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The dUTPase is a key DNA repair enzyme in Mycobacterium tuberculosis, and it may serve as a novel promising anti-tuberculosis target. Stl repressor from Staphylococcus aureus was shown to bind to and inhibit dUTPases from various sources, and its expression in mycobacterial cells interfered with cell growth. To fine-tune and optimize Stl-induced inhibition of mycobacterial dUTPase, we aimed to decipher the molecular details of this interaction. Structural background of the complex between dUTPase and a truncated Stl lacking the repressor C-terminal homodimerization domain has been described, however, the effects of this truncation of Stl on enzyme binding and inhibition are still not known. Using several independent biophysical, structural and enzyme kinetic methods, here we show that lack of the repressor homodimerization domain strongly perturbs both enzyme binding and inhibition. We also investigated the role of a mycobacteria-specific loop in the Stl-interaction. Our results show that removal of this loop leads to a ten-fold increase in the apparent inhibition constant of Stl. We present a high-resolution three-dimensional structure of mycobacterial dUTPase lacking the genus-specific loop for structural insight. Our present data suggest that potent inhibition of mycobacterial dUTPase by Stl requires the wild-type full-length protein context., (© 2024. The Author(s).)

Enzymatic processes play an increasing role in synthetic organic chemistry which requires the access to a broad and diverse set of enzymes. Metagenome mining is a valuable and efficient way to discover novel enzymes with unique properties for biotechnological applications. Here, we report the discovery and biocatalytic characterization of six novel metagenomic opine dehydrogenases from a hot spring environment (mODHs) (EC 1.5.1.X). These enzymes catalyze the asymmetric reductive amination between an amino acid and a keto acid resulting in opines which have defined biochemical roles and represent promising building blocks for pharmaceutical applications. The newly identified enzymes exhibit unique substrate specificity and higher thermostability compared to known examples. The feature that they preferably utilize negatively charged polar amino acids is so far unprecedented for opine dehydrogenases. We have identified two spatially correlated positions in their active sites that govern this substrate specificity and demonstrated a switch of substrate preference by site-directed mutagenesis. While they still suffer from a relatively narrow substrate scope, their enhanced thermostability and the orthogonality of their substrate preference make them a valuable addition to the toolbox of enzymes for reductive aminations. Importantly, enzymatic reductive aminations with highly polar amines are very rare in the literature. Thus, the preparative-scale enzymatic production, purification, and characterization of three highly functionalized chiral secondary amines lend a special significance to our work in filling this gap. KEY POINTS: • Six new opine dehydrogenases have been discovered from a hot spring metagenome • The newly identified enzymes display a unique substrate scope • Substrate specificity is governed by two correlated active-site residues., (© 2024. The Author(s).)

Hemozoin is a natural biomarker formed during the hemoglobin metabolism of Plasmodium parasites, the causative agents of malaria. The rotating-crystal magneto-optical detection (RMOD) has been developed for its rapid and sensitive detection both in cell cultures and patient samples. In the current article we demonstrate that, besides quantifying the overall concentration of hemozoin produced by the parasites, RMOD can also track the size distribution of the hemozoin crystals. We establish the relations between the magneto-optical signal, the mean parasite age and the median crystal size throughout one erythrocytic cycle of Plasmodium falciparum parasites, where the latter two are determined by optical and scanning electron microscopy, respectively. The significant correlation between the magneto-optical signal and the stage distribution of the parasites indicates that the RMOD method can be utilized for species-specific malaria diagnosis and for the quick assessment of drug efficacy., (© 2024. The Author(s).)

The enzyme dUTPase has an essential role in maintaining genomic integrity. In mouse, nuclear and mitochondrial isoforms of the enzyme have been described. Here we present the isoform-specific mRNA expression levels in different murine organs during development using RT-qPCR. In this study, we analyzed organs of 14.5-day embryos and of postnatal 2-, 4-, 10-week- and 13-month-old mice. We demonstrate organ-, sex- and developmental stage-specific differences in the mRNA expression levels of both isoforms. We found high mRNA expression level of the nuclear isoform in the embryo brain, and the expression level remained relatively high in the adult brain as well. This was surprising, since dUTPase is known to play an important role in proliferating cells, and mass production of neural cells is completed by adulthood. Thus, we investigated the pattern of the dUTPase protein expression specifically in the adult brain with immunostaining and found that dUTPase is present in the germinative zones, the subventricular and the subgranular zones, where neurogenesis occurs and in the rostral migratory stream where neuroblasts migrate to the olfactory bulb. These novel findings suggest that dUTPase may have a role in cell differentiation and indicate that accurate dTTP biosynthesis can be vital, especially in neurogenesis., (© 2024. The Author(s).)

Gemcitabine is a potent inhibitor of DNA replication and is a mainstay therapeutic for diverse cancers, particularly pancreatic ductal adenocarcinoma (PDAC). However, most tumors remain refractory to gemcitabine therapies. Here, to define the cancer cell response to gemcitabine, we performed genome-scale CRISPR-Cas9 chemical-genetic screens in PDAC cells and found selective loss of cell fitness upon disruption of the cytidine deaminases APOBEC3C and APOBEC3D. Following gemcitabine treatment, APOBEC3C and APOBEC3D promote DNA replication stress resistance and cell survival by deaminating cytidines in the nuclear genome to ensure DNA replication fork restart and repair in PDAC cells. We provide evidence that the chemical-genetic interaction between APOBEC3C or APOBEC3D and gemcitabine is absent in nontransformed cells but is recapitulated across different PDAC cell lines, in PDAC organoids and in PDAC xenografts. Thus, we uncover roles for APOBEC3C and APOBEC3D in DNA replication stress resistance and offer plausible targets for improving gemcitabine-based therapies for PDAC., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Polycyclic aromatic hydrocarbons (PAHs) are highly toxic, carcinogenic substances. On soils contaminated with PAHs, crop cultivation, animal husbandry and even the survival of microflora in the soil are greatly perturbed, depending on the degree of contamination. Most microorganisms cannot tolerate PAH-contaminated soils, however, some microbial strains can adapt to these harsh conditions and survive on contaminated soils. Analysis of the metagenomes of contaminated environmental samples may lead to discovery of PAH-degrading enzymes suitable for green biotechnology methodologies ranging from biocatalysis to pollution control. In the present study, our goal was to apply a metagenomic data search to identify efficient novel enzymes in remediation of PAH-contaminated soils. The metagenomic hits were further analyzed using a set of bioinformatics tools to select protein sequences predicted to encode well-folded soluble enzymes. Three novel enzymes (two dioxygenases and one peroxidase) were cloned and used in soil remediation microcosms experiments. The experimental design of the present study aimed at evaluating the effectiveness of the novel enzymes on short-term PAH degradation in the soil microcosmos model. The novel enzymes were found to be efficient for degradation of naphthalene and phenanthrene. Adding the inorganic oxidant CaO 2 further increased the degrading potential of the novel enzymes for anthracene and pyrene. We conclude that metagenome mining paired with bioinformatic predictions, structural modelling and functional assays constitutes a powerful approach towards novel enzymes for soil remediation., (© 2024. The Author(s).)

