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The MobM relaxase domain of plasmid pMV158: thermal stability and activity upon Mn2+ and specific DNA binding.

Lorenzo-Díaz F, Dostál L, Coll M, Schildbach JF, Menéndez M, Espinosa M - Nucleic Acids Res. (2011)

Bottom Line: However, whereas Mn(2+) strongly stabilized MobMN199 against thermal denaturation, no protective effect was observed for Mg(2+).The structure of MobMN199 was strongly stabilized by binding to the defined target DNA, indicating the formation of a tight protein-DNA complex.We demonstrate that the oriT recognition by MobMN199 was highly specific and suggest that this protein most probably employs Mn(2+) during pMV158 transfer.

View Article: PubMed Central - PubMed

Affiliation: Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain.

ABSTRACT
Protein MobM, the relaxase involved in conjugative transfer of the streptococcal plasmid pMV158, is the prototype of the MOB(V) superfamily of relaxases. To characterize the DNA-binding and nicking domain of MobM, a truncated version of the protein (MobMN199) encompassing its N-terminal region was designed and the protein was purified. MobMN199 was monomeric in contrast to the dimeric form of the full-length protein, but it kept its nicking activity on pMV158 DNA. The optimal relaxase activity was dependent on Mn(2+) or Mg(2+) cations in a dosage-dependent manner. However, whereas Mn(2+) strongly stabilized MobMN199 against thermal denaturation, no protective effect was observed for Mg(2+). Furthermore, MobMN199 exhibited a high affinity binding for Mn(2+) but not for Mg(2+). We also examined the binding-specificity and affinity of MobMN199 for several substrates of single-stranded DNA encompassing the pMV158 origin of transfer (oriT). The minimal oriT was delimited to a stretch of 26 nt which included an inverted repeat located eight bases upstream of the nick site. The structure of MobMN199 was strongly stabilized by binding to the defined target DNA, indicating the formation of a tight protein-DNA complex. We demonstrate that the oriT recognition by MobMN199 was highly specific and suggest that this protein most probably employs Mn(2+) during pMV158 transfer.

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DNA nicking by the MobMN199 relaxase in the presence of different metal concentrations. (a) Supercoiled pMV158 DNA (8 nM) was incubated with MobMN199 (250 nM) in the presence of different concentrations of MgCl2 or MnCl2 (0.015, 0.2, 0.4, 0.8, 1.5, 4, 8, 15 and 20 mM) at 30°C for 20 min. Samples were analysed as indicated in Figure 1d. (b) Histogram comparing the percentage of FII plasmid forms generated by MobMN199 nicking activity in presence of MgCl2 or MnCl2. (c) Best fit of nicking activity data as function of metal ion concentration assuming a single set of metal ion-binding sites.
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Figure 7: DNA nicking by the MobMN199 relaxase in the presence of different metal concentrations. (a) Supercoiled pMV158 DNA (8 nM) was incubated with MobMN199 (250 nM) in the presence of different concentrations of MgCl2 or MnCl2 (0.015, 0.2, 0.4, 0.8, 1.5, 4, 8, 15 and 20 mM) at 30°C for 20 min. Samples were analysed as indicated in Figure 1d. (b) Histogram comparing the percentage of FII plasmid forms generated by MobMN199 nicking activity in presence of MgCl2 or MnCl2. (c) Best fit of nicking activity data as function of metal ion concentration assuming a single set of metal ion-binding sites.

Mentions: MobM relaxation activity, measured by conversion of FI to FII forms, was previously tested in presence of different metal ions, being Mn2+ and Mg2+ the more efficient (19). Here we analysed the effect of Mn2+ and Mg2+ dosage on the activity of MobMN199, while keeping protein–DNA ratio (250 and 8 nM, respectively) and reaction conditions fixed. As shown in Figure 7, the nicking activity of MobMN199 strongly depended on the divalent cation concentration. Relaxed plasmid forms were not observed in the presence of EDTA (10 mM) or in the cation-free control. Maximum activity was found above 8 mM of divalent cation, with conversion levels to FII forms of 46 or 62% in the presence of saturating concentrations of MgCl2 or MnCl2, respectively. Interestingly, this difference in activity was also observed at 150 µM, a cation concentration high enough to provide full saturation of the high-affinity site of Mn2+ according to ITC data. Increase in MobMN199 activity with Mg2+ can be reasonably described assuming a single class of binding site with an apparent affinity of 1.8 mM (Figure 7c). For Mn2+, however, a first increase accounting for ∼15% FII forms, followed by a further increase of ∼48% at higher cation concentrations, was required to fit well the experimental data (Figure 7c). These results indicate that MobMN199 may contain two or more different classes of Mn2+ binding sites, at least in the presence of DNA, and that full nicking activity would be observed when the high and low affinity sites become saturated. Finally, based on this and on previously reported results (19), we can establish that the hierarchy of preference for cation usage by MobM is: Mn2+ > Mg2+ > Ca2+ > Zn2+ ≥ Ba2+.Figure 7.


