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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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MobMN199 binding competitions between IR3-Cy5 and several unlabeled oligonucleotides. Different concentrations of competitor unlabeled IR3 (a), ORIT (b), IR1 + 8 (c), IR1, IR3 − R or NIC − 10 (d) oligonucleotides were mixed with 2 nM Cy5-labelled IR3. Then, purified MobMN199 protein (80 nM) was added to the reaction as indicated. Samples were incubated for 20 min at 24°C and loaded onto native PAA (10%) gels. Free (F) and complexed (C) DNA are indicated.
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Figure 9: MobMN199 binding competitions between IR3-Cy5 and several unlabeled oligonucleotides. Different concentrations of competitor unlabeled IR3 (a), ORIT (b), IR1 + 8 (c), IR1, IR3 − R or NIC − 10 (d) oligonucleotides were mixed with 2 nM Cy5-labelled IR3. Then, purified MobMN199 protein (80 nM) was added to the reaction as indicated. Samples were incubated for 20 min at 24°C and loaded onto native PAA (10%) gels. Free (F) and complexed (C) DNA are indicated.

Mentions: Competition binding assays between IR3-Cy5 (2 nM) and different amounts of the same unlabelled DNA (Figure 9a) or the ORIT oligonucleotide (Figure 9b) were performed. The results obtained at a fixed MobMN199 concentration (80 nM) showed a comparable competition ability of both oligonucleotides. This indicated that the recognition sites of MobMN199 for oligonucleotides IR3 and ORIT were equivalent. Thus, the minimal oriT region able to bind MobMN199 with high affinity could be located within the IR3 sequence. To test this hypothesis, new competition experiments were done using DNA fragments shorter than IR3. The results showed that a DNA including the IR1 hairpin sequence plus 8 nt downstream (IR1 + 8), just up to the nick site, was able to compete for binding of MobMN199 to IR3 with, at least, the same efficiencies than IR3 or ORIT (Figure 9c). However, other oligonucleotides harbouring only the IR1 sequence (IR1), the right-arm of IR3 (IR3-R), or 10 nt upstream the nick site (10-NIC), did not compete effectively with IR3-Cy5 for binding to MobMN199 at the concentrations employed (Figure 9d). The same behaviour was observed with oligonucleotides containing the left-arm of IR3 (IR3-L), right-arm of IR1 (IR1-R), or IR2 sequences (not shown).Figure 9.


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)

MobMN199 binding competitions between IR3-Cy5 and several unlabeled oligonucleotides. Different concentrations of competitor unlabeled IR3 (a), ORIT (b), IR1 + 8 (c), IR1, IR3 − R or NIC − 10 (d) oligonucleotides were mixed with 2 nM Cy5-labelled IR3. Then, purified MobMN199 protein (80 nM) was added to the reaction as indicated. Samples were incubated for 20 min at 24°C and loaded onto native PAA (10%) gels. Free (F) and complexed (C) DNA are indicated.
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Related In: Results  -  Collection

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Figure 9: MobMN199 binding competitions between IR3-Cy5 and several unlabeled oligonucleotides. Different concentrations of competitor unlabeled IR3 (a), ORIT (b), IR1 + 8 (c), IR1, IR3 − R or NIC − 10 (d) oligonucleotides were mixed with 2 nM Cy5-labelled IR3. Then, purified MobMN199 protein (80 nM) was added to the reaction as indicated. Samples were incubated for 20 min at 24°C and loaded onto native PAA (10%) gels. Free (F) and complexed (C) DNA are indicated.
Mentions: Competition binding assays between IR3-Cy5 (2 nM) and different amounts of the same unlabelled DNA (Figure 9a) or the ORIT oligonucleotide (Figure 9b) were performed. The results obtained at a fixed MobMN199 concentration (80 nM) showed a comparable competition ability of both oligonucleotides. This indicated that the recognition sites of MobMN199 for oligonucleotides IR3 and ORIT were equivalent. Thus, the minimal oriT region able to bind MobMN199 with high affinity could be located within the IR3 sequence. To test this hypothesis, new competition experiments were done using DNA fragments shorter than IR3. The results showed that a DNA including the IR1 hairpin sequence plus 8 nt downstream (IR1 + 8), just up to the nick site, was able to compete for binding of MobMN199 to IR3 with, at least, the same efficiencies than IR3 or ORIT (Figure 9c). However, other oligonucleotides harbouring only the IR1 sequence (IR1), the right-arm of IR3 (IR3-R), or 10 nt upstream the nick site (10-NIC), did not compete effectively with IR3-Cy5 for binding to MobMN199 at the concentrations employed (Figure 9d). The same behaviour was observed with oligonucleotides containing the left-arm of IR3 (IR3-L), right-arm of IR1 (IR1-R), or IR2 sequences (not shown).Figure 9.

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