Human tyrosine hydroxylase (hTH) has key role in the production of catecholamine neurotransmitters. The structure, function and regulation of hTH has been extensively researched area and the possibility of enzyme replacement therapy (ERT) involving hTH through nanocarriers has been raised as well. However, our understanding on how hTH may interact with nanocarriers is still lacking. In this work, we attempted to investigate the immobilization of hTH on magnetic nanoparticles (MNPs) with various surface linkers in quantitative and mechanistic detail. Our results showed that the activity of hTH was retained after immobilization via secondary and covalent interactions as well. The colloidal stability of hTH could be also enhanced proved by Dynamic light scattering and Zeta potential analysis and a homogenous enzyme layer could be achieved, which was investigated by Raman mapping. The covalent attachment of hTH on MNPs via aldehyde or epoxy linkers provide irreversible immobilization and 38.1 % and 16.5 % recovery (ER). The hTH-MNPs catalyst had 25 % ER in average in simulated nasal electrolyte solution (SNES). This outcome highlights the relevance of immobilization applying MNPs as a potential formulation tool of sensitive therapeutic enzymes offering new opportunities for ERT related to neurodegenerative disorders., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Nuclear Piwi/Piwi-interacting RNA complexes mediate co-transcriptional silencing of transposable elements by inducing local heterochromatin formation. In Drosophila, sumoylation plays an essential role in the assembly of the silencing complex; however, the molecular mechanism by which the sumoylation machinery is recruited to the transposon loci is poorly understood. Here, we show that the Drosophila E3 SUMO-ligase Su(var)2-10 directly binds to the Piwi protein. This interaction is mediated by the SUMO-interacting motif-like (SIM-like) structure in the C-terminal domain of Su(var)2-10. We demonstrated that the SIM-like structure binds to a special region found in the MID domain of the Piwi protein, the structure of which is highly similar to the SIM-binding pocket of SUMO proteins. Abrogation of the Su(var)2-10-binding surface of the Piwi protein resulted in transposon derepression in the ovary of adult flies. Based on our results, we propose a model in which the Piwi protein initiates local sumoylation in the silencing complex by recruiting Su(var)2-10 to the transposon loci., (© 2024 HUN‐REN Biological Research Centre and The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Mutated genes may lead to cancer development in numerous tissues. While more than 600 cancer-causing genes are known today, some of the most widespread mutations are connected to the RAS gene; RAS mutations are found in approximately 25% of all human tumors. Specifically, KRAS mutations are involved in the three most lethal cancers in the U.S., namely pancreatic ductal adenocarcinoma, colorectal adenocarcinoma, and lung adenocarcinoma. These cancers are among the most difficult to treat, and they are frequently excluded from chemotherapeutic attacks as hopeless cases. The mutated KRAS proteins have specific three-dimensional conformations, which perturb functional interaction with the GAP protein on the GAP-RAS complex surface, leading to a signaling cascade and uncontrolled cell growth. Here, we describe a gluing docking method for finding small molecules that bind to both the GAP and the mutated KRAS molecules. These small molecules glue together the GAP and the mutated KRAS molecules and may serve as new cancer drugs for the most lethal, most difficult-to-treat, carcinomas. As a proof of concept, we identify two new, drug-like small molecules with the new method; these compounds specifically inhibit the growth of the PANC-1 cell line with KRAS mutation G12D in vitro and in vivo. Importantly, the two new compounds show significantly lower IC 50 and higher specificity against the G12D KRAS mutant human pancreatic cancer cell line PANC-1, as compared to the recently described selective G12D KRAS inhibitor MRTX-1133.

The excision and replication, thus the life cycle of pathogenicity islands in staphylococci are regulated by Stl master repressors that form strong dimers. It has been recently shown that SaPIbov1-Stl dimers are separated during the activation of the Staphylococcus aureus pathogenicity island (SaPI) transcription via helper phage proteins. To understand the mechanism of this regulation, a quantitative analysis of the dimerization characteristics is required. Due to the highly efficient dimerization process, such an analysis has to involve specific solutions that permit relevant experiments to be performed. In the present work, we focused on two staphylococcal Stls associated with high biomedical interest, namely Stl proteins of Staphylococcus aureus bov1 and Staphylococcus hominis ShoCI794_SEPI pathogenicity islands. Exploiting the interactions of these two Stl proteins with their antirepressor-mimicking interaction partners allowed precise determination of the Stl dimerization constant in the subnanomolar range., (© 2024. The Author(s).)

Due to the cyclical nature and changing water levels in the sequencing batch reactor (SBR), oxygen diffusion and utilization can be difficult to control particularly in light of the need to conserve the limited quantity of carbon source required to optimize biological nutrient removal. During the fill period, oxygen penetration may be undesirable since heterotrophic and autotrophic organisms cause a reduction in the readily biodegradable carbon source (rbCOD). This carbon source is essential and often limited in the anaerobic and anoxic periods. As a consequence, unwanted oxygen penetration can hinder efficient biological phosphorus and nitrogen removal. The purpose of the present research was to verify the advantage of a floating seal on the continuously moving surface of an SBR reactor to minimize undesirable oxygen penetration. In the floating seal-covered SBR both nitrification and denitrification efficiency proved to be higher due to insulation, and even during wintertime biological phosphorus removal met target removals without chemical dosing. The SVI values in the two SBR trains proved to be close to each other, despite the high difference in chemical dosing. Having experienced the higher efficiency of the seal-covered train, microbiome compositions of the two differently operated systems were investigated to determine potential differences via 16S rRNA gene amplicon sequencing experiments. In the samples taken from the seal-covered system, higher ratios of fermentative bacteria and phosphate accumulating organisms (PAOs) as well as glycogen accumulating organisms (GAOs) could be observed as compared to the samples deriving from the uncovered system. Highlights Seal-covering the periodically decreasing open water surface increased SBR efficiencySeal-covering the open water surface increased nitrification efficiency by insulationNo chemical dosing was necessary for phosphorous removal in the Test systemMetagenome investigations provided almost doubled amount of fermentative bacteriaProduction of GAOs indicated nutrient deficiency due to phosphorous removal.

