A user-friendly, high-throughput tool for the precise fluorescent quantification of deoxyribonucleoside triphosphates from biological samples

Cells maintain a fine-tuned, dynamic concentration balance in the pool of deoxyribonucleoside 5′-triphosphates (dNTPs). This balance is essential for physiological processes including cell cycle control or antiviral defense. Its perturbation results in increased mutation frequencies, replication arrest and may promote cancer development. An easily accessible and relatively high-throughput method would greatly accelerate the exploration of the diversified consequences of dNTP imbalances. The dNTP incorporation based, fluorescent TaqMan-like assay published by Wilson et al. has the aforementioned advantages over mass spectrometry, radioactive or chromatography based dNTP quantification methods. Nevertheless, the assay failed to produce reliable data in several biological samples. Therefore, we applied enzyme kinetics analysis on the fluorescent dNTP incorporation curves and found that the Taq polymerase exhibits a dNTP independent exonuclease activity that decouples signal generation from dNTP incorporation. Furthermore, we found that both polymerization and exonuclease activities are unpredictably inhibited by the sample matrix. To resolve these issues, we established a kinetics based data analysis method which identifies the signal generated by dNTP incorporation. We automated the analysis process in the nucleoTIDY software which enables even the inexperienced user to calculate the final and accurate dNTP amounts in a 96-well-plate setup within minutes

Differential control of dNTP biosynthesis and genome integrity maintenance by dUTPases

dUTPase superfamily enzymes generate dUMP, the obligate precursor for de novo dTTP biosynthesis, from either dUTP (monofunctional dUTPase, Dut) or dCTP (bifunctional dCTP deaminase/dUTPase, Dcd:dut). In addition, the elimination of dUTP by these enzymes prevents harmful uracil incorporation into DNA. These two beneficial outcomes have been thought to be related. Here we determined the relationship between dTTP biosynthesis (dTTP/dCTP balance) and the prevention of DNA uracilation in a mycobacterial model that encodes both the Dut and Dcd:dut enzymes, and has no other ways to produce dUMP. We show that, in dut mutant¬¬¬¬¬ mycobacteria, the dTTP/dCTP balance remained unchanged, but the uracil content of DNA and the mutation rate increased in parallel with the in vitro activity-loss of Dut. Conversely, dcd:dut inactivation resulted in perturbed dTTP/dCTP balance and two-fold increased mutation rate, but did not increase the uracil content of DNA. Thus, unexpectedly, the regulation of dNTP balance and the prevention of DNA uracilation are decoupled and separately brought about by the Dcd:dut and Dut enzymes, respectively. Available evidence suggests that the discovered functional separation is conserved in humans and other organisms

An improved and widely accessible dNTP quantitation tool

Cells maintain a fine-tuned concentration balance in the pool of deoxyribonucleoside 5’- triphosphates (dNTPs). The perturbation of this balance results in increased mutation frequencies suggested to promote cancer development and drug resistance. To study dNTP imbalances and their consequences, an accurate and relatively high-throughput method is necessary. The dNTP quantitation method of our choice is a fluorescence-based, TaqMan-like polymerase assay published by Wilson et al, NAR 2011. This assay has the advantages of being accessible in a standard molecular biology laboratory and having the potential to be automated in contrast to mass spectrometry or radioactive measurements. Although this method works well in diluted samples with high dNTP levels, we observed that the sample matrix largely decreases assay performance. Upon thorough kinetic analysis of the fluorescent dNTP incorporation curves, we found that the Taq polymerase exhibits a dNTP independent, signal generating exonuclease activity and that the polymerization and exonuclease activity are partially inhibited by the sample matrix. Based on our kinetic investigations we suggest several assay modifications and a novel, kineticsbased and automated analysis method. Using these modifications, we measured dNTP pools in widely different organisms including Mycobacterium smegmatis, Staphylococcus aureus and human cancer cells. We found that our improved method is capable of i) determining dNTP concentrations in samples previously proved to be unmeasurable by eliminating the interfering matrix effect, and ii) improving the quantitation limits of the assay. Fundings: NKFIH-PD 124330, NKFIH-K 115993, János Bolyai Research Scholarshi

The Role of a Key Amino Acid Position in Species-Specific Proteinaceous dUTPase Inhibition

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

Exploiting a Phage-Bacterium Interaction System as a Molecular Switch to Decipher Macromolecular Interactions in the Living Cell

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