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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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Interactions between MobMN199 and ss-oligonucleotides harbouring regions of the pMV158-oriT. (a) DNA sequence of the oriT, indicating the three inverted repeats (top) and the oligonucleotides used for affinity and competition analyses (bottom). The nic site is depicted by a vertical arrowhead. (b–d) MobMN199 DNA-binding measured by EMSA; the protein was incubated with Cy5-labelled DNA fragments containing the IR3 (b), the entire oriT (c) or the IR2 (d) sequences. MobMN199 concentrations were 0, 5, 10, 20, 40, 80, 160 and 320 nM from lane 1 to 8, respectively. Positions of the free (F) DNA and of the MobMN199–DNA complexes (C) are indicated.
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Figure 8: Interactions between MobMN199 and ss-oligonucleotides harbouring regions of the pMV158-oriT. (a) DNA sequence of the oriT, indicating the three inverted repeats (top) and the oligonucleotides used for affinity and competition analyses (bottom). The nic site is depicted by a vertical arrowhead. (b–d) MobMN199 DNA-binding measured by EMSA; the protein was incubated with Cy5-labelled DNA fragments containing the IR3 (b), the entire oriT (c) or the IR2 (d) sequences. MobMN199 concentrations were 0, 5, 10, 20, 40, 80, 160 and 320 nM from lane 1 to 8, respectively. Positions of the free (F) DNA and of the MobMN199–DNA complexes (C) are indicated.

Mentions: The oriT of pMV158 was proposed to be included within a DNA region that contains IR1 and IR2 which encompassed the MobM-mediated nick site (located between coordinates 3591 and 3592) (20). A closer inspection of the sequence allowed us to find a third 31-bp IR (IR3) that includes IR1 plus 5 and 8 bases up- and downstream, respectively (Figure 8a). To reveal the requirements of the DNA target for recognition by MobMN199, we designed a set of Cy5-labelled oligonucleotides that contained part or all of the oriT sequence (Figure 8a). EMSA analysis using 2 nM of DNA showed that MobMN199 recognized specifically the oriT sequence, since it generated single complexes with the IR3-Cy5 and ORIT-Cy5 oligonucleotides (Figure 8b and c, respectively). However, MobMN199 did not bind to IR2-Cy5, even at high protein concentration (Figure 8d). After quantification of the bound DNA fraction, it was possible to estimate the dissociation constants (Kd) of MobMN199 as 60±7 and 58±6 nM for IR3 and ORIT oligonucleotides, respectively. These results indicate that the region encompassing IR1/3 was able to bind MobMN199 with high affinity. However, loss of the 5′-region of the oriT sequence (represented by oligonucleotide IR2) resulted in, at least, a 5-fold decrease in binding affinity (Kd > 320 nM). We also analysed the stoichiometry of MobMN199 binding to IR3 oligonucleotide. Graphic representation of the percentage of bound DNA showed that saturation was reached at an approximately 2:1 molar ratio of MobMN199:DNA (Supplementary Figure S2), which suggested the presence of two monomers of MobMN199 per DNA molecule in the complex. The same result was obtained with the ORIT oligonucleotide (data not shown).Figure 8.


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)

Interactions between MobMN199 and ss-oligonucleotides harbouring regions of the pMV158-oriT. (a) DNA sequence of the oriT, indicating the three inverted repeats (top) and the oligonucleotides used for affinity and competition analyses (bottom). The nic site is depicted by a vertical arrowhead. (b–d) MobMN199 DNA-binding measured by EMSA; the protein was incubated with Cy5-labelled DNA fragments containing the IR3 (b), the entire oriT (c) or the IR2 (d) sequences. MobMN199 concentrations were 0, 5, 10, 20, 40, 80, 160 and 320 nM from lane 1 to 8, respectively. Positions of the free (F) DNA and of the MobMN199–DNA complexes (C) are indicated.
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Related In: Results  -  Collection

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Figure 8: Interactions between MobMN199 and ss-oligonucleotides harbouring regions of the pMV158-oriT. (a) DNA sequence of the oriT, indicating the three inverted repeats (top) and the oligonucleotides used for affinity and competition analyses (bottom). The nic site is depicted by a vertical arrowhead. (b–d) MobMN199 DNA-binding measured by EMSA; the protein was incubated with Cy5-labelled DNA fragments containing the IR3 (b), the entire oriT (c) or the IR2 (d) sequences. MobMN199 concentrations were 0, 5, 10, 20, 40, 80, 160 and 320 nM from lane 1 to 8, respectively. Positions of the free (F) DNA and of the MobMN199–DNA complexes (C) are indicated.
Mentions: The oriT of pMV158 was proposed to be included within a DNA region that contains IR1 and IR2 which encompassed the MobM-mediated nick site (located between coordinates 3591 and 3592) (20). A closer inspection of the sequence allowed us to find a third 31-bp IR (IR3) that includes IR1 plus 5 and 8 bases up- and downstream, respectively (Figure 8a). To reveal the requirements of the DNA target for recognition by MobMN199, we designed a set of Cy5-labelled oligonucleotides that contained part or all of the oriT sequence (Figure 8a). EMSA analysis using 2 nM of DNA showed that MobMN199 recognized specifically the oriT sequence, since it generated single complexes with the IR3-Cy5 and ORIT-Cy5 oligonucleotides (Figure 8b and c, respectively). However, MobMN199 did not bind to IR2-Cy5, even at high protein concentration (Figure 8d). After quantification of the bound DNA fraction, it was possible to estimate the dissociation constants (Kd) of MobMN199 as 60±7 and 58±6 nM for IR3 and ORIT oligonucleotides, respectively. These results indicate that the region encompassing IR1/3 was able to bind MobMN199 with high affinity. However, loss of the 5′-region of the oriT sequence (represented by oligonucleotide IR2) resulted in, at least, a 5-fold decrease in binding affinity (Kd > 320 nM). We also analysed the stoichiometry of MobMN199 binding to IR3 oligonucleotide. Graphic representation of the percentage of bound DNA showed that saturation was reached at an approximately 2:1 molar ratio of MobMN199:DNA (Supplementary Figure S2), which suggested the presence of two monomers of MobMN199 per DNA molecule in the complex. The same result was obtained with the ORIT oligonucleotide (data not shown).Figure 8.

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