Cells maintain a fine-tuned balance of deoxyribonucleoside 5'-triphosphates (dNTPs), a crucial factor in preserving genomic integrity. Any alterations in the nucleotide pool's composition or chemical modifications to nucleotides before their incorporation into DNA can lead to increased mutation frequency and DNA damage. In addition to the chemical modification of canonical dNTPs, the cellular de novo dNTP metabolism pathways also produce noncanonical dNTPs. To keep their levels low and prevent them from incorporating into the DNA, these noncanonical dNTPs are removed from the dNTP pool by sanitizing enzymes. In this study, we introduce innovative protocols for the high-throughput fluorescence-based quantification of dUTP, 5-methyl-dCTP, and 5-hydroxymethyl-dCTP. To distinguish between noncanonical dNTPs and their canonical counterparts, specific enzymes capable of hydrolyzing either the canonical or noncanonical dNTP analogs are employed. This approach provides a more precise understanding of the composition and noncanonical constituents of dNTP pools, facilitating a deeper comprehension of DNA metabolism and repair. It is also crucial for accurately interpreting mutational patterns generated through the next-generation sequencing of biological samples.

Opines and opine-type chemicals are valuable natural products with diverse biochemical roles, and potential synthetic building blocks of bioactive compounds. Their synthesis involves reductive amination of ketoacids with amino acids. This transformation has high synthetic potential in producing enantiopure secondary amines. Nature has evolved opine dehydrogenases for this chemistry. To date, only one enzyme has been used as biocatalyst, however, analysis of the available sequence space suggests more enzymes to be exploited in synthetic organic chemistry. This review summarizes the current knowledge of this underexplored enzyme class, highlights key molecular, structural, and catalytic features with the aim to provide a comprehensive general description of opine dehydrogenases, thereby supporting future enzyme discovery and protein engineering studies., (© 2023 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC.)

Ras GTPases play a crucial role in cell signaling pathways. Mutations of the Ras gene occur in about one third of cancerous cell lines and are often associated with detrimental clinical prognosis. Hot spot residues Gly12, Gly13, and Gln61 cover 97% of oncogenic mutations, which impair the enzymatic activity in Ras. Using QM/MM free energy calculations, we present a two-step mechanism for the GTP hydrolysis catalyzed by the wild-type Ras.GAP complex. We found that the deprotonation of the catalytic water takes place via the Gln61 as a transient Brønsted base. We also determined the reaction profiles for key oncogenic Ras mutants G12D and G12C using QM/MM minimizations, matching the experimentally observed loss of catalytic activity, thereby validating our reaction mechanism. Using the optimized reaction paths, we devised a fast and accurate procedure to design GAP mutants that activate G12D Ras. We replaced GAP residues near the active site and determined the activation barrier for 190 single mutants. We furthermore built a machine learning for ultrafast screening, by fast prediction of the barrier heights, tested both on the single and double mutations. This work demonstrates that fast and accurate screening can be accomplished via QM/MM reaction path optimizations to design protein sequences with increased catalytic activity. Several GAP mutations are predicted to re-enable catalysis in oncogenic G12D, offering a promising avenue to overcome aberrant Ras-driven signal transduction by activating enzymatic activity instead of inhibition. The outlined computational screening protocol is readily applicable for designing ligands and cofactors analogously.

In human cells two dUTPase isoforms have been described: one nuclear (DUT-N) and one mitochondrial (DUT-M), with cognate localization signals. In contrast, here we identified two additional isoforms; DUT-3 without any localization signal and DUT-4 with the same nuclear localization signal as DUT-N. Based on an RT-qPCR method for simultaneous isoform-specific quantification we analysed the relative expression patterns in 20 human cell lines of highly different origins. We found that the DUT-N isoform is expressed by far at the highest level, followed by the DUT-M and the DUT-3 isoform. A strong correlation between expression levels of DUT-M and DUT-3 suggests that these two isoforms may share the same promoter. We analysed the effect of serum starvation on the expression of dUTPase isoforms compared to non-treated cells and found that the mRNA levels of DUT-N decreased in A-549 and MDA-MB-231 cells, but not in HeLa cells. Surprisingly, upon serum starvation DUT-M and DUT-3 showed a significant increase in the expression, while the expression level of the DUT-4 isoform did not show any changes. Taken together our results indicate that the cellular dUTPase supply may also be provided in the cytoplasm and starvation stress induced expression changes are cell line dependent., (© 2023. The Author(s).)

Understanding and characterizing the molecular background of the maintenance of genomic integrity might be a major factor in comprehending the exceptional ability of the malaria parasite, Plasmodium falciparum to adapt at a fast pace to antimalarials. A balanced nucleotide pool is an essential factor for high-fidelity replication. The lack of detailed studies on deoxynucleotide-triphosphate (dNTP) pools in various intraerythrocytic stages of Plasmodium falciparum motivated our present study. Here, we focused on the building blocks of DNA and utilized an EvaGreen-based dNTP incorporation assay to successfully measure the temporal dynamics of dNTPs in every intraerythrocytic stage and in drug-treated trophozoites. Our findings show that the ratio of dNTPs in the ring-stage parasites significantly differs from the more mature trophozoite and schizont stages. We were also able to detect dGTP levels that have never been shown before and found it to be the least abundant dNTP in all stages. Treatment with WR99210, a TS-DHFR inhibitor drug, affected not only dTTP, but also dGTP levels, despite its presumed selective action on pyrimidine biosynthesis. Results from our studies might assist in a better understanding of genome integrity mechanisms and may potentially lead to novel drug related aspects involving purine and pyrimidine metabolic targets., (© 2022. The Author(s).)

Although malaria has been known for more than 4 thousand years 1 , it still imposes a global burden with approx. 240 million annual cases 2 . Improvement in diagnostic techniques is a prerequisite for its global elimination. Despite its main limitations, being time-consuming and subjective, light microscopy on Giemsa-stained blood smears is still the gold-standard diagnostic method used worldwide. Autonomous computer assisted recognition of malaria infected red blood cells (RBCs) using neural networks (NNs) has the potential to overcome these deficiencies, if a fast, high-accuracy detection can be achieved using low computational power and limited sets of microscopy images for training the NN. Here, we report on a novel NN-based scheme that is capable of the high-speed classification of RBCs into four categories-healthy ones and three classes of infected ones according to the parasite age-with an accuracy as high as 98%. Importantly, we observe that a smart reduction of data dimension, using characteristic one-dimensional cross-sections of the RBC images, not only speeds up the classification but also significantly improves its performance with respect to the usual two-dimensional NN schemes. Via comparative studies on RBC images recorded by two additional techniques, fluorescence and atomic force microscopy, we demonstrate that our method is universally applicable for different types of microscopy images. This robustness against imaging platform-specific features is crucial for diagnostic applications. Our approach for the reduction of data dimension could be straightforwardly generalised for the classification of different parasites, cells and other types of objects., (© 2022. The Author(s).)

