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Highly potent dUTPase inhibition by a bacterial repressor protein reveals a novel mechanism for gene expression control.

Szabó JE, Németh V, Papp-Kádár V, Nyíri K, Leveles I, Bendes AÁ, Zagyva I, Róna G, Pálinkás HL, Besztercei B, Ozohanics O, Vékey K, Liliom K, Tóth J, Vértessy BG - Nucleic Acids Res. (2014)

Bottom Line: We found that Φ11 helper phage dUTPase eliminates SaPIbov1 Stl binding to its cognate DNA by binding tightly to Stl protein.Our results disprove the previously proposed G-protein-like mechanism of SaPI transfer activation.We propose that the transfer only occurs if dUTP is cleared from the nucleotide pool, a condition promoting genomic stability of the virulence elements.

View Article: PubMed Central - PubMed

Affiliation: Institutes of Enzymology and Organic Chemistry, RCNS, Hungarian Academy of Sciences, Budapest, Hungary vertessy@mail.bme.hu vertessy.beata@ttk.mta.hu.

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dUTPase:Stl complex formation eliminates the physiological function of both proteins. (A) Inhibitory effect of Stl on Φ11DUTWT (10 nM) catalytic activity. Data represent average and error of three parallel measurements. Solid line represents fit of quadratic binding equation to the data, yielding IC50 26.64 ± 5.07 nM. (B) Shows titration of Φ11DUTF164W (1.5 μM) and Φ11DUTF164W (1.5 μM): dUPNPP (3 mM)/dUMP (2 mM) complex with Stl. Error bars represents SD for n = 3. Solid lines represent quadratic fits to the data (see Equation (1)). Dissociation constants from the fitted model are shown in Table 1. (C) Shows transient kinetic investigation of the mixing order dependency of Stl inhibition. 2 μM d Φ11DUTF164W, 3 μM Stl and 50 μM dUTP was mixed (post-mixing concentrations: X indicates the mixing of species in syringe A and B (syringe A X syringe B), parenthesis indicates that the components were pre-mixed. The curves are shown from 0.002 s (after the dead time). (D) Effect of dUPNPP on dUTPase derepression activity characterized by EMSA.
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Figure 2: dUTPase:Stl complex formation eliminates the physiological function of both proteins. (A) Inhibitory effect of Stl on Φ11DUTWT (10 nM) catalytic activity. Data represent average and error of three parallel measurements. Solid line represents fit of quadratic binding equation to the data, yielding IC50 26.64 ± 5.07 nM. (B) Shows titration of Φ11DUTF164W (1.5 μM) and Φ11DUTF164W (1.5 μM): dUPNPP (3 mM)/dUMP (2 mM) complex with Stl. Error bars represents SD for n = 3. Solid lines represent quadratic fits to the data (see Equation (1)). Dissociation constants from the fitted model are shown in Table 1. (C) Shows transient kinetic investigation of the mixing order dependency of Stl inhibition. 2 μM d Φ11DUTF164W, 3 μM Stl and 50 μM dUTP was mixed (post-mixing concentrations: X indicates the mixing of species in syringe A and B (syringe A X syringe B), parenthesis indicates that the components were pre-mixed. The curves are shown from 0.002 s (after the dead time). (D) Effect of dUPNPP on dUTPase derepression activity characterized by EMSA.

Mentions: We measured the enzymatic activity of dUTPase in the dUTPase:Stl complex and found that Stl exerts highly potent inhibition of dUTPase activity with an IC50 value that approximates the Kd of the protein–protein complex (Figure 2A, Table 1). This inhibition is only observed if dUTPase is pre-incubated with Stl prior to dUTP addition. Such behavior is typical for a slow and tight binding inhibitor (23) and is in excellent agreement with data obtained for the formation of the dUTPase:Stl complex (Figure 1, Table 1) as well as with the previously published kinetics of dUTP binding (20).


