SirA inhibits the essential DnaA:DnaD interaction to block helicase recruitment during<i>Bacillus subtilis</i>sporulation

Winterhalter, Charles; Stevens, Daryl; Fenyk, Stepan; Pelliciari, Simone; Marchand, Elie; Soultanas, Panos; Ilangovan, Aravindan; Murray, Heath

doi:10.1093/nar/gkac1060

Cited by 3 publications

(11 citation statements)

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“…DnaD is conserved in Firmicute pathogens and an alignment of homologous DnaD CTT sequences indicated the recurrence of positively charged and aromatic residues within this region (Figure 1C ). However, because alanine scanning mutagenesis did not identify single residues within the DnaD CTT that were essential for DNA binding ( 20 ), we hypothesized that the DnaD CTT contains a redundant set of residues capable of recognising nucleic acid substrates.…”

Section: Resultsmentioning

confidence: 99%

“…Next, DnaD variants with alterations to the C-terminal tail were purified: DnaD 7A and two truncations, which removed either the DnaB interaction region (DnaD 1-224 ( 20 )) or the entire C-terminal tail containing the putative ssDNA binding residues (DnaD 1-205 ). Both DnaD 7A and DnaD 1-205 were unable to interact with a fluorescently labelled dT 40 substrate, whereas DnaD 1-224 retained activity similar to wild-type (Figure 3E ).…”

Section: Resultsmentioning

confidence: 99%

“…It has been found that DnaD requires a direct interaction with DnaA to be recruited to oriC ( 20 ). We speculated that this protein:protein interaction could guide DnaD to the B. subtilis chromosome origin for it to bind DNA (Figure 4A ) ( 55 ).…”

Section: Resultsmentioning

confidence: 99%

“…However, the mechanism for loading a helicase onto the opposite strand is unclear. Recently, a region of DnaA domain I was identified to be essential for the recruitment of DnaD to oriC ( 20 ), raising the possibility that this interaction might guide the deposition of a second helicase at the replication origin.…”

Section: Introductionmentioning

confidence: 99%

“…The DnaD protein is comprised of an N-terminal domain (DnaD NTD ), a C-terminal domain (DnaD CTD ) and a C-terminal tail (DnaD CTT ). A recent alanine scan characterised the residues of DnaD necessary for its functions in vivo , identifying those required for tetramerization and protein:protein interactions with DnaA and DnaB (Figure 1B ) ( 20 ). Conspicuously however, this analysis did not identify any residues required for DNA binding.…”

Section: Introductionmentioning

confidence: 99%

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The DNA replication initiation protein DnaD recognises a specific strand of the Bacillus subtilis chromosome origin

Winterhalter

Pelliciari

Stevens

et al. 2023

Nucleic Acids Research

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Genome replication is a fundamental biological activity shared by all organisms. Chromosomal replication proceeds bidirectionally from origins, requiring the loading of two helicases, one for each replisome. However, the molecular mechanisms underpinning helicase loading at bacterial chromosome origins (oriC) are unclear. Here we investigated the essential DNA replication initiation protein DnaD in the model organism Bacillus subtilis. A set of DnaD residues required for ssDNA binding was identified, and photo-crosslinking revealed that this ssDNA binding region interacts preferentially with one strand of oriC. Biochemical and genetic data support the model that DnaD recognizes a new single-stranded DNA (ssDNA) motif located in oriC, the DnaD Recognition Element (DRE). Considered with single particle cryo-electron microscopy (cryo-EM) imaging of DnaD, we propose that the location of the DRE within oriC orchestrates strand-specific recruitment of helicase during DNA replication initiation. These findings significantly advance our mechanistic understanding of bidirectional replication from a bacterial chromosome origin.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

The DNA replication initiation protein DnaD recognises a specific strand of the Bacillus subtilis chromosome origin

Winterhalter

Pelliciari

Stevens

et al. 2023

Nucleic Acids Research

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The bacterial replication origin BUS promotes nucleobase capture

Pelliciari,

Bodet-Lefèvre,

Fenyk

et al. 2023

Nat Commun

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Genome duplication is essential for the proliferation of cellular life and this process is generally initiated by dedicated replication proteins at chromosome origins. In bacteria, DNA replication is initiated by the ubiquitous DnaA protein, which assembles into an oligomeric complex at the chromosome origin (oriC) that engages both double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) to promote DNA duplex opening. However, the mechanism of DnaA specifically opening a replication origin was unknown. Here we show that Bacillus subtilis DnaAATP assembles into a continuous oligomer at the site of DNA melting, extending from a dsDNA anchor to engage a single DNA strand. Within this complex, two nucleobases of each ssDNA binding motif (DnaA-trio) are captured within a dinucleotide binding pocket created by adjacent DnaA proteins. These results provide a molecular basis for DnaA specifically engaging the conserved sequence elements within the bacterial chromosome origin basal unwinding system (BUS).

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Time-Course Transcriptome Analysis of Bacillus subtilis DB104 during Growth

et al. 2023

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Bacillus subtilis DB104, an extracellular protease-deficient derivative of B. subtilis 168, is widely used for recombinant protein expression. An understanding of the changes in gene expression during growth is essential for the commercial use of bacterial strains. Transcriptome and proteome analyses are ideal methods to study the genomic response of microorganisms. In this study, transcriptome analysis was performed to monitor changes in the gene expression level of B. subtilis DB104 while growing on a complete medium. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, K-mean cluster analysis, gene ontology (GO) enrichment analysis, and the function of sigma factors were used to divide 2122 differentially expressed genes (DEGs) into 10 clusters and identified gene functions according to expression patterns. The results of KEGG pathway analysis indicated that ABC transporter is down-regulated during exponential growth and metabolic changes occur at the transition point where sporulation starts. At this point, several stress response genes were also turned on. The genes involved in the lipid catabolic process were up-regulated briefly at 15 h as an outcome of the programmed cell death that postpones sporulation. The results suggest that changes in the gene expression of B. subtilis DB104 were dependent on the initiation of sporulation. However, the expression timing of the spore coat gene was only affected by the relevant sigma factor. This study can help to understand gene expression and regulatory mechanisms in B. subtilis species by providing an overall view of transcriptional changes during the growth of B. subtilis DB104.

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SirA inhibits the essential DnaA:DnaD interaction to block helicase recruitment duringBacillus subtilissporulation

Cited by 3 publications

References 99 publications

The DNA replication initiation protein DnaD recognises a specific strand of the Bacillus subtilis chromosome origin

The DNA replication initiation protein DnaD recognises a specific strand of the Bacillus subtilis chromosome origin

The bacterial replication origin BUS promotes nucleobase capture

Time-Course Transcriptome Analysis of Bacillus subtilis DB104 during Growth

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