Aspartate ammonia-lyases (AALs) catalyze the non-oxidative elimination of ammonia from l-aspartate to give fumarate and ammonia. In this work the AAL coding gene from Pseudomonas fluorescens R124 was identified, isolated, and cloned into the pET-15b expression vector and expressed in E. coli. The purified enzyme (PfAAL) showed optimal activity at pH 8.8, Michaelis-Menten kinetics in the ammonia elimination from l-aspartate, and no strong dependence on divalent metal ions for its activity. The purified PfAAL was covalently immobilized on epoxy-functionalized magnetic nanoparticles (MNP), and effective kinetics of the immobilized PfAAL-MNP was compared to the native solution form. Glycerol addition significantly enhanced the storability of PfAAL-MNP. Inhibiting effect of the growing viscosity (modulated by addition of glycerol or glucose) on the enzymatic activity was observed for the native and immobilized form of PfAAL, as previously described for other free enzymes. The storage stability and recyclability of PfAAL-MNP is promising for further biocatalytic applications., (© 2022 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

The clonal composition of a malignant tumor strongly depends on cellular dynamics influenced by the asynchronized loss of DNA repair mechanisms. Here, our aim was to identify founder mutations leading to subsequent boosts in mutation load. The overall mutation burden in 591 colorectal cancer tumors was analyzed, including the mutation status of DNA-repair genes. The number of mutations was first determined across all patients and the proportion of genes having mutation in each percentile was ranked. Early mutations in DNA repair genes preceding a mutational expansion were designated as founder mutations. Survival analysis for gene expression was performed using microarray data with available relapse-free survival. Of the 180 genes involved in DNA repair, the top five founder mutations were in PRKDC ( n = 31), ATM ( n = 26), POLE ( n = 18), SRCAP ( n = 18), and BRCA2 ( n = 15). PRKDC expression was 6.4-fold higher in tumors compared to normal samples, and higher expression led to longer relapse-free survival in 1211 patients (HR = 0.72, p = 4.4 × 10 -3 ). In an experimental setting, the mutational load resulting from UV radiation combined with inhibition of PRKDC was analyzed. Upon treatments, the mutational load exposed a significant two-fold increase. Our results suggest PRKDC as a new key gene driving tumor heterogeneity.

Reverse transcription-quantitative real-time PCR (RT-qPCR) is a ubiquitously used method in biological research, however, finding appropriate reference genes for normalization is challenging. We aimed to identify genes characterized with low expression variability among human cancer and normal cell lines. For this purpose, we investigated the expression of 12 candidate reference genes in 13 widely used human cancer cell lines (HeLa, MCF-7, A-549, K-562, HL-60(TB), HT-29, MDA-MB-231, HCT 116, U-937, SH-SY5Y, U-251MG, MOLT-4 and RPMI-8226) and, in addition, 7 normal cell lines (HEK293, MRC-5, HUVEC/TERT2, HMEC, HFF-1, HUES 9, XCL-1). In our set of genes, we included SNW1 and CNOT4 as novel candidate reference genes based on the RNA HPA cell line gene data from The Human Protein Atlas. HNRNPL and PCBP1 were also included along with the "classical" reference genes ACTB, GAPDH, IPO8, PPIA, PUM1, RPL30, TBP and UBC. Results were evaluated using GeNorm, NormFiner, BestKeeper and the Comparative ΔCt methods. In conclusion, we propose IPO8, PUM1, HNRNPL, SNW1 and CNOT4 as stable reference genes for comparing gene expression between different cell lines. CNOT4 was also the most stable gene upon serum starvation., (© 2021. The Author(s).)

Rediscoveries are not rare in biology. A recent example is the re-birth of the "fluctuation fit" concept developed by F. B. Straub and G. Szabolcsi in the sixties of the last century, under various names, the most popular of which is the "conformational selection". This theory offers an alternative to the "induced fit" concept by Koshland for the interpretation of the mechanism of protein-ligand interactions. A central question is whether the ligand induces a conformational change (as described by the induced fit model) or rather selects and stabilizes a complementary conformation from a pre-existing equilibrium of various states of the protein (according to the fluctuation fit/conformational selection model). Straub and Szabolcsi's role and the factors hindering the spread of the fluctuation fit theory are discussed in the context of the history of the Hungarian biology in the 1950s and 1960s., (© 2021. The Author(s).)

Deoxyuridine in DNA has recently been in the focus of research due to its intriguing roles in several physiological and pathophysiological situations. Although not an orthodox DNA base, uracil may appear in DNA via either cytosine deamination or thymine-replacing incorporations. Since these alterations may induce mutation or may perturb DNA-protein interactions, free living organisms from bacteria to human contain several pathways to counteract uracilation. These efficient and highly specific repair routes uracil-directed excision repair initiated by representative of uracil-DNA glycosylase families. Interestingly, some bacteriophages exist with thymine-lacking uracil-DNA genome. A detailed understanding of the strategy by which such phages can replicate in bacteria where an efficient repair pathway functions for uracil-excision from DNA is expected to reveal novel inhibitors that can also be used for biotechnological applications. Here, we also review the several potential biotechnological applications already implemented based on inhibitors of uracil-excision repair, such as Crispr-base-editing and detection of nascent uracil distribution pattern in complex genomes.

The appearance of uracil in the deoxyuridine moiety of DNA is among the most frequently occurring genomic modifications. Three different routes can result in genomic uracil, two of which do not require specific enzymes: spontaneous cytosine deamination due to the inherent chemical reactivity of living cells, and thymine-replacing incorporation upon nucleotide pool imbalances. There is also an enzymatic pathway of cytosine deamination with multiple DNA (cytosine) deaminases involved in this process. In order to describe potential roles of genomic uracil, it is of key importance to utilize efficient uracil-DNA detection methods. In this review, we provide a comprehensive and critical assessment of currently available uracil detection methods with special focus on genome-wide mapping solutions. Recent developments in PCR-based and in situ detection as well as the quantitation of genomic uracil are also discussed.

