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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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Growth-dependent expression of dnaD, dnaI and dnaB genes. (A) Growth curve of B. subtilis strain 168 in LB at 30°C over time. Time points t1, t2 and t3 when samples were analysed are indicated. The top bar graph shows levels of dnaB, dnaD, dnaI and sigA as CT values from RT-PCR over time. The bottom bar graph shows changes in gene expression calculated from the formula 2−ΔΔCt for dnaB, dnaD and dnaI, using sigA as control and t1 as calibrator, over time. In both graphs each bar represents the mean of five independent experiments. The error bars indicate standard deviations. (B) Western blot analysis of B. subtilis soluble extracts from overnight cultures. A typical SDS–PAGE gel with decreasing amounts of extracts (72, 36, 18, 9, 4.5, 2.2, 1.1, 0.5 and 0.25 μg) is shown (top left), with western blots for DnaD, DnaI and DnaB, as indicated. DnaB is truncated to smaller versions. Pre-stained molecular weight markers from New England Biolabs are indicated in kDa.
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Figure 1: Growth-dependent expression of dnaD, dnaI and dnaB genes. (A) Growth curve of B. subtilis strain 168 in LB at 30°C over time. Time points t1, t2 and t3 when samples were analysed are indicated. The top bar graph shows levels of dnaB, dnaD, dnaI and sigA as CT values from RT-PCR over time. The bottom bar graph shows changes in gene expression calculated from the formula 2−ΔΔCt for dnaB, dnaD and dnaI, using sigA as control and t1 as calibrator, over time. In both graphs each bar represents the mean of five independent experiments. The error bars indicate standard deviations. (B) Western blot analysis of B. subtilis soluble extracts from overnight cultures. A typical SDS–PAGE gel with decreasing amounts of extracts (72, 36, 18, 9, 4.5, 2.2, 1.1, 0.5 and 0.25 μg) is shown (top left), with western blots for DnaD, DnaI and DnaB, as indicated. DnaB is truncated to smaller versions. Pre-stained molecular weight markers from New England Biolabs are indicated in kDa.

Mentions: Regulation of initiation of DNA replication is linked to the balance between DnaA levels and the number of DnaA-binding sites in the cell (38, 39). DnaA is regulated at the transcriptional level (39). The fate of the mRNAs of other primosomal genes during growth is unknown. We investigated the mRNA levels of dnaD, dnaB and dnaI during growth of asynchronous B. subtilis cultures by quantitative RT-PCR. Control experiments to verify direct proportionality between amounts of RNA and PCR signals were carried out and data were normalized against the sigA mRNA (data not shown). SigA codes for σA which is essential for transcription of housekeeping genes. Its mRNA remains constant during exponential growth (40). Comparison of CT values from RT-PCR experiments revealed that mRNA levels of dnaB, dnaI and dnaD were constant throughout growth (Figure 1A). The dnaB and dnaI genes are juxtaposed in the same operon (41) while dnaD is located in a distant operon. CT values indicated that the mRNA levels of dnaB and dnaI were identical and dnaD slightly higher (Figure 1A). Normalization against sigA mRNA and direct comparisons of ΔCT values indicated similar levels for each of the three mRNAs at t1 (early growth-phase) and t2 (middle growth phase) and only a marginal decrease at t3 (late growth phase) that was statistically insignificant. It was not possible to normalize samples from late stationary growth phase relative to each other as control sigA mRNA levels were not constant in these samples (data not shown). Attempts to use a different control, 16S rRNA, were unsuccessful because of growth dependent variability (data not shown). From these data we conclude that the mRNAs of dnaD, dnaB and dnaI are maintained throughout asynchronous growth.Figure 1.


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)

Growth-dependent expression of dnaD, dnaI and dnaB genes. (A) Growth curve of B. subtilis strain 168 in LB at 30°C over time. Time points t1, t2 and t3 when samples were analysed are indicated. The top bar graph shows levels of dnaB, dnaD, dnaI and sigA as CT values from RT-PCR over time. The bottom bar graph shows changes in gene expression calculated from the formula 2−ΔΔCt for dnaB, dnaD and dnaI, using sigA as control and t1 as calibrator, over time. In both graphs each bar represents the mean of five independent experiments. The error bars indicate standard deviations. (B) Western blot analysis of B. subtilis soluble extracts from overnight cultures. A typical SDS–PAGE gel with decreasing amounts of extracts (72, 36, 18, 9, 4.5, 2.2, 1.1, 0.5 and 0.25 μg) is shown (top left), with western blots for DnaD, DnaI and DnaB, as indicated. DnaB is truncated to smaller versions. Pre-stained molecular weight markers from New England Biolabs are indicated in kDa.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2874997&req=5

Figure 1: Growth-dependent expression of dnaD, dnaI and dnaB genes. (A) Growth curve of B. subtilis strain 168 in LB at 30°C over time. Time points t1, t2 and t3 when samples were analysed are indicated. The top bar graph shows levels of dnaB, dnaD, dnaI and sigA as CT values from RT-PCR over time. The bottom bar graph shows changes in gene expression calculated from the formula 2−ΔΔCt for dnaB, dnaD and dnaI, using sigA as control and t1 as calibrator, over time. In both graphs each bar represents the mean of five independent experiments. The error bars indicate standard deviations. (B) Western blot analysis of B. subtilis soluble extracts from overnight cultures. A typical SDS–PAGE gel with decreasing amounts of extracts (72, 36, 18, 9, 4.5, 2.2, 1.1, 0.5 and 0.25 μg) is shown (top left), with western blots for DnaD, DnaI and DnaB, as indicated. DnaB is truncated to smaller versions. Pre-stained molecular weight markers from New England Biolabs are indicated in kDa.
Mentions: Regulation of initiation of DNA replication is linked to the balance between DnaA levels and the number of DnaA-binding sites in the cell (38, 39). DnaA is regulated at the transcriptional level (39). The fate of the mRNAs of other primosomal genes during growth is unknown. We investigated the mRNA levels of dnaD, dnaB and dnaI during growth of asynchronous B. subtilis cultures by quantitative RT-PCR. Control experiments to verify direct proportionality between amounts of RNA and PCR signals were carried out and data were normalized against the sigA mRNA (data not shown). SigA codes for σA which is essential for transcription of housekeeping genes. Its mRNA remains constant during exponential growth (40). Comparison of CT values from RT-PCR experiments revealed that mRNA levels of dnaB, dnaI and dnaD were constant throughout growth (Figure 1A). The dnaB and dnaI genes are juxtaposed in the same operon (41) while dnaD is located in a distant operon. CT values indicated that the mRNA levels of dnaB and dnaI were identical and dnaD slightly higher (Figure 1A). Normalization against sigA mRNA and direct comparisons of ΔCT values indicated similar levels for each of the three mRNAs at t1 (early growth-phase) and t2 (middle growth phase) and only a marginal decrease at t3 (late growth phase) that was statistically insignificant. It was not possible to normalize samples from late stationary growth phase relative to each other as control sigA mRNA levels were not constant in these samples (data not shown). Attempts to use a different control, 16S rRNA, were unsuccessful because of growth dependent variability (data not shown). From these data we conclude that the mRNAs of dnaD, dnaB and dnaI are maintained throughout asynchronous growth.Figure 1.

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.

Show MeSH
Related in: MedlinePlus