cellular response to efficient dUTPase RNAi silencing in stable HeLa cell lines perturbs expression levels of genes involved in thymidylate metabolism.

dUTPase is involved in preserving DNA integrity in cells. We report an efficient dUTPase silencing by RNAi-based system in stable human cell line. Repression of dUTPase induced specific expression level increments for thymidylate kinase and thymidine kinase, and also an increased sensitization to 5-fluoro-2'-deoxyuridine and 5-fluoro-uracil. The catalytic mechanism of dUTPase was investigated for 5-fluoro-dUTP. The 5F-substitution on the uracil ring of the substrate did not change the kinetic mechanism of dUTP hydrolysis by dUTPase. Results indicate that RNAi silencing of dUTPase induces a complex cellular response wherein sensitivity towards fluoropyrimidines and gene expression levels of related enzymes are both modulated

Sixty-one macrofungi species new to Hungary in Őrség National Park

In this paper, an annotated checklist of macrofungi from Őrség National Park, West Hungary, is provided. A total of 726 macrofungi taxa representing 214 genera, 84 classes and 2 phyla (Asco- and Basidiomycota) were revealed. Sixty-one macrofungi species were new to the mycobiota of Hungary. Sporocarps were collected three times (in May, August and September–October) between 2009 and 2010 in 35 (40 m × 40 m) forest stands with different tree species compositions. Preferred tree species compositions and substrata of registered macrofungi are also listed

Analysis of Drosophila Atg8 proteins reveals multiple lipidation-independent roles

Yeast Atg8 and its homologs are involved in autophagosome biogenesis in all eukaryotes. These are the most widely used markers for autophagy thanks to the association of their lipidated forms with autophagic membranes. The Atg8 protein family expanded in animals and plants, with most Drosophila species having two Atg8 homologs. In this Brief Report, we use clear-cut genetic analysis in Drosophila melanogaster to show that lipidated Atg8a is required for autophagy, while its non-lipidated form is essential for developmentally programmed larval midgut elimination and viability. In contrast, expression of Atg8b is restricted to the male germline and its loss causes male sterility without affecting autophagy. We find that high expression of non-lipidated Atg8b in the male germline is required for fertility. Consistent with these non-canonical functions of Atg8 proteins, loss of Atg genes required for Atg8 lipidation lead to autophagy defects but do not cause lethality or male sterility

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

Calpain-Catalyzed Proteolysis of Human dUTPase Specifically Removes the Nuclear Localization Signal Peptide

Calpain proteases drive intracellular signal transduction via specific proteolysis of multiple substrates upon Ca(2+)-induced activation. Recently, dUTPase, an enzyme essential to maintain genomic integrity, was identified as a physiological calpain substrate in Drosophila cells. Here we investigate the potential structural/functional significance of calpain-activated proteolysis of human dUTPase.Limited proteolysis of human dUTPase by mammalian m-calpain was investigated in the presence and absence of cognate ligands of either calpain or dUTPase. Significant proteolysis was observed only in the presence of Ca(II) ions, inducing calpain action. The presence or absence of the dUTP-analogue α,β-imido-dUTP did not show any effect on Ca(2+)-calpain-induced cleavage of human dUTPase. The catalytic rate constant of dUTPase was unaffected by calpain cleavage. Gel electrophoretic analysis showed that Ca(2+)-calpain-induced cleavage of human dUTPase resulted in several distinctly observable dUTPase fragments. Mass spectrometric identification of the calpain-cleaved fragments identified three calpain cleavage sites (between residues (4)SE(5); (7)TP(8); and (31)LS(32)). The cleavage between the (31)LS(32) peptide bond specifically removes the flexible N-terminal nuclear localization signal, indispensable for cognate localization.Results argue for a mechanism where Ca(2+)-calpain may regulate nuclear availability and degradation of dUTPase