Targeted covalent inhibition and the use of irreversible chemical probes are important strategies in chemical biology and drug discovery. To date, the availability and reactivity of cysteine residues amenable for covalent targeting have been evaluated by proteomic and computational tools. Herein, we present a toolbox of fragments containing a 3,5-bis(trifluoromethyl)phenyl core that was equipped with chemically diverse electrophilic warheads showing a range of reactivities. We characterized the library members for their reactivity, aqueous stability and specificity for nucleophilic amino acids. By screening this library against a set of enzymes amenable for covalent inhibition, we showed that this approach experimentally characterized the accessibility and reactivity of targeted cysteines. Interesting covalent fragment hits were obtained for all investigated cysteine-containing enzymes., (© 2020 Wiley-VCH GmbH.)

As a member of small GTPase family, KRAS protein is a key physiological modulator of various cellular activities including proliferation. However, mutations of KRAS present in numerous cancer types, most frequently in pancreatic (> 60%), colorectal (> 40%), and lung cancers, drive oncogenic processes through overactivation of proliferation. The G12C mutation of KRAS protein is especially abundant in the case of these types of malignancies. Despite its key importance in human disease, KRAS was assumed to be non-druggable for a long time since the protein seemingly lacks potential drug-binding pockets except the nucleotide-binding site, which is difficult to be targeted due to the high affinity of KRAS for both GDP and GTP. Recently, a new approach broke the ice and provided evidence that upon covalent targeting of the G12C mutant KRAS, a highly dynamic pocket was revealed. This novel targeting is especially important since it serves with an inherent solution for drug selectivity. Based on these results, various structure-based drug design projects have been launched to develop selective KRAS mutant inhibitors. In addition to the covalent modification strategy mostly applicable for G12C mutation, different innovative solutions have been suggested for the other frequently occurring oncogenic G12 mutants. Here we summarize the latest advances of this field, provide perspectives for novel approaches, and highlight the special properties of KRAS, which might issue some new challenges.

The phospholipid biosynthesis of the malaria parasite, Plasmodium falciparum is a key process for its survival and its inhibition is a validated antimalarial therapeutic approach. The second and rate-limiting step of the de novo phosphatidylcholine biosynthesis is catalysed by CTP: phosphocholine cytidylyltransferase (PfCCT), which has a key regulatory function within the pathway. Here, we investigate the functional impact of the key structural differences and their respective role in the structurally unique pseudo-heterodimer PfCCT protein in a heterologous cellular context using the thermosensitive CCT-mutant CHO-MT58 cell line. We found that a Plasmodium-specific lysine-rich insertion within the catalytic domain of PfCCT acts as a nuclear localization signal and its deletion decreases the nuclear propensity of the protein in the model cell line. We further showed that the putative membrane-binding domain also affected the nuclear localization of the protein. Moreover, activation of phosphatidylcholine biosynthesis by phospholipase C treatment induces the partial nuclear-to-cytoplasmic translocation of PfCCT. We additionally investigated the cellular function of several PfCCT truncated constructs in a CHO-MT58 based rescue assay. In absence of the endogenous CCT activity we observed that truncated constructs lacking the lysine-rich insertion, or the membrane-binding domain provided similar cell survival ratio as the full length PfCCT protein.

Numerous anti-cancer drugs perturb thymidylate biosynthesis and lead to genomic uracil incorporation contributing to their antiproliferative effect. Still, it is not yet characterized if uracil incorporations have any positional preference. Here, we aimed to uncover genome-wide alterations in uracil pattern upon drug treatments in human cancer cell line models derived from HCT116. We developed a straightforward U-DNA sequencing method (U-DNA-Seq) that was combined with in situ super-resolution imaging. Using a novel robust analysis pipeline, we found broad regions with elevated probability of uracil occurrence both in treated and non-treated cells. Correlation with chromatin markers and other genomic features shows that non-treated cells possess uracil in the late replicating constitutive heterochromatic regions, while drug treatment induced a shift of incorporated uracil towards segments that are normally more active/functional. Data were corroborated by colocalization studies via dSTORM microscopy. This approach can be applied to study the dynamic spatio-temporal nature of genomic uracil., Competing Interests: HP, AB, GR, LP, GP, GT, EH, ÁP, CG, SA, MM, ER, MP, DS, BG, BV No competing interests declared, (© 2020, Pálinkás et al.)

Emergence of resistant Plasmodium species makes drug efficacy testing a crucial part of malaria control. Here we describe a novel assay for sensitive, fast and simple drug screening via the magneto-optical detection of hemozoin, a natural biomarker formed during the hemoglobin metabolism of Plasmodium species. By quantifying hemozoin production over the intraerythrocytic cycle, we reveal that hemozoin formation is already initiated by ~ 6-12 h old ring-stage parasites. We demonstrate that the new assay is capable of drug efficacy testing with incubation times as short as 6-10 h, using synchronized P. falciparum 3D7 cultures incubated with chloroquine, piperaquine and dihydroartemisinin. The determined 50% inhibitory concentrations agree well with values established by standard assays requiring significantly longer testing time. Accordingly, we conclude that magneto-optical hemozoin detection provides a practical approach for the quick assessment of drug effect with short incubation times, which may also facilitate stage-specific assessment of drug inhibitory effects.

Clinical evidence shows that following initial response to treatment, drug-resistant cancer cells frequently evolve and, eventually, most tumors become resistant to all available therapies. We compiled a focused library consisting of >500 commercially available or newly synthetized 8-hydroxyquinoline (8OHQ) derivatives whose toxicity is paradoxically increased rather than decreased by the activity of P-glycoprotein (Pgp), a transporter conferring multidrug resistance (MDR). Here, we deciphered the mechanism of action of NSC297366 that shows exceptionally strong Pgp-potentiated toxicity. Treatment of cells with NSC297366 resulted in changes associated with the activity of potent anticancer iron chelators. Strikingly, iron depletion was more pronounced in MDR cells due to the Pgp-mediated efflux of NSC297366-iron complexes. Our results indicate that iron homeostasis can be targeted by MDR-selective compounds for the selective elimination of multidrug resistant cancer cells, setting the stage for a therapeutic approach to fight transporter-mediated drug resistance. SIGNIFICANCE: Modulation of the MDR phenotype has the potential to increase the efficacy of anticancer therapies. These findings show that the MDR transporter is a "double-edged sword" that can be turned against resistant cancer., (©2019 American Association for Cancer Research.)

The RASopathy consortium was built from research groups of the Budapest University of Technology and Economics, Eötvös Loránd University, Semmelweis University and two startups: KINETO Lab Ltd. and Fototronic Ltd. The goal was to design and test novel covalent and allele-specific KRAS small molecular inhibitors. KRAS is the most frequently mutated human oncogene which was unsuccessfully targeted until recently. The consortium established G12C-expressing bacterial and human cancer cell models (homo- and heterozygous variants) of lung, colorectal and pancreatic tumors. Using covalent fragment and acrylamide warhead libraries we were able to select novel candidates of small molecular G12C-specific inhibitors which were compared to published best-in-class drug candidates.

