Using Selective Enzymes to Measure Noncanonical DNA Building Blocks: dUTP, 5-Methyl-dCTP, and 5-Hydroxymethyl-dCTP.

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.

dNTPpoolDB: a manually curated database of experimentally determined dNTP pools and pool changes in biological samples.

Stimulated by the growing interest in the role of dNTP pools in physiological and malignant processes, we established dNTPpoolDB, the database that offers access to quantitative data on dNTP pools from a wide range of species, experimental and developmental conditions (https://dntppool.org/). The database includes measured absolute or relative cellular levels of the four canonical building blocks of DNA and of exotic dNTPs, as well. In addition to the measured quantity, dNTPpoolDB contains ample information on sample source, dNTP quantitation methods and experimental conditions including any treatments and genetic manipulations. Functions such as the advanced search offering multiple choices from custom-built controlled vocabularies in 15 categories in parallel, the pairwise comparison of any chosen pools, and control-treatment correlations provide users with the possibility to quickly recognize and graphically analyse changes in the dNTP pools in function of a chosen parameter. Unbalanced dNTP pools, as well as the balanced accumulation or depletion of all four dNTPs result in genomic instability. Accordingly, key roles of dNTP pool homeostasis have been demonstrated in cancer progression, development, ageing and viral infections among others. dNTPpoolDB is designated to promote research in these fields and fills a longstanding gap in genome metabolism research.

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.

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.

Differential control of dNTP biosynthesis and genome integrity maintenance by the dUTPase superfamily enzymes.

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 increased in parallel with the in vitro activity-loss of Dut accompanied with a considerable increase in the mutation rate. 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.