The MobM relaxase domain of plasmid pMV158: thermal stability and activity upon Mn2+ and specific DNA binding.

Lorenzo-Díaz F, Dostál L, Coll M, Schildbach JF, Menéndez M, Espinosa M - Nucleic Acids Res. (2011)

DNA nicking by the MobMN199 relaxase in the presence of different metal concentrations. (a) Supercoiled pMV158 DNA (8 nM) was incubated with MobMN199 (250 nM) in the presence of different concentrations of MgCl2 or MnCl2 (0.015, 0.2, 0.4, 0.8, 1.5, 4, 8, 15 and 20 mM) at 30°C for 20 min. Samples were analysed as indicated in Figure 1d. (b) Histogram comparing the percentage of FII plasmid forms generated by MobMN199 nicking activity in presence of MgCl2 or MnCl2. (c) Best fit of nicking activity data as function of metal ion concentration assuming a single set of metal ion-binding sites.
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Related In: Results  -  Collection

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Figure 7: DNA nicking by the MobMN199 relaxase in the presence of different metal concentrations. (a) Supercoiled pMV158 DNA (8 nM) was incubated with MobMN199 (250 nM) in the presence of different concentrations of MgCl2 or MnCl2 (0.015, 0.2, 0.4, 0.8, 1.5, 4, 8, 15 and 20 mM) at 30°C for 20 min. Samples were analysed as indicated in Figure 1d. (b) Histogram comparing the percentage of FII plasmid forms generated by MobMN199 nicking activity in presence of MgCl2 or MnCl2. (c) Best fit of nicking activity data as function of metal ion concentration assuming a single set of metal ion-binding sites.
Mentions: MobM relaxation activity, measured by conversion of FI to FII forms, was previously tested in presence of different metal ions, being Mn2+ and Mg2+ the more efficient (19). Here we analysed the effect of Mn2+ and Mg2+ dosage on the activity of MobMN199, while keeping protein–DNA ratio (250 and 8 nM, respectively) and reaction conditions fixed. As shown in Figure 7, the nicking activity of MobMN199 strongly depended on the divalent cation concentration. Relaxed plasmid forms were not observed in the presence of EDTA (10 mM) or in the cation-free control. Maximum activity was found above 8 mM of divalent cation, with conversion levels to FII forms of 46 or 62% in the presence of saturating concentrations of MgCl2 or MnCl2, respectively. Interestingly, this difference in activity was also observed at 150 µM, a cation concentration high enough to provide full saturation of the high-affinity site of Mn2+ according to ITC data. Increase in MobMN199 activity with Mg2+ can be reasonably described assuming a single class of binding site with an apparent affinity of 1.8 mM (Figure 7c). For Mn2+, however, a first increase accounting for ∼15% FII forms, followed by a further increase of ∼48% at higher cation concentrations, was required to fit well the experimental data (Figure 7c). These results indicate that MobMN199 may contain two or more different classes of Mn2+ binding sites, at least in the presence of DNA, and that full nicking activity would be observed when the high and low affinity sites become saturated. Finally, based on this and on previously reported results (19), we can establish that the hierarchy of preference for cation usage by MobM is: Mn2+ > Mg2+ > Ca2+ > Zn2+ ≥ Ba2+.Figure 7.

Bottom Line: However, whereas Mn(2+) strongly stabilized MobMN199 against thermal denaturation, no protective effect was observed for Mg(2+).The structure of MobMN199 was strongly stabilized by binding to the defined target DNA, indicating the formation of a tight protein-DNA complex.We demonstrate that the oriT recognition by MobMN199 was highly specific and suggest that this protein most probably employs Mn(2+) during pMV158 transfer.

View Article: PubMed Central - PubMed

Affiliation: Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain.

ABSTRACT
Protein MobM, the relaxase involved in conjugative transfer of the streptococcal plasmid pMV158, is the prototype of the MOB(V) superfamily of relaxases. To characterize the DNA-binding and nicking domain of MobM, a truncated version of the protein (MobMN199) encompassing its N-terminal region was designed and the protein was purified. MobMN199 was monomeric in contrast to the dimeric form of the full-length protein, but it kept its nicking activity on pMV158 DNA. The optimal relaxase activity was dependent on Mn(2+) or Mg(2+) cations in a dosage-dependent manner. However, whereas Mn(2+) strongly stabilized MobMN199 against thermal denaturation, no protective effect was observed for Mg(2+). Furthermore, MobMN199 exhibited a high affinity binding for Mn(2+) but not for Mg(2+). We also examined the binding-specificity and affinity of MobMN199 for several substrates of single-stranded DNA encompassing the pMV158 origin of transfer (oriT). The minimal oriT was delimited to a stretch of 26 nt which included an inverted repeat located eight bases upstream of the nick site. The structure of MobMN199 was strongly stabilized by binding to the defined target DNA, indicating the formation of a tight protein-DNA complex. We demonstrate that the oriT recognition by MobMN199 was highly specific and suggest that this protein most probably employs Mn(2+) during pMV158 transfer.

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Related in: MedlinePlus