Highly potent dUTPase inhibition by a bacterial repressor protein reveals a novel mechanism for gene expression control.

Szabó JE, Németh V, Papp-Kádár V, Nyíri K, Leveles I, Bendes AÁ, Zagyva I, Róna G, Pálinkás HL, Besztercei B, Ozohanics O, Vékey K, Liliom K, Tóth J, Vértessy BG - Nucleic Acids Res. (2014)

dUTPase:Stl complex formation eliminates the physiological function of both proteins. (A) Inhibitory effect of Stl on Φ11DUTWT (10 nM) catalytic activity. Data represent average and error of three parallel measurements. Solid line represents fit of quadratic binding equation to the data, yielding IC50 26.64 ± 5.07 nM. (B) Shows titration of Φ11DUTF164W (1.5 μM) and Φ11DUTF164W (1.5 μM): dUPNPP (3 mM)/dUMP (2 mM) complex with Stl. Error bars represents SD for n = 3. Solid lines represent quadratic fits to the data (see Equation (1)). Dissociation constants from the fitted model are shown in Table 1. (C) Shows transient kinetic investigation of the mixing order dependency of Stl inhibition. 2 μM d Φ11DUTF164W, 3 μM Stl and 50 μM dUTP was mixed (post-mixing concentrations: X indicates the mixing of species in syringe A and B (syringe A X syringe B), parenthesis indicates that the components were pre-mixed. The curves are shown from 0.002 s (after the dead time). (D) Effect of dUPNPP on dUTPase derepression activity characterized by EMSA.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 2: dUTPase:Stl complex formation eliminates the physiological function of both proteins. (A) Inhibitory effect of Stl on Φ11DUTWT (10 nM) catalytic activity. Data represent average and error of three parallel measurements. Solid line represents fit of quadratic binding equation to the data, yielding IC50 26.64 ± 5.07 nM. (B) Shows titration of Φ11DUTF164W (1.5 μM) and Φ11DUTF164W (1.5 μM): dUPNPP (3 mM)/dUMP (2 mM) complex with Stl. Error bars represents SD for n = 3. Solid lines represent quadratic fits to the data (see Equation (1)). Dissociation constants from the fitted model are shown in Table 1. (C) Shows transient kinetic investigation of the mixing order dependency of Stl inhibition. 2 μM d Φ11DUTF164W, 3 μM Stl and 50 μM dUTP was mixed (post-mixing concentrations: X indicates the mixing of species in syringe A and B (syringe A X syringe B), parenthesis indicates that the components were pre-mixed. The curves are shown from 0.002 s (after the dead time). (D) Effect of dUPNPP on dUTPase derepression activity characterized by EMSA.
Mentions: We measured the enzymatic activity of dUTPase in the dUTPase:Stl complex and found that Stl exerts highly potent inhibition of dUTPase activity with an IC50 value that approximates the Kd of the protein–protein complex (Figure 2A, Table 1). This inhibition is only observed if dUTPase is pre-incubated with Stl prior to dUTP addition. Such behavior is typical for a slow and tight binding inhibitor (23) and is in excellent agreement with data obtained for the formation of the dUTPase:Stl complex (Figure 1, Table 1) as well as with the previously published kinetics of dUTP binding (20).

Bottom Line: We found that Φ11 helper phage dUTPase eliminates SaPIbov1 Stl binding to its cognate DNA by binding tightly to Stl protein.Our results disprove the previously proposed G-protein-like mechanism of SaPI transfer activation.We propose that the transfer only occurs if dUTP is cleared from the nucleotide pool, a condition promoting genomic stability of the virulence elements.

View Article: PubMed Central - PubMed

Affiliation: Institutes of Enzymology and Organic Chemistry, RCNS, Hungarian Academy of Sciences, Budapest, Hungary vertessy@mail.bme.hu vertessy.beata@ttk.mta.hu.

Show MeSH
Related in: MedlinePlus