EDTA is commonly used as an efficient chelator of metal ion enzyme cofactors. It is highly soluble, optically inactive and does not interfere with most chemicals used in standard buffers making EDTA a common choice to generate metal-free conditions for biochemical and biophysical investigations. However, the controversy in the literature on metal-free enzyme activities achieved using EDTA or by other means called our attention to a putative effect of EDTA beyond chelation. Here, we show that EDTA competes for the nucleotide binding site of the nucleotide hydrolase dUTPase by developing an interaction network within the active site similar to that of the substrate. To achieve these findings, we applied kinetics and molecular docking techniques using two different dUTPases. Furthermore, we directly measured the binding of EDTA to dUTPases and to two other dNTPases, the Taq polymerase and MutT using isothermal titration calorimetry. EDTA binding proved to be exothermic and mainly enthalpy driven with a submicromolar dissociation constant considerably lower than that of the enzyme:substrate or the Mg:EDTA complexes. Control proteins, including an ATPase, did not interact with EDTA. Our findings indicate that EDTA may act as a selective inhibitor against dNTP hydrolyzing enzymes and urge the rethinking of the utilization of EDTA in enzymatic experiments., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

The rotating-crystal magneto-optical diagnostic (RMOD) technique was developed as a sensitive and rapid platform for malaria diagnosis. Herein, we report a detailed in vivo assessment of the synchronized Plasmodium vinckei lentum strain blood-stage infections by the RMOD method and comparing the results to the unsynchronized Plasmodium yoelii 17X-NL (non-lethal) infections. Furthermore, we assess the hemozoin production and clearance dynamics in chloroquine-treated compared to untreated self-resolving infections by RMOD. The findings of the study suggest that the RMOD signal is directly proportional to the hemozoin content and closely follows the actual parasitemia level. The lack of long-term accumulation of hemozoin in peripheral blood implies a dynamic equilibrium between the hemozoin production rate of the parasites and the immune system's clearing mechanism. Using parasites with synchronous blood stage cycle, which resemble human malaria parasite infections with Plasmodium falciparum and Plasmodium vivax , we are demonstrating that the RMOD detects both hemozoin production and clearance rates with high sensitivity and temporal resolution. Thus, RMOD technique offers a quantitative tool to follow the maturation of the malaria parasites even on sub-cycle timescales.

The dUTPase enzyme family plays an essential role in maintaining the genome integrity and are represented by two distinct classes of proteins; the β-pleated homotrimeric and the all-α homodimeric dUTPases. Representatives of both trimeric and dimeric dUTPases are encoded by Staphylococcus aureus phage genomes and have been shown to interact with the Stl repressor protein of S. aureus pathogenicity island SaPIbov1. In the present work we set out to characterize the interactions between these proteins based on a range of biochemical and biophysical methods and shed light on the binding mechanism of the dimeric φNM1 phage dUTPase and Stl. Using hydrogen deuterium exchange mass spectrometry, we also characterize the protein regions involved in the dUTPase:Stl interactions. Based on these results we provide reasonable explanation for the enzyme inhibitory effect of Stl observed in both types of complexes. Our experiments reveal that Stl employs different peptide segments and stoichiometry for the two different phage dUTPases which allows us to propose a functional plasticity of Stl. The malleable character of Stl serves as a basis for the inhibition of both dimeric and trimeric dUTPases.

Protein inhibitors of key DNA repair enzymes play an important role in deciphering physiological pathways responsible for genome integrity, and may also be exploited in biomedical research. The staphylococcal repressor StlSaPIbov1 protein was described to be an efficient inhibitor of dUTPase homologues showing a certain degree of species-specificity. In order to provide insight into the inhibition mechanism, in the present study we investigated the interaction of StlSaPIbov1 and Escherichia coli dUTPase. Although we observed a strong interaction of these proteins, unexpectedly the E. coli dUTPase was not inhibited. Seeking a structural explanation for this phenomenon, we identified a key amino acid position where specific mutations sensitized E. coli dUTPase to StlSaPIbov1 inhibition. We solved the three-dimensional (3D) crystal structure of such a mutant in complex with the substrate analogue dUPNPP and surprisingly found that the C-terminal arm of the enzyme, containing the P-loop-like motif was ordered in the structure. This segment was never localized before in any other E. coli dUTPase crystal structures. The 3D structure in agreement with solution phase experiments suggested that ordering of the flexible C-terminal segment upon substrate binding is a major factor in defining the sensitivity of E. coli dUTPase for StlSaPIbov1 inhibition., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

The coupling of nucleotide biosynthesis and genome integrity plays an important role in ensuring faithful maintenance and transmission of genetic information. The enzyme dUTPase is a prime example of such coupling, as it generates dUMP for thymidylate biosynthesis and removes dUTP for synthesis of uracil-free DNA. Despite its significant role, the expression patterns of dUTPase isoforms in animals have not yet been described. Here, we developed a detailed optimization procedure for RT-qPCR-based isoform-specific analysis of dUTPase expression levels in various organs of adult mice. Primer design, optimal annealing temperature, and primer concentrations were specified for both nuclear and mitochondrial dUTPase isoforms, as well as two commonly used reference genes, GAPDH and PPIA. The linear range of the RNA concentration for the reverse transcription reaction was determined. The PCR efficiencies were calculated using serial dilutions of cDNA. Our data indicate that organs involved in lymphocyte production, as well as reproductive organs, are characterized by high levels of expression of the nuclear dUTPase isoform. On the other hand, we observed that expression of the mitochondrial dUTPase isoform is considerably increased in heart, kidney, and ovary. Despite the differences in expression levels among the various organs, we also found that the mitochondrial dUTPase isoform shows a much more uniform expression pattern as compared to the reference genes GAPDH and PPIA., (© 2019 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

Sanitization of nucleotide pools is essential for genome maintenance. Deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase) is a key enzyme in this pathway since it catalyzes the cleavage of 2'-deoxyuridine 5'-triphosphate (dUTP) into 2'-deoxyuridine 5'-monophosphate (dUMP) and inorganic pyrophosphate. Through its action dUTPase efficiently prevents uracil misincorporation into DNA and at the same time provides dUMP, the substrate for de novo thymidylate biosynthesis. Despite its physiological significance, knock-out models of dUTPase have not yet been investigated in mammals, but only in unicellular organisms, such as bacteria and yeast. Here we generate CRISPR/Cas9-mediated dUTPase knock-out in mice. We find that heterozygous dut +/- animals are viable while having decreased dUTPase levels. Importantly, we show that dUTPase is essential for embryonic development since early dut -/- embryos reach the blastocyst stage, however, they die shortly after implantation. Analysis of pre-implantation embryos indicates perturbed growth of both inner cell mass (ICM) and trophectoderm (TE). We conclude that dUTPase is indispensable for post-implantation development in mice.

