Cross-species inhibition of dUTPase via the Staphylococcal Stl protein perturbs dNTP pool and colony formation in Mycobacterium

Hirmondó, Rita; Szabó, J.; Nyíri, Kinga; Tarjányi, Szilvia; Dobrotka, Paula; Tóth, Judit; Vértessy, Beáta G.

doi:10.1016/j.dnarep.2015.03.005

Cited by 21 publications

(45 citation statements)

References 58 publications

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“…This value is in good agreement with the IC 50 of Stl determined for phage and mycobacterial dUTPase. However, the total inhibition observed at saturating Stl concentrations was ~ 40%, to be compared with almost 100% inhibition for Φ11 phage dUTPase and 80% inhibition for the mycobacterial dUTPase . These characteristics indicate that although the complexation proceeds similarly, the actual inhibitory capacity of Stl within the different complexes reflects some species‐specific differences.…”

Section: Resultsmentioning

confidence: 99%

“…1C,D,E) presents largely varied distribution of polar, charged, and hydrophobic surfaces. Despite this variation, the interaction among the mycobacterial dUTPase and Stl shows similar characteristics to the staphylococcal phage dUTPase-Stl interaction [16].…”

Section: Resultsmentioning

confidence: 99%

“…Several small molecular dUTPase inhibitors have been identified in the literature [12][13][14][15]. A proteinaceous dUTPase inhibitor, namely the staphylococcal Stl repressor, has also been discovered recently and it was shown to be active against trimeric dUTPases of several staphylococcal phages, as well as against the trimeric mycobacterial dUTPase [16][17][18].…”

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confidence: 99%

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The Stl repressor from Staphylococcus aureus is an efficient inhibitor of the eukaryotic fruitfly dUTPase

et al. 2017

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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 dUTP ase 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 dUTP ase. We provide experimental evidence for the formation of a strong complex between Stl and Drosophila melanogaster dUTP ase. We also find that dUTP ase activity is strongly diminished in this complex. Our results suggest that the dUTP ase protein sequences involved in binding to Stl are at least partially conserved through evolution from bacteria to eukaryotes.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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confidence: 99%

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The Stl repressor from Staphylococcus aureus is an efficient inhibitor of the eukaryotic fruitfly dUTPase

et al. 2017

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“…dNTP levels were measured using a DNA polymerase assay with minor modifications (16)(17)(18). Extracts were incubated in 40 µL of dUTPase buffer (34 mM Tris-HCl, pH 7.8, 5 mM MgCl 2 ) or dUTPase buffer plus 30 ng of human dUTPase (19) for 20 min at 37…”

Section: Intracellular Dntp Pool Size Determinationmentioning

confidence: 99%

The nucleotidohydrolases DCTPP1 and dUTPase are involved in the cellular response to decitabine

Requena

Pérez-Moreno

Horváth

et al. 2016

Biochemical Journal

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Decitabine (5-aza-2′-deoxycytidine, aza-dCyd) is an anti-cancer drug used clinically for the treatment of myelodysplastic syndromes and acute myeloid leukaemia that can act as a DNA-demethylating or genotoxic agent in a dose-dependent manner. On the other hand, DCTPP1 (dCTP pyrophosphatase 1) and dUTPase are two ‘house-cleaning’ nucleotidohydrolases involved in the elimination of non-canonical nucleotides. In the present study, we show that exposure of HeLa cells to decitabine up-regulates the expression of several pyrimidine metabolic enzymes including DCTPP1, dUTPase, dCMP deaminase and thymidylate synthase, thus suggesting their contribution to the cellular response to this anti-cancer nucleoside. We present several lines of evidence supporting that, in addition to the formation of aza-dCTP (5-aza-2′-deoxycytidine-5′-triphosphate), an alternative cytotoxic mechanism for decitabine may involve the formation of aza-dUMP, a potential thymidylate synthase inhibitor. Indeed, dUTPase or DCTPP1 down-regulation enhanced the cytotoxic effect of decitabine producing an accumulation of nucleoside triphosphates containing uracil as well as uracil misincorporation and double-strand breaks in genomic DNA. Moreover, DCTPP1 hydrolyses the triphosphate form of decitabine with similar kinetic efficiency to its natural substrate dCTP and prevents decitabine-induced global DNA demethylation. The data suggest that the nucleotidohydrolases DCTPP1 and dUTPase are factors involved in the mode of action of decitabine with potential value as enzymatic targets to improve decitabine-based chemotherapy.

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“…Different strains of S. aureus encode numerous mutated versions of SAUGI [18]. While the exact biological role of SAUGI is still unclear, it is highly interesting to note that S. aureus also encodes an inhibitory protein for dUT-Pase, namely Stl [23][24][25][26][27].…”

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Mass spectrometry‐based analysis of macromolecular complexes ofStaphylococcus aureusuracil‐DNA glycosylase and its inhibitor reveals specific variations due to naturally occurring mutations

et al. 2019

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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.

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Cross-species inhibition of dUTPase via the Staphylococcal Stl protein perturbs dNTP pool and colony formation in Mycobacterium

Cited by 21 publications

References 58 publications

The Stl repressor from Staphylococcus aureus is an efficient inhibitor of the eukaryotic fruitfly dUTPase

The Stl repressor from Staphylococcus aureus is an efficient inhibitor of the eukaryotic fruitfly dUTPase

The nucleotidohydrolases DCTPP1 and dUTPase are involved in the cellular response to decitabine

Mass spectrometry‐based analysis of macromolecular complexes ofStaphylococcus aureusuracil‐DNA glycosylase and its inhibitor reveals specific variations due to naturally occurring mutations

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