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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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Thermal denaturation of MobMN199 relaxase measured by DSC. (a) Influence of Mn2+ binding. Continuous grey and black lines are the thermograms of DNA-free protein monitored at 124 µM and 8 mM Mn2+ (83 µM MobMN199), respectively. Dashed red line is the fit of MobMN199 thermogram at low Mn2+ concentration to the two-state model. Dashed–dotted black lines are the elementary transitions under the thermogram at 8 mM MnCl2 assuming that MobMN199 denaturation takes place in three steps and the dashed–dotted green line is the theoretical envelope (‘Results’ section). (b) Influence of DNA binding. The presence of oligonucleotide IR1 + 8 shifted the unfolding endotherms of MobMN199 toward higher temperatures. Curves monitored at fixed protein:DNA ratio of 2:1 are in dark yellow (13.3 µM:6.5 µM) and blue (40 µM:20 µM). Mn2+ concentrations were fixed to have a free metal concentration of 82 µM at half denaturation.
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Figure 5: Thermal denaturation of MobMN199 relaxase measured by DSC. (a) Influence of Mn2+ binding. Continuous grey and black lines are the thermograms of DNA-free protein monitored at 124 µM and 8 mM Mn2+ (83 µM MobMN199), respectively. Dashed red line is the fit of MobMN199 thermogram at low Mn2+ concentration to the two-state model. Dashed–dotted black lines are the elementary transitions under the thermogram at 8 mM MnCl2 assuming that MobMN199 denaturation takes place in three steps and the dashed–dotted green line is the theoretical envelope (‘Results’ section). (b) Influence of DNA binding. The presence of oligonucleotide IR1 + 8 shifted the unfolding endotherms of MobMN199 toward higher temperatures. Curves monitored at fixed protein:DNA ratio of 2:1 are in dark yellow (13.3 µM:6.5 µM) and blue (40 µM:20 µM). Mn2+ concentrations were fixed to have a free metal concentration of 82 µM at half denaturation.

Mentions: bData in parenthesis correspond to the fit based on DSC parameters (see Figure 5 and text for details).


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)

Thermal denaturation of MobMN199 relaxase measured by DSC. (a) Influence of Mn2+ binding. Continuous grey and black lines are the thermograms of DNA-free protein monitored at 124 µM and 8 mM Mn2+ (83 µM MobMN199), respectively. Dashed red line is the fit of MobMN199 thermogram at low Mn2+ concentration to the two-state model. Dashed–dotted black lines are the elementary transitions under the thermogram at 8 mM MnCl2 assuming that MobMN199 denaturation takes place in three steps and the dashed–dotted green line is the theoretical envelope (‘Results’ section). (b) Influence of DNA binding. The presence of oligonucleotide IR1 + 8 shifted the unfolding endotherms of MobMN199 toward higher temperatures. Curves monitored at fixed protein:DNA ratio of 2:1 are in dark yellow (13.3 µM:6.5 µM) and blue (40 µM:20 µM). Mn2+ concentrations were fixed to have a free metal concentration of 82 µM at half denaturation.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3105389&req=5

Figure 5: Thermal denaturation of MobMN199 relaxase measured by DSC. (a) Influence of Mn2+ binding. Continuous grey and black lines are the thermograms of DNA-free protein monitored at 124 µM and 8 mM Mn2+ (83 µM MobMN199), respectively. Dashed red line is the fit of MobMN199 thermogram at low Mn2+ concentration to the two-state model. Dashed–dotted black lines are the elementary transitions under the thermogram at 8 mM MnCl2 assuming that MobMN199 denaturation takes place in three steps and the dashed–dotted green line is the theoretical envelope (‘Results’ section). (b) Influence of DNA binding. The presence of oligonucleotide IR1 + 8 shifted the unfolding endotherms of MobMN199 toward higher temperatures. Curves monitored at fixed protein:DNA ratio of 2:1 are in dark yellow (13.3 µM:6.5 µM) and blue (40 µM:20 µM). Mn2+ concentrations were fixed to have a free metal concentration of 82 µM at half denaturation.
Mentions: bData in parenthesis correspond to the fit based on DSC parameters (see Figure 5 and text for details).

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.

Show MeSH
Related in: MedlinePlus