DNA replication initiation in <i>Bacillus subtilis</i>; Structural and functional characterisation of the essential DnaA-DnaD interaction

Martin, Eleyna M.; Williams, Huw E. L.; Pitoulias, Matthaios; Stevens, Daryl; Winterhalter, Charles; Craggs, Timothy D.; Murray, Heath; Searle, Mark S.; Soultanas, Panos

doi:10.1101/444885

Cited by 4 publications

(6 citation statements)

References 41 publications

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“…This may occur either directly, as for Escherichia coli DnaA (35,36) or indirectly, as for B. subtilis DnaA (Figure 1A) (37,38). Interestingly, in both cases the same surface of DnaA domain I is suggested to be involved (Supplementary Figure S1B, C) (34,37,(39)(40)(41).…”

Section: Introductionmentioning

confidence: 94%

“…Having identified the essential surface of DnaD required to interact with DnaA, we next determined essential residues in DnaA required to interact with DnaD. Previous twohybrid and NMR studies implicated residues on the surface of DnaA DI that interact with DnaD (37,40). To investigate the physiological relevance of the proposed DnaA DI interface, we replaced the endogenous dnaA gene with mutants encoding alanine substitutions at key residues (dnaA T26A , dnaA W27A , dnaA F49A ) (Figure 5A).…”

Section: The Interaction Between Dnaa DI and Dnad Is Necessary For Dn...mentioning

confidence: 99%

“…The DnaD protein:protein interactions required for B. subtilis DNA replication initiation have yet to be clearly defined. An interaction with DnaA was proposed to involve residues located in both the DnaD NTD (Phe51, Ile83, Glu95) and the DnaD CTD (Ile132, Glu134, Glu169, Val171) (37,40), while an interaction with DnaB was suggested to involve the DnaD NTD (37,43). Critically however, the functional significance of specific DnaD interactions with DnaA and DnaB in vivo was unclear.…”

Section: Introductionmentioning

confidence: 99%

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

Winterhalter

Stevens

Fenyk

et al. 2022

Nucleic Acids Research

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Bidirectional DNA replication from a chromosome origin requires the asymmetric loading of two helicases, one for each replisome. Our understanding of the molecular mechanisms underpinning helicase loading at bacterial chromosome origins is incomplete. Here we report both positive and negative mechanisms for directing helicase recruitment in the model organism Bacillus subtilis. Systematic characterization of the essential initiation protein DnaD revealed distinct protein interfaces required for homo-oligomerization, interaction with the master initiator protein DnaA, and interaction with the helicase co-loader protein DnaB. Informed by these properties of DnaD, we went on to find that the developmentally expressed repressor of DNA replication initiation, SirA, blocks the interaction between DnaD and DnaA, thereby restricting helicase recruitment from the origin during sporulation to inhibit further initiation events. These results advance our understanding of the mechanisms underpinning DNA replication initiation in B. subtilis, as well as guiding the search for essential cellular activities to target for antimicrobial drug design.

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Section: Introductionmentioning

confidence: 94%

Section: The Interaction Between Dnaa DI and Dnad Is Necessary For Dn...mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Winterhalter

Stevens

Fenyk

et al. 2022

Nucleic Acids Research

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“…Part of this control is mediated by protein-protein interactions and involves various protein regulators that bind DnaA and affect its activity [58][59][60][61][62]. In B. subtilis, four proteins, SirA, Soj, DnaD and YabA, have been identified to regulate DnaA activity or its assembly at oriC through direct interaction [58,[63][64][65].…”

Section: Introductionmentioning

confidence: 99%

Prophage-encoded small protein YqaH counteracts the activities of the replication initiator DnaA in Bacillus subtilis

