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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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Overview of the IncP-1 system. (a) Alignment of the NTDs of several ParA homologues. The HTH motif is marked by a light gray box. Significant identical or conserved positions are marked with a black or dark gray box. (b) Map of the partitioning operon of RK2 showing the KorB binding sites (OBs) studied previously as possible centromere-like sequences. (c) Cartoon of IncC1 and IncC2 proteins showing the possible HTH and Walker ATPase motifs. (d) Western blotting of extracts of E. coli and P. putida probed with antibodies to IncC as described in Materials and Methods.
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fig1: Overview of the IncP-1 system. (a) Alignment of the NTDs of several ParA homologues. The HTH motif is marked by a light gray box. Significant identical or conserved positions are marked with a black or dark gray box. (b) Map of the partitioning operon of RK2 showing the KorB binding sites (OBs) studied previously as possible centromere-like sequences. (c) Cartoon of IncC1 and IncC2 proteins showing the possible HTH and Walker ATPase motifs. (d) Western blotting of extracts of E. coli and P. putida probed with antibodies to IncC as described in Materials and Methods.

Mentions: Alignment of the ParA protein sequences identifies two major forms depending on whether the protein starts just before the Walker ATPase box A or includes an N-terminal extension of approximately 100 aa, which generally contains a helix–turn–helix (HTH) motif (Fig. 1a). These longer ParA proteins are plasmid encoded and normally bind DNA specifically to autoregulate their cognate par operon—for example, ParA from P117 and SopA from plasmid F.18 Specific DNA binding by ParA proteins is stimulated by the presence of ADP and inhibited by ATP.19 This ATP–ADP switch in ParA activity is hypothesised to enable ParA to respond to the cell cycle of the host, segregating the plasmid DNA in the presence of ATP, whose levels are higher in exponential phase cells, and repressing expression from the par operon once this ATP is hydrolysed to ADP.20 This extra domain could therefore be entirely devoted to an autoregulatory function.


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)

Overview of the IncP-1 system. (a) Alignment of the NTDs of several ParA homologues. The HTH motif is marked by a light gray box. Significant identical or conserved positions are marked with a black or dark gray box. (b) Map of the partitioning operon of RK2 showing the KorB binding sites (OBs) studied previously as possible centromere-like sequences. (c) Cartoon of IncC1 and IncC2 proteins showing the possible HTH and Walker ATPase motifs. (d) Western blotting of extracts of E. coli and P. putida probed with antibodies to IncC as described in Materials and Methods.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2666795&req=5

fig1: Overview of the IncP-1 system. (a) Alignment of the NTDs of several ParA homologues. The HTH motif is marked by a light gray box. Significant identical or conserved positions are marked with a black or dark gray box. (b) Map of the partitioning operon of RK2 showing the KorB binding sites (OBs) studied previously as possible centromere-like sequences. (c) Cartoon of IncC1 and IncC2 proteins showing the possible HTH and Walker ATPase motifs. (d) Western blotting of extracts of E. coli and P. putida probed with antibodies to IncC as described in Materials and Methods.
Mentions: Alignment of the ParA protein sequences identifies two major forms depending on whether the protein starts just before the Walker ATPase box A or includes an N-terminal extension of approximately 100 aa, which generally contains a helix–turn–helix (HTH) motif (Fig. 1a). These longer ParA proteins are plasmid encoded and normally bind DNA specifically to autoregulate their cognate par operon—for example, ParA from P117 and SopA from plasmid F.18 Specific DNA binding by ParA proteins is stimulated by the presence of ADP and inhibited by ATP.19 This ATP–ADP switch in ParA activity is hypothesised to enable ParA to respond to the cell cycle of the host, segregating the plasmid DNA in the presence of ATP, whose levels are higher in exponential phase cells, and repressing expression from the par operon once this ATP is hydrolysed to ADP.20 This extra domain could therefore be entirely devoted to an autoregulatory function.

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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