Src homology 3 (SH3) domains bind proline-rich linear motifs in eukaryotes. By mediating inter- and intramolecular interactions, they regulate the functions of many proteins involved in a wide variety of signal transduction pathways. Phosphorylation at different tyrosine residues in SH3 domains has been reported previously. In several cases, the functional consequences have also been investigated. However, a full understanding of the effects of tyrosine phosphorylation on the ligand interactions and cellular functions of SH3 domains requires detailed structural, atomic-resolution studies along with biochemical and biophysical analyses. Here, we present the first crystal structures of tyrosine-phosphorylated human SH3 domains derived from the Abelson-family kinases ABL1 and ABL2 at 1.6 and 1.4 Å resolutions, respectively. The structures revealed that simultaneous phosphorylation of Tyr 89 and Tyr 134 in ABL1 or the homologous residues Tyr 116 and Tyr 161 in ABL2 induces only minor structural perturbations. Instead, the phosphate groups sterically blocked the ligand-binding grooves, thereby strongly inhibiting the interaction with proline-rich peptide ligands. Although some crystal contact surfaces involving phosphotyrosines suggested the possibility of tyrosine phosphorylation-induced dimerization, we excluded this possibility by using small-angle X-ray scattering (SAXS), dynamic light scattering (DLS), and NMR relaxation analyses. Extensive analysis of relevant databases and literature revealed not only that the residues phosphorylated in our model systems are well-conserved in other human SH3 domains, but that the corresponding tyrosines are known phosphorylation sites in vivo in many cases. We conclude that tyrosine phosphorylation might be a mechanism involved in the regulation of the human SH3 interactome., (© 2019 Merő et al.)

The base excision repair pathway plays an important role in correcting damage induced by either physiological or external effects. This repair pathway removes incorrect bases from the DNA. The uracil base is among the most frequently occurring erroneous bases in DNA, and is cut out from the phosphodiester backbone via the catalytic action of uracil-DNA glycosylase. Uracil excision repair is an evolutionarily highly conserved pathway and can be specifically inhibited by a protein inhibitor of uracil-DNA glycosylase. Interestingly, both uracil-DNA glycosylase ( Staphylococcus aureus uracil-DNA glycosylase; SAUDG) and its inhibitor ( S. aureus uracil-DNA glycosylase inhibitor; SAUGI) are present in the staphylococcal cell. The interaction of these two proteins effectively decreases the efficiency of uracil-DNA excision repair. The physiological relevance of this complexation has not yet been addressed in detailed; however, numerous mutations have been identified within SAUGI. Here, we investigated whether these mutations drastically perturb the interaction with SAUDG. To perform quantitative analysis of the macromolecular interactions, we applied native mass spectrometry and demonstrated that this is a highly efficient and specific method for determination of dissociation constants. Our results indicate that several naturally occurring mutations of SAUGI do indeed lead to appreciable changes in the dissociation constants for complex formation. However, all of these K d values remain in the nanomolar range and therefore the association of these two proteins is preserved. We conclude that complexation is most likely preserved even with the naturally occurring mutant uracil-DNA glycosylase inhibitor proteins., Competing Interests: The authors declare no conflict of interest.

The enzyme catalyzed degradation of poly(3-hydroxybutyrate) (PHB) is a two-step process consisting of the adsorption of the enzyme on the surface of a PHB substrate and the cleavage of ester bonds. A deactivated enzyme was prepared by point mutagenesis to separate the two steps from each other. Measurements carried out with active and inactive enzymes on PHB particles proved that mutagenesis was successful and the modified enzyme did not catalyze degradation. Based on the Michaelis-Menten approach a kinetic model was proposed which could describe the processes quantitatively, the agreement between prediction and the measured data was excellent. The separation of the two processes allowed the determination of the adsorption kinetics of the enzyme; the rate constants of the adsorption and desorption process were determined for the first time. Comparison of these constants to reaction rates showed that adsorption is not instantaneous and can be the rate-determining step. The area occupied by an enzyme molecule was also determined (13.1 nm 2 ) and it was found to be smaller than the value published in the literature (17 ± 8 nm 2 ). The separation of the two steps makes possible the prediction and control of the degradation process., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Plasmodium falciparum parasites undergo multiple genome duplication events during their development. Within the intraerythrocytic stages, parasites encounter an oxidative environment and DNA synthesis necessarily proceeds under these circumstances. In addition to these conditions, the extreme AT bias of the P. falciparum genome poses further constraints for DNA synthesis. Taken together, these circumstances may allow appearance of damaged bases in the Plasmodium DNA. Here, we focus on uracil that may arise in DNA either via oxidative deamination or thymine-replacing incorporation. We determine the level of uracil at the ring, trophozoite, and schizont intraerythrocytic stages and evaluate the base-excision repair potential of P. falciparum to deal with uracil-DNA repair. We find approximately 7-10 uracil per million bases in the different parasite stages. This level is considerably higher than found in other wild-type organisms from bacteria to mammalian species. Based on a systematic assessment of P. falciparum genome and transcriptome databases, we conclude that uracil-DNA repair relies on one single uracil-DNA glycosylase and proceeds through the long-patch base-excision repair route. Although potentially efficient, the repair route still leaves considerable level of uracils in parasite DNA, which may contribute to mutation rates in P. falciparum .

The development of the malaria parasite, Plasmodium falciparum, in the human erythrocyte, relies on phospholipid metabolism to fulfil the massive need for membrane biogenesis. Phosphatidylcholine (PC) is the most abundant phospholipid in Plasmodium membranes. PC biosynthesis is mainly ensured by the de novo Kennedy pathway that is considered as an antimalarial drug target. The CTP:phosphocholine cytidylyltransferase (CCT) catalyses the rate-limiting step of the Kennedy pathway. Here we report a series of structural snapshots of the PfCCT catalytic domain in its free, substrate- and product-complexed states that demonstrate the conformational changes during the catalytic mechanism. Structural data show the ligand-dependent conformational variations of a flexible lysine. Combined kinetic and ligand-binding analyses confirm the catalytic roles of this lysine and of two threonine residues of the helix αE. Finally, we assessed the variations in active site residues between Plasmodium and mammalian CCT which could be exploited for future antimalarial drug design.

