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DnaB proteolysis in vivo regulates oligomerization and its localization at oriC in Bacillus subtilis.

Grainger WH, Machón C, Scott DJ, Soultanas P - Nucleic Acids Res. (2010)

Bottom Line: Proteolysis is confined to cytosolic, not to membrane-associated DnaB, and affects oligomerization.Truncated DnaB is depleted at the oriC relative to the native protein.It encompasses an area from the middle of dnaA to the end of yaaA that includes the AT-rich region melted during the initiation stage of DNA replication.

View Article: PubMed Central - PubMed

Affiliation: Centre for Biomolecular Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, UK.

ABSTRACT
Initiation of bacterial DNA replication at oriC is mediated by primosomal proteins that act cooperatively to melt an AT-rich region where the replicative helicase is loaded prior to the assembly of the replication fork. In Bacillus subtilis, the dnaD, dnaB and dnaI genes are essential for initiation of DNA replication. We established that their mRNAs are maintained in fast growing asynchronous cultures. DnaB is truncated at its C-terminus in a growth phase-dependent manner. Proteolysis is confined to cytosolic, not to membrane-associated DnaB, and affects oligomerization. Truncated DnaB is depleted at the oriC relative to the native protein. We propose that DNA-induced oligomerization is essential for its action at oriC and proteolysis regulates its localization at oriC. We show that DnaB has two separate ssDNA-binding sites one located within residues 1-300 and another between residues 365-428, and a dsDNA-binding site within residues 365-428. Tetramerization of DnaB is mediated within residues 1-300, and DNA-dependent oligomerization within residues 365-428. Finally, we show that association of DnaB with the oriC is asymmetric and extensive. It encompasses an area from the middle of dnaA to the end of yaaA that includes the AT-rich region melted during the initiation stage of DNA replication.

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DnaB–ssDNA interactions. Gel shifts showing binding of DnaB polypeptides to ssDNA (dT45, dT90 or dT135). The concentrations (µM) of proteins in the binding reactions are shown underneath the lanes of the gels. Lanes M are internal controls showing binding reactions with 45 µM DnaBN. The positions of the slowest migrating bands of DnaB(1–365) are marked by asterisks. The equivalent position with native DnaB is also marked by an asterisk in the dT45 gel shift.
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Figure 4: DnaB–ssDNA interactions. Gel shifts showing binding of DnaB polypeptides to ssDNA (dT45, dT90 or dT135). The concentrations (µM) of proteins in the binding reactions are shown underneath the lanes of the gels. Lanes M are internal controls showing binding reactions with 45 µM DnaBN. The positions of the slowest migrating bands of DnaB(1–365) are marked by asterisks. The equivalent position with native DnaB is also marked by an asterisk in the dT45 gel shift.

Mentions: DnaBN and DnaBC did not interact with each other (data not shown) but both bound ssDNA. DnaBN formed defined complexes with oligonucleotides dT45, dT90 and dT135, (Figure 4). The patterns of shifted bands with increasing oligonucleotide sizes suggest that a small fraction of DnaBN binds as a dimer forming a minor band shifted the least with all three substrates, while most DnaBN binds as a tetramer to form one major shifted band with dT45, two with dT90 (two bound tetramers) and three with dT135 (three bound tetramers). Therefore, the apparent DnaBN footprints are ∼45 and 22 nucleotides for the tetramer and dimer, respectively. DnaBC formed mixtures of bigger complexes retarded at the top of the gel higher than it would be anticipated by its monomeric state and size (Figure 4). Native DnaB produced undefined diffuse shifts of decreased mobility with dT45 indicative of mixed higher order oligomers. These oligomers appeared to be more defined with the larger dT90 and dT135 oligonucleotides suggesting that they are more stable with longer ssDNA substrates.Figure 4.


DnaB proteolysis in vivo regulates oligomerization and its localization at oriC in Bacillus subtilis.

Grainger WH, Machón C, Scott DJ, Soultanas P - Nucleic Acids Res. (2010)

DnaB–ssDNA interactions. Gel shifts showing binding of DnaB polypeptides to ssDNA (dT45, dT90 or dT135). The concentrations (µM) of proteins in the binding reactions are shown underneath the lanes of the gels. Lanes M are internal controls showing binding reactions with 45 µM DnaBN. The positions of the slowest migrating bands of DnaB(1–365) are marked by asterisks. The equivalent position with native DnaB is also marked by an asterisk in the dT45 gel shift.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2874997&req=5

Figure 4: DnaB–ssDNA interactions. Gel shifts showing binding of DnaB polypeptides to ssDNA (dT45, dT90 or dT135). The concentrations (µM) of proteins in the binding reactions are shown underneath the lanes of the gels. Lanes M are internal controls showing binding reactions with 45 µM DnaBN. The positions of the slowest migrating bands of DnaB(1–365) are marked by asterisks. The equivalent position with native DnaB is also marked by an asterisk in the dT45 gel shift.
Mentions: DnaBN and DnaBC did not interact with each other (data not shown) but both bound ssDNA. DnaBN formed defined complexes with oligonucleotides dT45, dT90 and dT135, (Figure 4). The patterns of shifted bands with increasing oligonucleotide sizes suggest that a small fraction of DnaBN binds as a dimer forming a minor band shifted the least with all three substrates, while most DnaBN binds as a tetramer to form one major shifted band with dT45, two with dT90 (two bound tetramers) and three with dT135 (three bound tetramers). Therefore, the apparent DnaBN footprints are ∼45 and 22 nucleotides for the tetramer and dimer, respectively. DnaBC formed mixtures of bigger complexes retarded at the top of the gel higher than it would be anticipated by its monomeric state and size (Figure 4). Native DnaB produced undefined diffuse shifts of decreased mobility with dT45 indicative of mixed higher order oligomers. These oligomers appeared to be more defined with the larger dT90 and dT135 oligonucleotides suggesting that they are more stable with longer ssDNA substrates.Figure 4.

Bottom Line: Proteolysis is confined to cytosolic, not to membrane-associated DnaB, and affects oligomerization.Truncated DnaB is depleted at the oriC relative to the native protein.It encompasses an area from the middle of dnaA to the end of yaaA that includes the AT-rich region melted during the initiation stage of DNA replication.

View Article: PubMed Central - PubMed

Affiliation: Centre for Biomolecular Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, UK.

ABSTRACT
Initiation of bacterial DNA replication at oriC is mediated by primosomal proteins that act cooperatively to melt an AT-rich region where the replicative helicase is loaded prior to the assembly of the replication fork. In Bacillus subtilis, the dnaD, dnaB and dnaI genes are essential for initiation of DNA replication. We established that their mRNAs are maintained in fast growing asynchronous cultures. DnaB is truncated at its C-terminus in a growth phase-dependent manner. Proteolysis is confined to cytosolic, not to membrane-associated DnaB, and affects oligomerization. Truncated DnaB is depleted at the oriC relative to the native protein. We propose that DNA-induced oligomerization is essential for its action at oriC and proteolysis regulates its localization at oriC. We show that DnaB has two separate ssDNA-binding sites one located within residues 1-300 and another between residues 365-428, and a dsDNA-binding site within residues 365-428. Tetramerization of DnaB is mediated within residues 1-300, and DNA-dependent oligomerization within residues 365-428. Finally, we show that association of DnaB with the oriC is asymmetric and extensive. It encompasses an area from the middle of dnaA to the end of yaaA that includes the AT-rich region melted during the initiation stage of DNA replication.

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