The History of Colonel Simonyi and the Baron Simonyi of Vitézvár Family

A „vitézvári” nemesi előnevet, illetve igen sok dokumentumban – nem teljesen jogszerűen – a „barbácsi” predikátumot is viselő báró Simonyi család nem tartozik a magyar arisztokrácia legismertebb famíliái közé. Ennek pedig az a legfőbb magyarázata, hogy a család már második generációjában – illetve a harmadik generációba tartozó két személy korai, még gyermekkorban bekövetkezett halála miatt – mind fi-, mind leányágon kihalt, így nevét nem volt módjában különösebben ismerté tenni a széles közönség előtt. Két felnőttkort megélt tagja, József, az ezredes és Lajos, a miniszter azonban oly tettekkel írták be nevüket a magyar történelem „arany oldalaira”, hogy emlékük felidézése kötelességünk. A két említett férfiről, báró Simonyi Józsefről – közismertebb nevén Simonyi óbesterről – (1771–1832) és fiáról, báró Simonyi Lajosról (1824–1894) igen jelentős mennyiségű publikáció látott eddig napvilágot. Ezek szinte mindegyikében található azonban olyan tévedés, hiba, elírás, vagy csúsztatás, amelyeket – az általam felkutatott, jelentős mennyiségű új levéltári forrás alapján – korrigálni tudtam, illetve megfogalmazódtak bennük olyan kérdések, amelyekre talán most sikerül választ találnom. Ez fokozottan vonatkozik az egyébként nem túl bőséges genealógiai szakirodalomra is, ahol a család történetének, leszármazásának közlésekor szintén sok hibás adat került ismertetésre. Pedig a báró Simonyi család történetében – rövidsége ellenére – e tévedések nélkül is előfordulnak homályos pontok, rejtélyek, amelyeket tisztázni kell az utókor számára. Írásomban tehát arra törekedtem, hogy komplexen bemutassam Simonyi óbester és családjának, a vitézvári báró Simonyi famíliának a történetét. Így az értekezés egyben egy életrajz – jelentős hadtörténeti, valamint némi jogtörténeti részekkel – és egy család története is. A hangsúlyt természetesen Simonyi származására, születésére, katona előmenetelére, bárói címére, családalapítására, hadbírósági perére és halálára fektettem. De ezek mellett természetesen bőven szó esik majd Simonyi óbester őseinek, közvetlen családtagjainak – feleségének, gyermekeinek, unokáinak – és rokonságának a történetéről is. The Baron Simonyi family, wearing the nobility prefix “of Vitézvár” as well as – though not quite lawfully – the predicate “of Barbács”, which appears in a number of documents, does not belong to the most renowned families of the Hungarian aristocracy. This is mainly because already in the second generation of the family – not counting the two members of the third generation who died at an early age – both its male and female lines became extinct, so its name could not become famous among a wide audience. However, two adult members of the family, József the colonel and Lajos the minister, must be remembered because of their great and historically important deeds. A number of works have been published so far on the two men, Baron József Simonyi (1771–1832) – better known as Colonel Simonyi – and his son, Baron Lajos Simonyi (1824–1894). Yet, almost all of the publications contain errors, mistakes, spelling mistakes or obscurings that I was able to correct on the basis of the data collected from newly-found sources, and they also raise questions which I was able to answer. The same applies to the low amount of genealogical literature to an even greater extent, in which many incorrect data are presented in relation to the history and the descent of the family. Yet, apart from these mistakes, the history of the Baron Simonyi family, despite its shortness, contains vague points, mysteries, which should be clarified for posterity. Therefore, in my writing I strive to present the history of Colonel Simonyi and his family, the Baron Simonyi of Vitézvár family, in a complex way. As a result, the present dissertation is a biography and a family history at the same time, completed with extensive parts on military history and some legal history. The emphasis, evidently, was put on the descent, birth, military career, baronial title, settling down, military court trial and death of the Colonel. Besides these issues the thesis also contains details on the history of the ancestors, immediate family members (wife, children, grandchildren) and relatives of Colonel Simonyi.d