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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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The oligomeric states of the DnaB domains. Sedimentation velocity AUC data for DnaBC (A velocity sedimentation at 40 000 rpm, B: equilibrium sedimentation at 17 000, 24 000 and 29 000 rpm) and DnaBN (C: velocity sedimentation at 40 000 rpm, D: equilibrium sedimentation at 12 000, 16 000 and 20 000 rpm) reveal that DnaBC is a monomer, while DnaBN is tetrameric at higher concentrations but in a dimer–tetramer equilibrium at lower concentrations (Kd = 500 nM). Experiments were carried out at 12.96, 11.25 and 8.07 μM DnaBC and 21.3, 15.6 and 7.8 μM DnaBN.
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Figure 3: The oligomeric states of the DnaB domains. Sedimentation velocity AUC data for DnaBC (A velocity sedimentation at 40 000 rpm, B: equilibrium sedimentation at 17 000, 24 000 and 29 000 rpm) and DnaBN (C: velocity sedimentation at 40 000 rpm, D: equilibrium sedimentation at 12 000, 16 000 and 20 000 rpm) reveal that DnaBC is a monomer, while DnaBN is tetrameric at higher concentrations but in a dimer–tetramer equilibrium at lower concentrations (Kd = 500 nM). Experiments were carried out at 12.96, 11.25 and 8.07 μM DnaBC and 21.3, 15.6 and 7.8 μM DnaBN.

Mentions: MALDI-PMF of truncated DnaB polypeptides purified from E. coli revealed peptides covering residues 1–411 (Supplementary Figure 3S). The smaller polypeptides, labelled 2–5, produced peptides covering residues 1–289 suggesting that proteolysis takes place at the C-terminus (Supplementary Figure 3S). Previous studies identified two domains at each end (residues 1–184 and 297–472) with a small central domain (residues 204–296), (23). Interestingly, the same studies reported proteolytic sensitivity of the C-terminal region, residues 430–472. We constructed two domains of DnaB encompassing residues 1–300 (DnaBN; N-terminal domain plus the central domain, MW 35 157 Da) and 301–472 (DnaBC; C-terminal domain, MW 20 445 Da), (Figure 2C). DnaBC was found to be monomeric by analytical ultracentrifugation (Figure 3A and B) whilst DnaBN was a tetramer at high concentrations and in a dimer-tetramer equilibrium at low concentrations (Kd = 500 nM) (Figure 3C and D). To unequivocally show that the C-terminal region of DnaB was being truncated we raised a sheep polyclonal antibody against a peptide encompassing the last twenty two C-terminal residues (DLEEQKKKMMEEMQKLKKYSAY). We argued that if proteolysis takes place at the C-terminus this antibody will recognize native DnaB but not truncated polypeptides. The antibody was significantly weaker than rabbit anti-DnaB and was not sensitive enough to detect DnaB in B. subtilis extracts (data not shown), but it recognized purified native DnaB, DnaBC and not DnaBN (Supplementary Figure 2S). It was only useful in establishing that over-expressed DnaB in E. coli was truncated from its C-terminus. The combined data show that DnaB is proteolytically truncated in vivo from its C-terminus in a growth-dependent manner and analogous truncation is observed when DnaB is heterologously expressed in E. coli. It is not clear whether E. coli uses the same or different proteases to process DnaB. However, proteolysis of DnaB in E. coli is less efficient probably because of its large over-expression in a short period of time.Figure 3.


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)

The oligomeric states of the DnaB domains. Sedimentation velocity AUC data for DnaBC (A velocity sedimentation at 40 000 rpm, B: equilibrium sedimentation at 17 000, 24 000 and 29 000 rpm) and DnaBN (C: velocity sedimentation at 40 000 rpm, D: equilibrium sedimentation at 12 000, 16 000 and 20 000 rpm) reveal that DnaBC is a monomer, while DnaBN is tetrameric at higher concentrations but in a dimer–tetramer equilibrium at lower concentrations (Kd = 500 nM). Experiments were carried out at 12.96, 11.25 and 8.07 μM DnaBC and 21.3, 15.6 and 7.8 μM DnaBN.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 3: The oligomeric states of the DnaB domains. Sedimentation velocity AUC data for DnaBC (A velocity sedimentation at 40 000 rpm, B: equilibrium sedimentation at 17 000, 24 000 and 29 000 rpm) and DnaBN (C: velocity sedimentation at 40 000 rpm, D: equilibrium sedimentation at 12 000, 16 000 and 20 000 rpm) reveal that DnaBC is a monomer, while DnaBN is tetrameric at higher concentrations but in a dimer–tetramer equilibrium at lower concentrations (Kd = 500 nM). Experiments were carried out at 12.96, 11.25 and 8.07 μM DnaBC and 21.3, 15.6 and 7.8 μM DnaBN.
Mentions: MALDI-PMF of truncated DnaB polypeptides purified from E. coli revealed peptides covering residues 1–411 (Supplementary Figure 3S). The smaller polypeptides, labelled 2–5, produced peptides covering residues 1–289 suggesting that proteolysis takes place at the C-terminus (Supplementary Figure 3S). Previous studies identified two domains at each end (residues 1–184 and 297–472) with a small central domain (residues 204–296), (23). Interestingly, the same studies reported proteolytic sensitivity of the C-terminal region, residues 430–472. We constructed two domains of DnaB encompassing residues 1–300 (DnaBN; N-terminal domain plus the central domain, MW 35 157 Da) and 301–472 (DnaBC; C-terminal domain, MW 20 445 Da), (Figure 2C). DnaBC was found to be monomeric by analytical ultracentrifugation (Figure 3A and B) whilst DnaBN was a tetramer at high concentrations and in a dimer-tetramer equilibrium at low concentrations (Kd = 500 nM) (Figure 3C and D). To unequivocally show that the C-terminal region of DnaB was being truncated we raised a sheep polyclonal antibody against a peptide encompassing the last twenty two C-terminal residues (DLEEQKKKMMEEMQKLKKYSAY). We argued that if proteolysis takes place at the C-terminus this antibody will recognize native DnaB but not truncated polypeptides. The antibody was significantly weaker than rabbit anti-DnaB and was not sensitive enough to detect DnaB in B. subtilis extracts (data not shown), but it recognized purified native DnaB, DnaBC and not DnaBN (Supplementary Figure 2S). It was only useful in establishing that over-expressed DnaB in E. coli was truncated from its C-terminus. The combined data show that DnaB is proteolytically truncated in vivo from its C-terminus in a growth-dependent manner and analogous truncation is observed when DnaB is heterologously expressed in E. coli. It is not clear whether E. coli uses the same or different proteases to process DnaB. However, proteolysis of DnaB in E. coli is less efficient probably because of its large over-expression in a short period of time.Figure 3.

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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