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Bacterial genome partitioning: N-terminal domain of IncC protein encoded by broad-host-range plasmid RK2 modulates oligomerisation and DNA binding.

Batt SM, Bingle LE, Dafforn TR, Thomas CM - J. Mol. Biol. (2008)

Bottom Line: ParA proteins normally occur in one of two forms, differing by their N-terminal domain (NTD) of approximately 100 aa, which is generally associated with site-specific DNA binding.The IncC1 NTD does not dimerise or bind DNA alone, but it does bind IncC2 in the presence of nucleotides.Mixing IncC1 and IncC2 improved polymerisation and DNA binding.

View Article: PubMed Central - PubMed

Affiliation: School of Biosciences, University of Birmingham, Edgbaston, Birmingham, UK.

ABSTRACT
ParA Walker ATPases form part of the machinery that promotes better-than-random segregation of bacterial genomes. ParA proteins normally occur in one of two forms, differing by their N-terminal domain (NTD) of approximately 100 aa, which is generally associated with site-specific DNA binding. Unusually, and for as yet unknown reasons, parA (incC) of IncP-1 plasmids is translated from alternative start codons producing two forms, IncC1 (364 aa) and IncC2 (259 aa), whose ratio varies between hosts. IncC2 could be detected as an oligomeric form containing dimers, tetramers and octamers, but the N-terminal extension present in IncC1 favours nucleotide-stimulated dimerisation as well as high-affinity and ATP-dependent non-specific DNA binding. The IncC1 NTD does not dimerise or bind DNA alone, but it does bind IncC2 in the presence of nucleotides. Mixing IncC1 and IncC2 improved polymerisation and DNA binding. Thus, the NTD may modulate the polymerisation interface, facilitating polymerisation/depolymerisation and DNA binding, to promote the cycle that drives partitioning.

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DNA binding activity of IncC1. (a) Competition band shifts of IncC1 with linear and supercoiled DNAs plus and minus the central control region from RK2 (pSMB201, plasmid with central control region from RK2; pSMB200, pSMB201 without the central control region). Triangles represent increasing concentrations of each IncC1 in the retardation (in 1.5-fold steps, beginning with 18 nM). Band shift assays performed with supercoiled and digested pSMB200, same as pSMB201 but without the central control region and supercoiled pSMB200 on agarose gel (0.7%). (b-i) Velocity sedimentation of IncC1 titrated onto DNA. A 15 nM mixture of supercoiled (CCC) and open circular (OC) pSMB201 DNAs was titrated with IncC1. (ii) Velocity sedimentation of DNA titrated onto IncC1. IncC1 (34 μM) was titrated with supercoiled and open circular pSMB201 DNAs. A concentration of ATP was added such that there was a 1:1 ratio of IncC1 monomer/dimer. (c) Band shifts of IncC1 with no nucleotide, ATP and ADP. Band shift assays performed with supercoiled pSMB201 DNA on agarose gel (0.7%) with increasing concentrations of IncC1 (in 1.5-fold steps, beginning with 12 nM). (d) Band shifts of IncC2 with no nucleotide, ATP and ADP. Band shift assays performed with supercoiled pSMB201 DNA on agarose gel (0.7%) with increasing concentrations of IncC2 (in 1.5-fold steps, beginning with 12 nM). (e) Graphical representation of band shifts of IncC1 and IncC2 with various nucleotides. For band shifts with ATP and ATPγS, the percentage of change in mobility was calculated as the percentage of reduction in the distance of the DNA band from the well. For band shifts with no nucleotide and ADP, the percentage of retardation was calculated by the reduction of the DNA band's intensity, which was analysed using Quantity One (BioRad).
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fig4: DNA binding activity of IncC1. (a) Competition band shifts of IncC1 with linear and supercoiled DNAs plus and minus the central control region from RK2 (pSMB201, plasmid with central control region from RK2; pSMB200, pSMB201 without the central control region). Triangles represent increasing concentrations of each IncC1 in the retardation (in 1.5-fold steps, beginning with 18 nM). Band shift assays performed with supercoiled and digested pSMB200, same as pSMB201 but without the central control region and supercoiled pSMB200 on agarose gel (0.7%). (b-i) Velocity sedimentation of IncC1 titrated onto DNA. A 15 nM mixture of supercoiled (CCC) and open circular (OC) pSMB201 DNAs was titrated with IncC1. (ii) Velocity sedimentation of DNA titrated onto IncC1. IncC1 (34 μM) was titrated with supercoiled and open circular pSMB201 DNAs. A concentration of ATP was added such that there was a 1:1 ratio of IncC1 monomer/dimer. (c) Band shifts of IncC1 with no nucleotide, ATP and ADP. Band shift assays performed with supercoiled pSMB201 DNA on agarose gel (0.7%) with increasing concentrations of IncC1 (in 1.5-fold steps, beginning with 12 nM). (d) Band shifts of IncC2 with no nucleotide, ATP and ADP. Band shift assays performed with supercoiled pSMB201 DNA on agarose gel (0.7%) with increasing concentrations of IncC2 (in 1.5-fold steps, beginning with 12 nM). (e) Graphical representation of band shifts of IncC1 and IncC2 with various nucleotides. For band shifts with ATP and ATPγS, the percentage of change in mobility was calculated as the percentage of reduction in the distance of the DNA band from the well. For band shifts with no nucleotide and ADP, the percentage of retardation was calculated by the reduction of the DNA band's intensity, which was analysed using Quantity One (BioRad).