The plasmodial CTP:phosphocholine cytidylyltransferase (PfCCT) is a promising antimalarial target, which can be inhibited to exploit the need for increased lipid biosynthesis during the erythrocytic life stage of Plasmodium falciparum. Notable structural and regulatory differences of plasmodial and mammalian CCTs offer the possibility to develop species-specific inhibitors. The aim of this study was to use CHO-MT58 cells expressing a temperature-sensitive mutant CCT for the functional characterization of PfCCT. We show that heterologous expression of wild type PfCCT restores the viability of CHO-MT58 cells at non-permissive (40 °C) temperatures, whereas catalytically perturbed or structurally destabilized PfCCT variants fail to provide rescue. Detailed in vitro characterization indicates that the H630N mutation diminishes the catalytic rate constant of PfCCT. The flow cytometry-based rescue assay provides a quantitative readout of the PfCCT function opening the possibility for the functional analysis of PfCCT and the high throughput screening of antimalarial compounds targeting plasmodial CCT.

Poly-[(R)-3-hydroxybutyrate] (PHB) films prepared by compression molding and solvent casting, respectively, were degraded with the intracellular depolymerase enzyme natively synthetized by the strain Bacillus megaterium. Quantitative analysis proved that practically only (R)-3-hydroxybutyric acid (3-HBA) forms in the enzyme catalyzed reaction, the amount of other metabolites or side products is negligible. The purity of the product was verified by several methods (UV-VIS spectroscopy, liquid chromatography, mass spectroscopy). Degradation was followed as a function of time to determine the rate of enzymatic degradation. Based on the Michaelis-Menten equation a completely new kinetic model has been derived which takes into consideration the heterogeneous nature of the enzymatic reaction. Degradation proceeds in two steps, the adsorption of the enzyme onto the surface of the PHB film and the subsequent degradation reaction. The rate of both steps depend on the preparation method of the samples, degradation proceed almost twice as fast in compression molded films than in solvent cast samples. The model can describe and predict the formation of the reaction product as a function of time. The approach can be used even for the commercial production of 3-HBA, the chemical synthesis of which is complicated and expensive., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Pathogenicity islands of Staphylococcus aureus are under the strong control of helper phages, where regulation is communicated at the gene expression level via a family of specific repressor proteins. The repressor proteins are crucial to phage-host interactions and, based on their protein characteristics, may also be exploited as versatile molecular tools. The Stl repressor from this protein family has been recently investigated and although the binding site of Stl on DNA was recently discovered, there is a lack of knowledge on the specific protein segments involved in this interaction. Here, we develop a generally applicable system to reveal the mechanism of the interaction between Stl and its cognate DNA within the cellular environment. Our unbiased approach combines random mutagenesis with high-throughput analysis based on the lac operon to create a well-characterized gene expression system. Our results clearly indicate that, in addition to a previously implicated helix-turn-helix segment, other protein moieties also play decisive roles in the DNA binding capability of Stl. Structural model-based investigations provided a detailed understanding of Stl:DNA complex formation. The robustness and reliability of our novel test system were confirmed by several mutated Stl constructs, as well as by demonstrating the interaction between Stl and dUTPase from the Staphylococcal ϕ11 phage. Our system may be applied to high-throughput studies of protein:DNA and protein:protein interactions., Competing Interests: The authors declare no conflict of interest.

Human deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase), essential for DNA integrity, acts as a survival factor for tumor cells and is a target for cancer chemotherapy. Here we report that the Staphylococcal repressor protein Stl SaPIBov1 (Stl) forms strong complex with human dUTPase. Functional analysis reveals that this interaction results in significant reduction of both dUTPase enzymatic activity and DNA binding capability of Stl. We conducted structural studies to understand the mechanism of this mutual inhibition. Small-angle X-ray scattering (SAXS) complemented with hydrogen-deuterium exchange mass spectrometry (HDX-MS) data allowed us to obtain 3D structural models comprising a trimeric dUTPase complexed with separate Stl monomers. These models thus reveal that upon dUTPase-Stl complex formation the functional homodimer of Stl repressor dissociates, which abolishes the DNA binding ability of the protein. Active site forming dUTPase segments were directly identified to be involved in the dUTPase-Stl interaction by HDX-MS, explaining the loss of dUTPase activity upon complexation. Our results provide key novel structural insights that pave the way for further applications of the first potent proteinaceous inhibitor of human dUTPase.

Ride the wave! Biocatalysis uses nature's catalysts, enzymes and whole cell systems, for synthetic purposes. In a biotransformation, the biocatalyst transforms a well-defined substrate to the desired product, in contrast to the fermentation process, which produces the desired product from a complex mixture of nutrients. Biocatalysis has reached an industrially established level through several waves of technological evolution; participants of the BioTrans 2017 conference in Budapest could witness the newest wave of this technology., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

DNA metabolism and repair is vital for the maintenance of genome integrity. Specific proteinaceous inhibitors of key factors in this process have high potential for deciphering pathways of DNA metabolism and repair. The dUTPase enzyme family is responsible for guarding against erroneous uracil incorporation into DNA. Here, we investigate whether the staphylococcal Stl repressor may interact with not only bacterial but also eukaryotic dUTPase. We provide experimental evidence for the formation of a strong complex between Stl and Drosophila melanogaster dUTPase. We also find that dUTPase activity is strongly diminished in this complex. Our results suggest that the dUTPase protein sequences involved in binding to Stl are at least partially conserved through evolution from bacteria to eukaryotes.

Cancer genome sequencing has implicated the cytosine deaminase activity of apolipoprotein B mRNA editing enzyme catalytic polypeptide-like (APOBEC) genes as an important source of mutations in diverse cancers, with APOBEC3B (A3B) expression especially correlated with such cancer mutations. To better understand the processes directing A3B over-expression in cancer, and possible therapeutic avenues for targeting A3B, we have investigated the regulation of A3B gene expression. Here, we show that A3B expression is inversely related to p53 status in different cancer types and demonstrate that this is due to a direct and pivotal role for p53 in repressing A3B expression. This occurs through the induction of p21 (CDKN1A) and the recruitment of the repressive DREAM complex to the A3B gene promoter, such that loss of p53 through mutation, or human papilloma virus-mediated inhibition, prevents recruitment of the complex, thereby causing elevated A3B expression and cytosine deaminase activity in cancer cells. As p53 is frequently mutated in cancer, our findings provide a mechanism by which p53 loss can promote cancer mutagenesis., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)