Ventroux

Noirot‐Gros

2022

Microbiology

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Bacterial genomes harbour cryptic prophages that are mostly transcriptionally silent with many unannotated genes. Still, cryptic prophages may contribute to their host fitness and phenotypes. In Bacillus subtilis, the yqaF-yqaN operon belongs to the prophage element skin, and is tightly repressed by the Xre-like repressor SknR. This operon contains several small ORFs (smORFs) potentially encoding small-sized proteins. The smORF-encoded peptide YqaH was previously reported to bind to the replication initiator DnaA. Here, using a yeast two-hybrid assay, we found that YqaH binds to the DNA binding domain IV of DnaA and interacts with Spo0A, a master regulator of sporulation. We isolated single amino acid substitutions in YqaH that abolished the interaction with DnaA but not with Spo0A. Then, using a plasmid-based inducible system to overexpress yqaH WT and mutant derivatives, we studied in B. subtilis the phenotypes associated with the specific loss-of-interaction with DnaA (DnaA_LOI). We found that expression of yqaH carrying DnaA_LOI mutations abolished the deleterious effects of yqaH WT expression on chromosome segregation, replication initiation and DnaA-regulated transcription. When YqaH was induced after vegetative growth, DnaA_LOI mutations abolished the drastic effects of YqaH WT on sporulation and biofilm formation. Thus, YqaH inhibits replication, sporulation and biofilm formation mainly by antagonizing DnaA in a manner that is independent of the cell cycle checkpoint Sda.

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“…Part of this control is mediated by protein-protein interactions and involves various protein regulators that bind DnaA and affect its activity (Felicori et al, 2016a; Jameson and Wilkinson, 2017; Katayama et al, 2017; Riber et al, 2016; Skarstad and Katayama, 2013). In B. subtilis , four proteins SirA, Soj, DnaD and YabA have been identified to regulate DnaA activity or its assembly at oriC through direct interaction (Bonilla and Grossman, 2012; Felicori et al, 2016a; Martin et al, 2019; Murray and Errington, 2008).…”

Section: Introductionmentioning

confidence: 99%

Prophage-encoded small protein YqaH counteracts the activities of the replication initiator DnaA inBacillus subtilis

Ventroux¹,

Noirot‐Gros²

2020

Preprint

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Bacteriophages are able to hijack host essential machineries to benefit their fitness and assemble their own progeny. Phage proteins targeting major bacterial pathways can be powerful tools to understand cell functions and have possible applications in human health and industry. Bacterial genomes also harbor cryptic prophages carrying genes that may contribute to their host fitness and properties. The cryptic prophages are mostly transcriptionally silent and most of the functions they encode are not annotated. In B. subtilis, the 48 kb-long skin element is a prophage carrying the yqaF-yqaN operon, which is tightly regulated by the Xre-like repressor sknR. The small yqaH gene potentially encodes the protein YqaH in absence of SknR. It was previously reported that YqaH interacts with the replication initiator DnaA in yeast two-hybrid assay and its expression in B. subtilis causes defects in the chromosomal cycle. In this study, we report that, in addition to DnaA, YqaH interacts with Spo0A, a master regulator of sporulation. To decipher yqaH mode of action, we used the yeast two-hybrid to isolate single mutations in yqaH that separate interactions with DnaA and Spo0A. We isolated mutations that caused loss-of-interaction (LOI) with DnaA but not Spo0A. However, all mutations disrupting the interaction with Spo0A were also DnaA-LOI functions, suggesting that these functions could not be separated. We found that expression YqaH carrying DnaA-LOI mutations affects both chromosome integrity and DnaA-mediated transcription, leading to growth inhibition as well as preventing bacterial development such as sporulation and biofilm formation. These results show that YqaH acts as an antimicrobial peptide in B. subtilis and pave the way for the structural design of mutants with improved antibacterial action.

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DNA replication initiation in Bacillus subtilis; Structural and functional characterisation of the essential DnaA-DnaD interaction

Cited by 4 publications

References 41 publications

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

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

Prophage-encoded small protein YqaH counteracts the activities of the replication initiator DnaA in Bacillus subtilis

Prophage-encoded small protein YqaH counteracts the activities of the replication initiator DnaA inBacillus subtilis

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