Mentions: Although no DNA binding by IncC was observed previously,30 we reinvestigated this using a mixture of whole plasmids with and without the central control region of RK2, which was thought most likely to contain binding sites. In the absence of ATP, IncC1 (Fig. 4ai) and IncC2 (data not shown) were found to favour binding to supercoiled DNA, which was fully retarded in EMSAs at twofold lower protein concentrations than the linear DNA. No preference for plasmids containing RK2 central control region DNA with pSMB200 versus pSMB201 was observed. Velocity sedimentation performed with a 15 nM mixture of supercoiled and open circular pSMB201 DNAs (Fig. 4bi) also indicated an IncC1 preference for supercoiled DNA: the sedimentation coefficient of the peak corresponding to supercoiled DNA was more sensitive to increasing IncC1 concentrations than that of the open circular form. A change in sedimentation rate was not observed until the concentration of IncC1 reached between 0.9 and 3 μM, consistent with the concentration at which EMSA showed the transition from unretarded state to complete retardation (Fig. 4ai).


Bacterial genome partitioning: N-terminal domain of IncC protein encoded by broad-host-range plasmid RK2 modulates oligomerisation and DNA binding.

Batt SM, Bingle LE, Dafforn TR, Thomas CM - J. Mol. Biol. (2008)

DNA binding activity of IncC1. (a) Competition band shifts of IncC1 with linear and supercoiled DNAs plus and minus the central control region from RK2 (pSMB201, plasmid with central control region from RK2; pSMB200, pSMB201 without the central control region). Triangles represent increasing concentrations of each IncC1 in the retardation (in 1.5-fold steps, beginning with 18 nM). Band shift assays performed with supercoiled and digested pSMB200, same as pSMB201 but without the central control region and supercoiled pSMB200 on agarose gel (0.7%). (b-i) Velocity sedimentation of IncC1 titrated onto DNA. A 15 nM mixture of supercoiled (CCC) and open circular (OC) pSMB201 DNAs was titrated with IncC1. (ii) Velocity sedimentation of DNA titrated onto IncC1. IncC1 (34 μM) was titrated with supercoiled and open circular pSMB201 DNAs. A concentration of ATP was added such that there was a 1:1 ratio of IncC1 monomer/dimer. (c) Band shifts of IncC1 with no nucleotide, ATP and ADP. Band shift assays performed with supercoiled pSMB201 DNA on agarose gel (0.7%) with increasing concentrations of IncC1 (in 1.5-fold steps, beginning with 12 nM). (d) Band shifts of IncC2 with no nucleotide, ATP and ADP. Band shift assays performed with supercoiled pSMB201 DNA on agarose gel (0.7%) with increasing concentrations of IncC2 (in 1.5-fold steps, beginning with 12 nM). (e) Graphical representation of band shifts of IncC1 and IncC2 with various nucleotides. For band shifts with ATP and ATPγS, the percentage of change in mobility was calculated as the percentage of reduction in the distance of the DNA band from the well. For band shifts with no nucleotide and ADP, the percentage of retardation was calculated by the reduction of the DNA band's intensity, which was analysed using Quantity One (BioRad).
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fig4: DNA binding activity of IncC1. (a) Competition band shifts of IncC1 with linear and supercoiled DNAs plus and minus the central control region from RK2 (pSMB201, plasmid with central control region from RK2; pSMB200, pSMB201 without the central control region). Triangles represent increasing concentrations of each IncC1 in the retardation (in 1.5-fold steps, beginning with 18 nM). Band shift assays performed with supercoiled and digested pSMB200, same as pSMB201 but without the central control region and supercoiled pSMB200 on agarose gel (0.7%). (b-i) Velocity sedimentation of IncC1 titrated onto DNA. A 15 nM mixture of supercoiled (CCC) and open