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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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Heparin-induced oligomerization. Chromatograms on the left of panels A–C show elution profiles of DnaB (27 µM), DnaB(1–428) (10 µM) or DnaBC (30 µM) from a HiTrap Heparin column (GE Healthcare) with a 0–2 M NaCl gradient over 20 ml. Letters L and E mark loading and elution points, respectively. Fractions (2 ml) were collected and numbered 1–3 or 1–4. The conductivity at each peak is indicated. DnaBN and DnaB(1–365) did not bind to the heparin column under our conditions (data not shown). Gel filtration profiles of the heparin fractions are shown on the right of A–C. Samples (0.5 ml) from heparin fractions (1–3) were loaded onto a Superose 6 (HR 10/30, GE Healthcare), except for DnaBC where Superdex 200 (HR 10/30, GE Healthcare) was used instead. Traces are superimposed and numbered the same as the heparin fractions. Higher order DnaB oligomers are marked accordingly.
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Figure 5: Heparin-induced oligomerization. Chromatograms on the left of panels A–C show elution profiles of DnaB (27 µM), DnaB(1–428) (10 µM) or DnaBC (30 µM) from a HiTrap Heparin column (GE Healthcare) with a 0–2 M NaCl gradient over 20 ml. Letters L and E mark loading and elution points, respectively. Fractions (2 ml) were collected and numbered 1–3 or 1–4. The conductivity at each peak is indicated. DnaBN and DnaB(1–365) did not bind to the heparin column under our conditions (data not shown). Gel filtration profiles of the heparin fractions are shown on the right of A–C. Samples (0.5 ml) from heparin fractions (1–3) were loaded onto a Superose 6 (HR 10/30, GE Healthcare), except for DnaBC where Superdex 200 (HR 10/30, GE Healthcare) was used instead. Traces are superimposed and numbered the same as the heparin fractions. Higher order DnaB oligomers are marked accordingly.

Mentions: To obtain further evidence that DNA binding induces DnaB oligomerization we utilized heparin affinity chromatography combined with size exclusion chromatography. DnaB, DnaBC and DnaB(1–428) were loaded to heparin and eluted with an increasing NaCl gradient (Figure 5). DnaB and DnaB(1–428) were eluted as two overlapping peaks (Figure 5A and B). Subsequent analysis of samples from fractions 1–3 by size exclusion chromatography revealed that fraction 1 contained tetramers, whereas fractions 2 and 3 mixtures of higher oligomers (Figure 5A and B). Velocity sedimentation analysis of fractions 1–3 from native DnaB verified the presence of tetramers in fraction 1 with sedimentation coefficient s* = 6.8 and mixtures of higher oligomers in fractions 2 and 3 with a wide distribution of sedimentation coefficients s* = 10–50 (Supplementary Figure 5S). DnaBC eluted as two separated peaks from the heparin column indicating formation of oligomers. However, subsequent analysis of fractions 1–4 by size exclusion chromatography revealed that they were all monomeric (Figure 5C). The simplest explanation is that DnaBC oligomers are formed in the presence of DNA but are somewhat unstable in the absence of DNA. Therefore, the presence of DnaBN may contribute towards greater stability of higher order oligomers in the absence of DNA. DnaB(1–365) did not bind to heparin (data not shown) indicating that the heparin interaction site is between residues 365–428, likely to coincide with the second ssDNA binding site in DnaBC. Since high salt does not induce oligomerization (data not shown) we conclude that binding to heparin mimics binding to DNA and induces the formation of higher order DnaB oligomers. The ssDNA binding and oligomerization sites in DnaBC are located within residues 365–428.Figure 5.


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)

Heparin-induced oligomerization. Chromatograms on the left of panels A–C show elution profiles of DnaB (27 µM), DnaB(1–428) (10 µM) or DnaBC (30 µM) from a HiTrap Heparin column (GE Healthcare) with a 0–2 M NaCl gradient over 20 ml. Letters L and E mark loading and elution points, respectively. Fractions (2 ml) were collected and numbered 1–3 or 1–4. The conductivity at each peak is indicated. DnaBN and DnaB(1–365) did not bind to the heparin column under our conditions (data not shown). Gel filtration profiles of the heparin fractions are shown on the right of A–C. Samples (0.5 ml) from heparin fractions (1–3) were loaded onto a Superose 6 (HR 10/30, GE Healthcare), except for DnaBC where Superdex 200 (HR 10/30, GE Healthcare) was used instead. Traces are superimposed and numbered the same as the heparin fractions. Higher order DnaB oligomers are marked accordingly.
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Figure 5: Heparin-induced oligomerization. Chromatograms on the left of panels A–C show elution profiles of DnaB (27 µM), DnaB(1–428) (10 µM) or DnaBC (30 µM) from a HiTrap Heparin column (GE Healthcare) with a 0–2 M NaCl gradient over 20 ml. Letters L and E mark loading and elution points, respectively. Fractions (2 ml) were collected and numbered 1–3 or 1–4. The conductivity at each peak is indicated. DnaBN and DnaB(1–365) did not bind to the heparin column under our conditions (data not shown). Gel filtration profiles of the heparin fractions are shown on the right of A–C. Samples (0.5 ml) from heparin fractions (1–3) were loaded onto a Superose 6 (HR 10/30, GE Healthcare), except for DnaBC where Superdex 200 (HR 10/30, GE Healthcare) was used instead. Traces are superimposed and numbered the same as the heparin fractions. Higher order DnaB oligomers are marked accordingly.
Mentions: To obtain further evidence that DNA binding induces DnaB oligomerization we utilized heparin affinity chromatography combined with size exclusion chromatography. DnaB, DnaBC and DnaB(1–428) were loaded to heparin and eluted with an increasing NaCl gradient (Figure 5). DnaB and DnaB(1–428) were eluted as two overlapping peaks (Figure 5A and B). Subsequent analysis of samples from fractions 1–3 by size exclusion chromatography revealed that fraction 1 contained tetramers, whereas fractions 2 and 3 mixtures of higher oligomers (Figure 5A and B). Velocity sedimentation analysis of fractions 1–3 from native DnaB verified the presence of tetramers in fraction 1 with sedimentation coefficient s* = 6.8 and mixtures of higher oligomers in fractions 2 and 3 with a wide distribution of sedimentation coefficients s* = 10–50 (Supplementary Figure 5S). DnaBC eluted as two separated peaks from the heparin column indicating formation of oligomers. However, subsequent analysis of fractions 1–4 by size exclusion chromatography revealed that they were all monomeric (Figure 5C). The simplest explanation is that DnaBC oligomers are formed in the presence of DNA but are somewhat unstable in the absence of DNA. Therefore, the presence of DnaBN may contribute towards greater stability of higher order oligomers in the absence of DNA. DnaB(1–365) did not bind to heparin (data not shown) indicating that the heparin interaction site is between residues 365–428, likely to coincide with the second ssDNA binding site in DnaBC. Since high salt does not induce oligomerization (data not shown) we conclude that binding to heparin mimics binding to DNA and induces the formation of higher order DnaB oligomers. The ssDNA binding and oligomerization sites in DnaBC are located within residues 365–428.Figure 5.

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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