circular (OC) pSMB201 DNAs was titrated with IncC1. (ii) Velocity sedimentation of DNA titrated onto IncC1. IncC1 (34 μM) was titrated with supercoiled and open circular pSMB201 DNAs. A concentration of ATP was added such that there was a 1:1 ratio of IncC1 monomer/dimer. (c) Band shifts of IncC1 with no nucleotide, ATP and ADP. Band shift assays performed with supercoiled pSMB201 DNA on agarose gel (0.7%) with increasing concentrations of IncC1 (in 1.5-fold steps, beginning with 12 nM). (d) Band shifts of IncC2 with no nucleotide, ATP and ADP. Band shift assays performed with supercoiled pSMB201 DNA on agarose gel (0.7%) with increasing concentrations of IncC2 (in 1.5-fold steps, beginning with 12 nM). (e) Graphical representation of band shifts of IncC1 and IncC2 with various nucleotides. For band shifts with ATP and ATPγS, the percentage of change in mobility was calculated as the percentage of reduction in the distance of the DNA band from the well. For band shifts with no nucleotide and ADP, the percentage of retardation was calculated by the reduction of the DNA band's intensity, which was analysed using Quantity One (BioRad).
Mentions: Although no DNA binding by IncC was observed previously,30 we reinvestigated this using a mixture of whole plasmids with and without the central control region of RK2, which was thought most likely to contain binding sites. In the absence of ATP, IncC1 (Fig. 4ai) and IncC2 (data not shown) were found to favour binding to supercoiled DNA, which was fully retarded in EMSAs at twofold lower protein concentrations than the linear DNA. No preference for plasmids containing RK2 central control region DNA with pSMB200 versus pSMB201 was observed. Velocity sedimentation performed with a 15 nM mixture of supercoiled and open circular pSMB201 DNAs (Fig. 4bi) also indicated an IncC1 preference for supercoiled DNA: the sedimentation coefficient of the peak corresponding to supercoiled DNA was more sensitive to increasing IncC1 concentrations than that of the open circular form. A change in sedimentation rate was not observed until the concentration of IncC1 reached between 0.9 and 3 μM, consistent with the concentration at which EMSA showed the transition from unretarded state to complete retardation (Fig. 4ai).

Bottom Line: ParA proteins normally occur in one of two forms, differing by their N-terminal domain (NTD) of approximately 100 aa, which is generally associated with site-specific DNA binding.The IncC1 NTD does not dimerise or bind DNA alone, but it does bind IncC2 in the presence of nucleotides.Mixing IncC1 and IncC2 improved polymerisation and DNA binding.

View Article: PubMed Central - PubMed

Affiliation: School of Biosciences, University of Birmingham, Edgbaston, Birmingham, UK.

ABSTRACT
ParA Walker ATPases form part of the machinery that promotes better-than-random segregation of bacterial genomes. ParA proteins normally occur in one of two forms, differing by their N-terminal domain (NTD) of approximately 100 aa, which is generally associated with site-specific DNA binding. Unusually, and for as yet unknown reasons, parA (incC) of IncP-1 plasmids is translated from alternative start codons producing two forms, IncC1 (364 aa) and IncC2 (259 aa), whose ratio varies between hosts. IncC2 could be detected as an oligomeric form containing dimers, tetramers and octamers, but the N-terminal extension present in IncC1 favours nucleotide-stimulated dimerisation as well as high-affinity and ATP-dependent non-specific DNA binding. The IncC1 NTD does not dimerise or bind DNA alone, but it does bind IncC2 in the presence of nucleotides. Mixing IncC1 and IncC2 improved polymerisation and DNA binding. Thus, the NTD may modulate the polymerisation interface, facilitating polymerisation/depolymerisation and DNA binding, to promote the cycle that drives partitioning.

Show MeSH